The administration of iodinated contrast material may be associated with AKI. Historically, when AKI occurred after administration of iodinated contrast material, it was referred to as contrast-induced nephropathy (CIN); however, because it is often not possible to exclude other causes of AKI, the term contrast-associated AKI (CA-AKI) has been more commonly used. The incidence of CA-AKI is controversial and varies from <1% to >30% with little consensus. In some patients with moderate to advanced CKD, the risk of CA-AKI may be as high as 50%. Other risk factors include diabetes mellitus, effective arterial volume depletion, use of high-osmolality contrast media, advanced age, proteinuria, and anemia. ^,

Unlike many other forms of intrinsic tubular injury, radiocontrast medium–induced AKI is usually associated with urinary sodium retention and fractional excretion of sodium (FE _Na ) of <1%. AKI resulting from iodinated contrast media is typically nonoliguric and rarely requires dialysis. However, requirements for renal support, prolonged hospitalization, and increased mortality are associated with (although not necessarily caused by) this condition.

The pathophysiology of CA-AKI likely consists of combined hypoxic and toxic renal tubular damage associated with renal endothelial dysfunction and alterations in the microcirculation. ^, The administration of radiocontrast media causes vasoconstriction and markedly affects renal parenchymal oxygenation, especially in the outer medulla, as documented in various studies where the cortical P O ₂ declined from 40 to 25 mm Hg and the medullary P o ₂ fell from 26 to 30 mm Hg to 9 to 15 mm Hg. Radiocontrast media injection leads to an abrupt but transient increase in renal plasma flow, GFR, and urinary output. This effect is due to the hyperosmolar radiocontrast medium–enhancing solute delivery to the distal nephron and leads to increased oxygen consumption by enhanced tubular sodium reabsorption. Video microscopy has shown that radiocontrast media markedly reduce inner medullary papillary blood flow, even to the extent of near-cessation of red blood cell (RBC) movement in papillary vessels, associated with RBC aggregation within the papillary vasa recta. In isolated vasa recta from rats and humans contrast medium applied to the lumen has led to constriction and enhanced vasa recta responses to Ang II. ^, However, it should be noted that there may be different patterns of response possibly related to the type, volume, and route of radiocontrast medium administration. Numerous neurohumoral mediators may contribute to the changes in renal microcirculation caused by radiocontrast medium injection. Intrarenal NO synthase activity, NO concentration, plasma endothelin, adenosine, prostaglandins, and vasopressin are all thought to play a role in altering the cortical and medullary microcirculation after radiocontrast medium injection. Mechanical factors may also play a role because radiocontrast media increase blood viscosity and may affect the flow in the complex, low-pressure medullary microcirculation. An increased plasma viscosity after radiocontrast medium administration can interfere with blood flow, particularly under the hypertonic conditions of the (inner) renal medulla, where the plasma viscosity is already increased as a result of hemoconcentration. Indeed, several animal studies have shown a correlation between experimental CIN and viscosity of the radiopaque compound. ^,

Evidence also suggests direct tubular toxicity from radiocontrast media. Radiocontrast media has direct toxic effects on proximal tubular cells (PTCs) in vitro. Radiocontrast media (e.g., diatrizoate and iopamidol) induced a decline in tubule K ⁺ , adenosine triphosphate (ATP), and total adenine nucleotide contents. At the same time, there was a decrease in the respiratory rate of the tubules and an increase in intracellular Ca ²⁺ content. These changes were more pronounced with the very high-osmolality ionic compound diatrizoate than the lower osmolality nonionic iopamidol. Importantly, cytotoxic effects were aggravated by hypoxia, indicating interactions between direct cellular mechanisms and vasoconstriction-mediated hypoxia. Radiocontrast media cause release of tubular marker enzymes, ultrastructural changes, and cell death in both Madin-Darby canine kidney (MDCK) and porcine kidney proximal tubule epithelial (LLC-PK ₁ ) cells. Radiocontrast medium–induced critical medullary hypoxia may lead to formation of reactive oxygen species (ROS), with subsequent membrane and DNA damage. A vicious cycle of hypoxia, free radical formation, and further hypoxic injury may be activated after radiocontrast medium exposure. Clinically, CA-AKI presents as an acute decline in the GFR within 24 to 48 hours of administration, with a peak serum creatinine concentration usually occurring in 3 to 5 days and return to baseline within 1 week, although return to baseline serum creatinine concentration may take longer in patients with moderate to advanced CKD. Existing CKD, diabetic nephropathy, advanced age, congestive heart failure, volume depletion, and coincident use of NSAIDs also increase the risk for CIN.

Unlike the first-generation contrast media, high-osmolar contrast media (HOCM; osmolality of 1500–1800 mOsm/kg), low-osmolar contrast media (LOCM; osmolality of 600–850 mOsm/kg), and isosmolar contrast media (IOCM; osmolality of 270–320 mOsm/kg) are associated with a lower incidence of contrast-induced AKI. However, whether the incidence of contrast-induced AKI is lower in IOCM than LOCM is still debated. One factor that may mitigate against the theoretic value of IOCM versus LOCM is that IOCM is a dimer and has a higher viscosity than LOCM. Viscosities for HOCM, LOCM, and IOCM are 0.00275, 0.00525, and 0.0114 pascal-second (Pa·s) at 37 ^o C, respectively. According to Poiseuille’s law, the greater the viscosity, the greater the resistance to fluid flow and the greater the shear stress on the vascular endothelium. Following intravenous injection, contrast medium becomes diluted in the bloodstream, the viscosity and osmolality are reduced, and therefore nonkidney organs are exposed to low concentrations of contrast medium. In the kidney, however, because of the increase in medullary osmolality, the concentration of contrast medium increases in the peritubular capillary and, following filtration, the concentration of contrast rises in the tubule lumen. The consequence is that 1. the distal tubules are exposed to increasing concentration and viscosity of contrast medium and 2. the tubule flow rate decreases, leading to prolonged exposure to contrast, potentially enhancing direct tubule nephrotoxicity. ^, Furthermore, when infused in animals, medullary P O ₂ is lower when IOCM is utilized when compared with LOCM.

The nephrotoxicity of aminoglycosides has best been characterized for gentamicin, a polar drug excreted by glomerular filtration. It is thought that cationic amino groups (NH ₃ ⁺ ) on the drug bind to anionic phospholipid residues on the brush border of PTCs, and the drug is then internalized by endocytosis. Although the precise cellular mechanisms responsible for renal accumulation of aminoglycosides have not been fully elucidated, binding to the endocytic complex formed by megalin and cubilin at the apical surface of PTCs appears important. ^, The complete elimination of aminoglycoside uptake in mice deficient in megalin suggests that this is the major pathway responsible for renal aminoglycoside accumulation. Chloride transporters including cystic fibrosis transmembrane conductance regulator (CFTR) and the ClC-5 have been implicated because mice lacking functional CFTR (Cftr ^ΔF/ΔF ) or deficient in the Cl ^– /H ⁺ exchanger (Clcn5 ^Y/– ) have decreased kidney accumulation of gentamicin. A three-dimensional (3D) model has described the complexity between megalin and gentamicin. Gentamicin binds to megalin with low affinity and exploits the common ligand-binding motif using the indole side chain of amino acids Trp-1126 and the negatively charged residues of Asp-1129, Asp-1131, and Asp-1133. Once endocytosed, aminoglycosides inhibit endosomal fusion. They are also directly trafficked to the Golgi apparatus and, through retrograde movement, to the endoplasmic reticulum (ER). From the ER, gentamicin moves into the cytosol in a size- and charge-dependent manner. Once in the cytosol, either from the ER or via lysosomal rupture, aminoglycosides distribute to various intracellular organelles and mediate organelle-specific toxicity, such as mitochondrial dysfunction. ^, Gentamicin acts on mitochondria, activating the intrinsic pathway of apoptosis, disrupting ATP production, ^, and producing hydroxyl radicals and superoxide anions. ^, Also, delivery to the ER via retrograde transport from the Golgi apparatus allows for the binding of aminoglycosides to the 16S rRNA subunit, resulting in a reduction of protein synthesis ^, and altering posttranslational protein folding. The number of cationic groups on the molecules determines the facility with which these drugs are transported across the cell membrane and is an important determinant of toxicity. ^, Neomycin is associated with the most nephrotoxicity, gentamicin, tobramycin, and amikacin are intermediate, and streptomycin is the least nephrotoxic.

Receptor-associated protein (RAP) and cilastin, known to associate with megalin and block proximal tubule endocytosis, reduce gentamicin nephrotoxicity. ^, Risk factors for aminoglycoside nephrotoxicity include the use of high or repeated doses or prolonged therapy, CKD, volume depletion, diabetes, advanced age, and the coexistence of renal ischemia, hypotension, sepsis or use of other nephrotoxins.

Vancomycin was discovered, developed, and approved by the U.S. Food and Drug Administration (FDA) in 1958 ^, and was found to be active against most gram-positive organisms including penicillin-resistant staphylococci. Initial formulations of the drug were dubbed “Mississippi mud” due to the brown color presumably due to impurities, which were thought to be responsible for nephrotoxicity and ototoxicity. Reformulation resulted in a drug that had improved purity and was called vancomycin (from the word vanquish).

The incidence of vancomycin nephrotoxicity is variable, ranging from as low as 0% to >40%, and those with vancomycin-associated nephrotoxicity are more likely to have higher trough levels and prolonged duration of treatment. Durations of 7 to more than 15 days of treatment are associated with nephrotoxicity. With modern formulations of vancomycin, the variability in nephrotoxicity is due to concomitant drug use, severity of illness, and variability in the definition of AKI. Daily total dose of vancomycin of more than 4 g and supratherapeutic levels (>30 mg/L) are a risk factor for nephrotoxicity. ^, Vancomycin-associated nephrotoxicity is thought to be more common when combined with antipseudomonal β-lactams. In a retrospective matched cohort study of patients receiving vancomycin and cefepime (588 patients) or vancomycin and piperacillin-tazobactam (3605 patients), the unadjusted incidence of AKI was 12.6% versus 21.4%, respectively ( P <.0001). Vancomycin and piperacillin-tazobactam were associated with a more than twofold increase in the risk of AKI, after matching for severity of illness. In non-intensive care unit (ICU) pediatric patients, serum trough levels of vancomycin >24.35 μg/mL and concomitant use of piperacillin-tazobactam predicted nephrotoxicity and longer hospital stay.

In a patient who received vancomycin without coadministration of an aminoglycoside, the biopsy showed obstructive tubular casts composed of noncrystal nanospheric vancomycin aggregates associated with uromodulin. In eight additional patients with AKI associated with high vancomycin levels, vancomycin-associated casts were also found. These findings, which were reproduced in mice given vancomycin, demonstrate a link between vancomycin and AKI.

Treatment with cisplatin (cis-platinum), a platinum-based chemotherapeutic agent, is commonly associated with nephrotoxicity. The pathophysiologic mechanisms of cisplatin-induced tubular damage are complex and involve a number of interconnected factors, such as accumulation of cisplatin mediated by membrane transport, conversion into nephrotoxins, DNA damage, mitochondrial dysfunction, oxidative stress, inflammatory response, activation of signal transducers and intracellular messengers, autophagy, cell cycle regulation, and activation of cell death pathways ( Fig. 27.1 ).

The pathophysiology of cisplatin nephrotoxicity.

Uptake of cisplatin in the PTCs is mediated by solute carrier family 22 member 2 (SLC22A2), SLC22A6, and SLC22A8 and its extrusion is mediated by SLC47A1 (MATE1). Upon entering PTCs, cisplatin is hydrated and activated to the toxic form by a γ-glutamyl transpeptidase (GGT) and kynurenine aminotransferase 1 (KYAT1)- dependent pathway. Cisplatin accumulation to toxic levels in PTCs induces DNA damage, mitochondrial damage, oxidative stress, endoplasmic reticulum (ER) stress, autophagy, and cell cycle regulation. Ultimately, these processes lead to cell death, acute kidney injury, and chronic kidney disease.

Created in BioRender. Okusa M. 2025. https://BioRender.com/bbf7fdx.

The S3 segment of the proximal tubule in the corticomedullary region is the primary target of cisplatin to AKI in rats. In contrast, more distal sites may be affected in humans; however, glomeruli remain unaffected. Cisplatin flux in renal tubular cells occurs through transporters in a basolateral to apical direction. The solute carrier family 22 member 2 (SLC22A2, also known as organic cation transporter 2 [OCT2]), which is expressed on the basolateral membrane of proximal convoluted tubules, and SLC31A1 (also known as the copper transport protein 1 [CTR1]) expressed on proximal and distal tubules are the main transporters mediating cisplatin uptake. Other transporters mediating uptake may include SLC22A6, SLC22A8. SLC47A1 (also known as multidrug and toxin extrusion 1 [MATE1]), expressed on the apical membrane of the proximal tubule cells, is responsible for the efflux of cisplatin. ^, When cisplatin was administered to Mate1 ^–/– mice, blood urea nitrogen (BUN) and creatinine levels were higher than in Mate1 ^+/+ mice. Furthermore, cisplatin levels were higher in plasma and in kidneys of Mate1 ^–/– mice compared with Mate1 ^+/+ mice, suggesting that MATE1 mediates the efflux of cisplatin and could contribute to cisplatin nephrotoxicity. The differential expression of SLC22A2, which is predominantly expressed in kidneys but only a few types of human tumor cells, provides the rationale for specifically targeting this transporter.

The effects on the kidneys of low but frequent doses of cisplatin given once a week for 4 weeks were studied. Mice who received multiple doses of cisplatin had increased kidney fibrotic markers including fibronectin, transforming growth factor (TGF)-β, and α-smooth muscle actin (SMA), as well as interstitial fibrosis. These studies in mice support observations in humans showing that adult patients with cancer who received multiple doses of cisplatin experience small but permanent declines in the estimated GFR (eGFR). ^,

Regulated necrosis contributes to cisplatin-induced AKI. In cisplatin-treated mice, necroptosis contributes to pathogenesis of cell death; receptor-interacting serine-threonine kinase (RIPK1), RIPK3, and mixed-lineage kinase domain–like protein (MLKL) expression were increased and deletion of Ripk3 or Mlkl or pharmacologic inhibition of RIPK1 attenuated kidney injury. ^, Pyroptosis, an inflammatory mode of regulated cell death, requires the formation of plasma membrane pores through Gasdermin D (GSDMD). Following cisplatin treatment, GSDMD is upregulated and mice deficient in Gsdmd are protected from cisplatin-induced AKI. Dysregulation of glutathione and iron metabolism by cisplatin leads to ferroptosis and AKI. A pharmacologic inhibitor of ferroptosis, namely Ferrostatin-1 (Fer-1), was reported to attenuate AKI.

Myoglobin and hemoglobin are the endogenous toxins most commonly associated with ATN. Myoglobin, a 17.8-kDa heme protein released during skeletal and cardiac muscle injury, is freely filtered and causes red-brown urine, with a dipstick result positive for heme in the absence of RBCs in the urine. Intravascular hemolysis results in circulating free hemoglobin, which, when excessive, is filtered, resulting in hemoglobinuria, hemoglobin cast formation, and heme uptake by PTCs. The uptake in the proximal tubule may be mediated via endocytosis by megalin and cubilin. Megalin knockout mice have reduced accumulation of injected myoglobin in tubule cells and reduced nephrotoxicity. The heme center of myoglobin may directly induce lipid peroxidation and, in addition, the liberation of free ferrous iron, depending on the redox potential, can promote hydroxyl radical formation by the Haber-Weiss (Fenton) reaction, resulting in the oxidation of lipids, proteins, and nucleic acids. ^, Iron is an intermediate accelerator in the generation of free radicals. Studies have suggested that there is increased formation of H ₂ O ₂ in rat kidney models of myohemoglobinuria. The subsequent hydroxyl (OH ⁻ ) radical plays a vital role in oxidative stress–induced AKI through mechanisms discussed in detail later in this chapter. In response, heme protein induces the heme-degradative enzyme, heme oxygenase, and increased synthesis of ferritin. Ferritin, a major factor in sequestering free iron, is made up of two types of 24 subunits, heavy chain and light chain. It is the ferritin heavy chain (FtH) that has ferroxidase activity necessary for iron incorporation and to limit toxicity. Proximal tubule-specific, FtH -knockout mice ( FtH ^PT-/- mice) have significant mortality in myoglobin-induced AKI, indicating the protective role of proximal tubule FtH in AKI. Various iron chelators such as deferoxamine and other scavengers of ROS, such as glutathione, provide protection against myohemoglobinuric AKI. Similarly, endothelin antagonists also prevent hypofiltration and proteinuria in rats that have undergone glycerol-induced rhabdomyolysis. NO supplementation may be beneficial by preventing heme-induced renal vasoconstriction perhaps, in part, because heme proteins scavenge NO. ^, Finally, precipitation of myoglobin with Tamm-Horsfall protein (THP) associated with shed PTCs leads to cast formation and tubular obstruction, which is enhanced in acidic urine. In human studies, volume expansion and perhaps alkalization of urine to limit cast formation are the preventive measures generally used because none of the experimental agents used in animal studies has been convincingly beneficial. This emphasizes the multifactorial nature of these conditions. It is unlikely that a single agent will be beneficial in this setting.

Regulated cell death pathways can be grouped into either a caspase-controlled cell death system (apoptosis, necroptosis, and pyroptosis) or a lipid peroxidation−autoxidation-controlled necrosis system (ferroptosis; Fig. 27.4 ).

Overview of the pathways of regulated cell death.

In general, two systems may be best differentiated when regulated cell death is considered. The caspase-controlled system includes apoptosis, necroptosis, and pyroptosis. In contrast, the peroxidation-controlled system of ferroptotic cell death functions entirely independently of the caspase-controlled network.

From Tonnus W, Gembardt F, Latk M, et al. The clinical relevance of necroinflammation highlighting the importance of acute kidney injury and the adrenal glands. Cell Death Differ. 2019;26[1]:68–82.

It is not dependent on caspase activation but results from a rise in intracellular calcium and the activation of membrane phospholipases. ^, Functionally, severe ATP depletion results first in mitochondrial injury, with subsequent arrest of oxidative phosphorylation, depletion of energy stores, and robust formation of ROS. This in turn mediates further cellular injury. In some cases, defined regulated molecular pathways may lead to necrotic cell death, a process referred to as necroptosis . Differentiation between necrosis and necroptosis requires that necroptosis-dependent cell death involves the receptor-interacting protein kinase 3 (RIPK3).

Cells undergoing sublethal or less severe injury have the capacity for functional and structural recovery if the insult is interrupted. Cells that suffer a more severe (or lethal) injury undergo apoptosis or necrosis. Apoptosis is an energy-dependent, programmed cell death due to both intrinsic (mitochondrial) and extrinsic activation of caspases resulting, either of which results in the condensation of nuclear and cytoplasmic material, forming apoptotic bodies. These apoptotic bodies, which are plasma membrane bound, are rapidly phagocytosed by macrophages and neighboring viable epithelial cells ( eFig. 27.2 ).

Morphologic features of necroptosis and apoptosis.

HT29 colon cancer cells treated with an anticancer drug for 48 hours were analyzed by transmission electron microscopy. The cell undergoing necroptosis shows plasma membrane rupture and permeabilization, compared with the intact plasma membrane, with blebbing in the apoptotic cell (red arrowheads). The necroptotic cell exhibits cytoplasm swelling and vacuolization, which are absent in the apoptotic cell (green arrowheads). The necroptotic cell has swelled mitochondria, in contrast to those in the apoptotic cells (yellow arrowheads). The necroptotic cell also lacks the condensed and fragmented nuclei seen in the apoptotic cell (blue arrowheads).

From Chen D, Yu J, Zhang L. Necroptosis: an alternative cell death program defending against cancer. Biochim Biophys Acta. 2016;1865[2]:228–236.

The caspase family of proteases is an important initiator and effector of apoptosis. ^, Both the intrinsic (mitochondrial) and extrinsic (death receptor) apoptotic pathways are activated in human AKI. Specifically, activation of procaspase-9 primarily depends on intrinsic mitochondrial pathways regulated by the Bcl-2 family of proteins, whereas that of procaspase-8 results from extrinsic signaling via cell surface death receptors, such as Fas and their ligand FADD (Fas-associated protein with death domain). There is also considerable crosstalk between the intrinsic and extrinsic pathways. The other group of caspases—3, 6, and 7 are effector caspases, which are more abundant and catalytically robust, cleaving many cellular proteins and resulting in the classic apoptotic phenotype.

Caspase activation in epithelial cells occurs due to ischemic and other cytotoxic insults, whereas inhibition of caspase activity is protective against such injury in cultured and in vivo renal epithelial tubular AKI. ^, Several pathways including the intrinsic (Bcl-2 family, cytochrome c , and caspase-9), extrinsic (Fas, FADD, and caspase-8), and regulatory (p53 and NF-κB) pathways appear to be activated during ischemic renal tubular cell injury. It has also been shown that the balance between cell survival and death depends on the relative concentrations of the proapoptotic (Bax, Bcl-2–associated death promoter [Bad], and Bid) and antiapoptotic (Bcl-2 and Bcl-xL) members of the Bcl-2 family of proteins. Overexpression of proapoptotic or relative deficiency of antiapoptotic proteins may lead to the formation of mitochondrial pores. Conversely, the inhibition of such pore formation may occur with the opposite imbalance.

Other proteins that have been shown to play a significant role in the apoptotic pathways include NF-κB and p53. ^, The kinase-mediated pathways such as ERKs and c-Jun N-terminal kinases (JNKs) are responsible for mediating cellular responses involved in apoptosis, survival, and repair through their interaction with other signals from growth factors, such as hepatocyte growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). ^, These independent mechanisms can inhibit proapoptotic proteins such as Bad and activate the antiapoptotic transcription of cyclic adenosine monophosphate response element binding (CREB) factors. There is rapid delivery of small interfering RNA (siRNA) to PTCs in AKI; targeting siRNA to minimize p53 production leads to a dose-dependent attenuation of apoptotic signaling and kidney function, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury. In vivo, microRNA-24 (miR-24) also regulates the HO-1 and H2A histone family, member X. Overall, these results indicate that miR-24 promotes renal ischemic injury by stimulating apoptosis in endothelial and tubular epithelial cells (TECs).

A novel Ying Yang 1-KIM1-death receptor 5 (YY1-KIM1-DR5) axis has been identified in the progression of AKI. The transcription factor YY1, a transcriptional repressor of KIM1, is downregulated upon AKI, leading to KIM1 secretion. KIM1 binds to DR5 and activates the caspase cascade inducing renal cell apoptosis and exacerbating AKI. Blocking the KIM1-DR5 interaction with rationally designed peptides exhibits kidney protection from AKI. Considering the various pathways available for blockade or modulation, the therapeutic implications of targeting apoptosis in preventing epithelial cell injury are significant. However, it is likely that the “window” to avert lethal injury and prevent cells from progressing to necrosis is in the early initiating apoptotic phases.

Numerous studies have shown that apoptosis lead to a rise in intracellular calcium through impairment of calcium ATPases, whereas inhibition of Na ⁺ -K ⁺ -ATPase activity resulting from ATP depletion potentiates calcium entry into the cell via the sodium-calcium exchanger. Increased cytosolic calcium causes further mitochondrial injury and cytoskeletal alterations. This chain of events results in the downstream activation of proteases such as calpain and phospholipases. Phospholipases such as phospholipase A2 cause direct hydrolytic damage to membranes and also release toxic free fatty acids. They also cause release of eicosanoids that have vasoactive and hemokinetic activities, resulting in an intense surrounding inflammatory response. Calpain activation mediates plasma membrane permeability and hydrolysis of the cytoskeletal proteins. ^, Finally, there is release of lysosomal enzymes and proteases that degrade histones, resulting in accessibility of the endonucleases to the entire segment, typically seen as the smear pattern on gel electrophoresis, in contrast to the typical ladder pattern seen in apoptosis.

Epithelial cell necrosis is a passive nonenergy-dependent process that develops secondary to severe ATP depletion from toxic or ischemic insult and is independent of caspase activation. In necrosis, there is cellular and organelle swelling, with loss of plasma membrane integrity and release of unprocessed intracellular contents, including cellular organelles, highly immunogenic proteins such as ATP, HMGB1, double-stranded DNA, and RNA components, also referred to as damage-associated molecular patterns (DAMPs), a concept known as necroinflammation . Differentiation between necrosis and necroptosis requires that necroptosis-dependent cell death involves the receptor-interacting protein kinase 3 (RIPK3) (see Fig. 27.4 ). Although tubular necrosis was thought to be accidental, work done over the past 2 decades has revealed several pathways of genetically determined and regulated necrosis in which receptor-interacting serine-threonine protein kinase 1 (RIPK1), ^, RIPK3 and its substrate, MLKL, have been directly implicated in the regulation of the novel cell death pathway known as necroptosis. This activation of unique signaling paradigms forms the basis for differentiating between necrosis and necroptosis. The signaling pathway that triggers necroptosis includes the engagement of death receptors in the presence of caspase inhibition, stimulation of Toll-like receptors, signaling through interferons, with the activation of kinase RIPK3, and phosphorylation of pseudokinase MLKL. Phosphorylation of MLKL by RIPK3 leads to a molecular switch mechanism that induces plasma membrane rupture. Demonstration of a protective effect on kidney function with the use of necrostatin-1, an inhibitor of RIPK1, suggests that necroptosis occurs in ischemic AKI. Necroptosis also occurs in the model of cisplatin-induced AKI, as demonstrated by the significant protection of kidney function when using RIPK3 and MLKL-deficient mice. Peptidylprolyl isomerase F (PPIF) and phosphorylated MLKL have been shown to be localized to injured tubules in human kidney biopsies of oxalosis-related AKI. Further, mice lacking PPIF or MLKL or given an inhibitor of mitochondrial permeability displayed attenuated oxalate-induced AKI.

Despite significant advances in necroptosis research in experimental systems, clinical studies on necroptosis inhibitors for the treatment of acute and chronic kidney disease are lacking. Few reports thus far could detect necroptosis in human ischemic kidney biopsies. One study of 10 transplanted living-donor kidneys, biopsied 1 hour after transplantation, identified phospho-MLKL staining on immunohistochemistry. A later study of kidney allografts with AKI identified phospho-MLKL in 10% to 15% of tubule cells. Interestingly, screening a panel of clinical plasma membrane channel blockers identified that an anticonvulsant drug, phenytoin, blocked upstream necrosome formation by attenuating RIPK1 kinase activity in vitro, due to the hydantoin scaffold that is also present in necrostatin-1. Phenytoin also protected mice from renal ischemia-reperfusion induced ( IRI) and tumor necrosis factor-α (TNF-α)-induced systemic inflammatory response syndrome (SIRS). These findings suggest that drugs used for other indications could be used in the future to target necroptosis for the benefit of patients with AKI.

Ferroptosis is a nonapoptotic form of regulated cell death that is iron dependent and characterized by increased lipid peroxidation resulting from lack of activity of the lipid repair selenoprotein enzyme glutathione peroxidase 4 (GPX4). This leads to the accumulation of lipid-based ROS including lipid hydroxyperoxides. This form of iron-dependent cell death is distinct from other forms of cell death such as apoptosis, unregulated necrosis, and necroptosis because it mediates cell death in a noncell autonomous and synchronized manner, providing a potential explanation for nephron loss during AKI. Erastin and RSL3 are ferroptosis-inducing compounds that induce cell death in the absence of apoptotic features. ^, Ferroptosis plays a key role in folic acid−induced AKI, resulting in increased inflammation. Ferroptosis is also importantly regulated by heme oxygenase-1 (HO-1), an enzyme discussed earlier that mediates the breakdown of heme. Immortalized proximal tubule cells from HO-1 ^+/+ mice were much more susceptible to erastin or RSL3 than cells from HO-1 ^–/– mice. Iron supplementation decreased cell viability further in HO-1 ^–/– compared with HO-1 ^+/+ cells. Finally, ferrostatin (a ferroptosis inhibitor), deferoxamine (an iron chelator), or N -acetyl- l -cysteine (a glutathione replenisher) attenuate erastin-induced ferroptosis (see Fig. 27.4 ).

Unbiased transcriptomics studies have identified novel targets for intervention in AKI. Charged multivesicular body protein 1a (CHMP1A) and dipeptidase 1 (DPEP1) regulate ferroptosis in tubular cells via altering cellular iron trafficking, and their deletion attenuated injury in cisplatin, folic acid, and UUO-induced kidney injury. Silencing the repressor element 1-silencing transcription factor (REST), a master regulator of gene repression under hypoxia, is upregulated in AKI patients, mice, and RTECs. Downregulation of REST attenuated ferroptosis in primary RTECs and AKI-to-CKD transition. Loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4cys/–) sensitizes kidneys to tubular ferroptosis. Linkermann and colleagues generated Nec-1f, which simultaneously targeted RIPK1 and ferroptosis. These findings warrant additional studies on the role of ferroptosis in AKI.

This highly inflammatory form of regulated cell death requires caspase 1,4, 5 (caspase 11 in mice) for activation ^, ( Fig. 27.4 ). Intracellular damage and release of DAMPs activate NLRP3 inflammasome leading to caspase activation and cleavage of pro−interleukin-1 β (IL-1β) and pro−IL-18 to their mature forms. Pyroptosis executioner, GSDMD, is activated, releasing the N-terminal fragment (GSDMD-NT), ^, which oligomerizes and forms a membrane pore on the cell membrane, leading to cell swelling, osmotic lysis, and release of IL-1β and IL-18 and other intracellular contents. ^, Unlike apoptosis, pyroptosis results in plasma membrane rupture and release of DAMPs, which activate innate immunity and recruitment of immune cells.

Multiple cell death pathways are involved in the regulation of successful and maladaptive repair; pyroptosis and NLRP3 inflammasomes are intertwined with AKI and fibrogenesis. Genes associated with pyroptosis ( Casp1, Casp3, Casp4, Il1b, Il18, and Gsdmd ) and ferroptosis ( Ptgs2, Chac1, Acsl4, Slc7a11, and Hmox1 ) were highly enriched in PT cells following long IRI. Attempts have been made to identify novel modulators of pyroptosis in AKI. During sepsis, inhibition with disulfiram or genetic deletion of GSDMD abrogated neutrophil activation formation, reducing multiorgan dysfunction and lethality. Another inhibitor, dapansutrile, was also found to inhibit the NLRP3 inflammasome and subsequent activation of IL-1β. The dsDNA sensor, absent in melanoma 2 (AIM2), forms an inflammasome and induces GSDMD cleavage, which activates pyroptosis. In a mouse model of rhabdomyolysis-induced AKI, AIM2 deficiency delayed recovery and worsened fibrosis in the kidney. Thus macrophage pyroptosis serves in an immunoregulatory capacity and determines the healing process in rhabdomyolysis-induced AKI.

CCAAT/enhancer-binding protein β (C/EBPβ), a critical regulator of the immunosuppressive environment in cancers, upregulates mitochondrial transcription factor A (TFAM) and induces the secretion of IL-1β and IL-18 in a model of sepsis-induced AKI, suggesting that an interaction between C/EBPβ and TFAM facilitated pyroptosis via NLRP3/caspase-1 signal. Other endogenous molecules, such as ketone bodies (e.g., β-hydroxybutyrate), were shown to protect against ischemic tissue injury via increasing the expression of forkhead transcription factor O3 (FOXO3), an upstream regulator of pyroptosis. Understanding cell death pathways can lead to specific interventions to preserve apoptosis and avoid inflammation while blocking the more inflammatory pathways such as pyroptosis, considered the most proinflammatory of the regulated pathways. During the COVID-19 pandemic, remdesivir (RDV, GS-5734), a broad-spectrum antiviral nucleotide prodrug, alleviated AKI by specifically inhibiting NLRP3 inflammasome activation in macrophages.

Autophagy is an essential mechanism for normal homeostasis, disease pathogenesis, and aging in kidneys. Fig. 27.5 reviews the three forms of autophagy—macroautophagy, microautophagy, and chaperone-mediated autophagy.

Autophagy.

The forms of autophagy are macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy starts with the de novo formation of a cup-shaped isolation double membrane that engulfs a portion of cytoplasm. Microautophagy involves the engulfment of cytoplasm instantly at the lysosomal membrane by invagination, protrusion, and separation. Chaperone-mediated autophagy is a process of direct transport of unfolded proteins via the lysosomal chaperonin hsc70 and LAMP-2A. All forms of autophagy subsequently lead to the degradation of intra-autophagosomal components by lysosomal hydrolases. PE, Phosphatidylethanolamine.

From Periyasamy-Thandavan S, Jiang M, Schoenlein P, Dong Z. Autophagy: molecular machinery, regulation, and implications for renal pathophysiology. Am J Physiol Renal Physiol. 2009;297[2]:F244–256.

Autophagy serves a protective function in renal tubular cells during ischemia-reperfusion injury; it precedes the appearance of apoptotic cells, and suppression of autophagy exacerbates renal injury, in part by inhibiting apoptosis. ^, The importance of autophagy has been demonstrated through the generation of proximal tubule-specific ATG5 or ATG7 knockout mice. The absence of ATG5 in proximal tubule cells led to the accumulation of deformed mitochondria and cytoplasmic inclusions or protein aggregates, ²⁰⁷ more proinflammatory cytokine production, and increased Ang II-induced phosphorylation and NF-κB activation, whereas overexpression of ATG5 attenuated activation of NF-κB and protected against renal inflammation. Similar results were observed in proximal tubule-specific ATG7 knockout mice. In contrast, the lack of ATG5 in distal tubules does not cause significant alterations in kidney function. Importantly, renal ischemia-reperfusion injury was exaggerated in proximal tubule-specific ATG5 or ATG7 knockout mice. ^{, ,} A number of AKI models demonstrate changes in the autophagy pathway. For instance, autophagy activation was also detected in cisplatin-induced AKI in mice. ATG7 ^–/– mice demonstrated worse kidney function and injury with cisplatin treatment. Chloroquine, an inhibitor of autophagy, blocked lysosomal degradation of microtubule associated protein LC3 in autophagosomes and worsened kidney function in a cisplatin-induced model of AKI. Similarly, autophagy was induced in the septic AKI model of LPS treatment of mice, and inhibition by chloroquine or tubular ATG7 ablation worsened LPS-induced AKI.

Mitophagy is a specialized form of autophagy, a cellular process that selectively targets and degrades damaged or dysfunctional mitochondria. Mediators of mitophagy are implicated in AKI: Pink1 and/or Park2-knockout mice had greater accumulation of damaged mitochondria, tubular cell death, ROS generation and inflammation, and worsening AKI outcomes. Of interest, PINK1/Parkin were downregulated in human CKD kidney samples and TGF-β1–treated human renal macrophages suggesting targets for intervention. Providing further evidence for a role of mitophagy in AKI, the induction of BNIP3-mediated mitophagy also provided renoprotection during ischemic AKI. Knockdown of prohibitin 2 (PHB2), a newly identified intracellular receptor of mitophagy that participates in the removal of damaged mitochondria, attenuated mitophagy leading to increased AKI, whereas overexpression of PHB2 ameliorated mitochondrial dysfunction and NLRP3 activation, highlighting PHB2 as a new target in AKI.

Caloric restriction mimics ischemic preconditioning (IPC) and stimulates the SIRT, SGK1–FOXO3a–(HIF1α) axis to prevent a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), leading to renoprotection. IPC induced autophagy in renal tubular cells in mice, and the protection was abolished by pharmacologic inhibitors of autophagy or PTC-specific deletion of Atg7. Treatment with Beclin1 peptide induced autophagy and protected against IRI. In sepsis-induced AKI, bone marrow–derived stem cells inhibited inflammation and upregulated expressions of Parkin and SIRT1, promoting mitophagy.

However, autophagy might also have a profibrotic role as chronic hypoxia after severe ischemic AKI; Atg5 deletion in the S3 segment inhibited tubular senescence, attenuated interstitial fibrosis, and improved kidney function. Chronic hypoxia after AKI activated FOXO3 in a HIF1α-dependent manner, and Foxo3 deletion attenuated autophagy and transition to CKD. Genetic deficiency in tubular autophagy diminished FGF2 in TGFB1/TGF-β1-treated renal tubular cells, and this effect was suppressed by FGF2 neutralizing antibody and reduced fibrosis. In humans, renal biopsies from post-AKI patients showed higher levels of autophagy and FGF2 in tubular cells that significantly correlated with renal fibrosis. The inhibition of mTOR could also induce persistent activation of autophagy and impaired proliferation. These findings indicate that autophagy is a dynamic process that is critical for survival of tubular cells initially after AKI and later stages may regulate fibrosis productive renal repair.

Thus taken together, these collective data, from both pharmacologic studies and genetically deficient mice, have provided strong evidence for a renoprotective role of autophagy. In contrast, dysregulated autophagy can result in increased renal degeneration, leading to progressive kidney disease, as characterized by interstitial fibrosis. ^,

The interstitial microenvironment, that region between the basement membranes of epithelial cells and peritubular capillaries, is a reactive compartment containing mononuclear phagocytes, interstitial fibroblasts, and pericytes, as well as various soluble mediators. Leukocytes, including but not limited to macrophages, NKT cells, B lymphocytes, and T lymphocytes, contribute to kidney IRI. Selective depletion, knockout mouse models, and specific blockade have shown that all of these cells interact and contribute to tubular injury at various phases. ^,

The mononuclear phagocytic system consists of bone marrow–derived macrophages and dendritic cells, which overlap in both functional characteristics and surface biomarkers. Macrophages are resident tissue phagocytic cells that function generally to clear dying cells and produce cytokines and growth factors. ^, Although the mononuclear phagocytic system is well known to be activated in response to foreign pathogens, the microenvironment responds to endogenous molecules released from dying cells following tissue injury or to changes produced by conditions, such as hypoxia, ischemia, or other forms of sterile inflammation. ^, Matzinger proposed the danger model to explain exceptions to the classical role of immune responsiveness to foreign antigens. Dendritic cells are activated by DAMPs or pathogen-associated molecular patterns (PAMPs) and are key initiators of the innate immune system following ischemia-reperfusion ( eFig. 27.3 ). Dendritic cells, a resident population of bone marrow–derived cells, and macrophages form a network between the basement membranes of tubular epithelial and peritubular endothelial cells. ^, Although dendritic cells and macrophages are often considered distinct cell types with characteristic functions, they share considerable overlap in cell surface markers and function ( Fig. 27.6 ).

Heterogeneity of mononuclear phagocytes within the microenvironment.

Mononuclear phagocytes within the microenvironment, identified as CX3CR1 ⁺ /GFP ⁺ cells (labeled green ), in the kidney are shown in panels A to D, and the heterogeneity of mononuclear phagocytes is shown in panel E. (A) A heterogeneous population of CX3CR1 ⁺ /GFP ⁺ mononuclear phagocytes in superficial cortex includes dendritic cells and macrophages and reside from the cortex and medulla. CX3CR1 ⁺ GFP ⁺ cells extend to the edge of the cortex (large arrow) and populate the entire interstitial space abundantly. The Bowman capsule is encased by CX3CR1 ⁺ /GFP ⁺ cells (small arrows). Also notable are CX3CR1 ⁺ GFP ⁺ cells that lie within each glomeruli. (B) Rhodamine-conjugated peanut agglutinin (red fluorescence) highlights distal tubules and collecting ducts, capsule (upper left) to the papilla (lower right). (C) Magnification of the boxed area in panel B showing CX3CR1 ⁺ GFP ⁺ cells in the interstitium of the medulla, including transition into pyramidal tracks. Note the same spatial regularity as in the cortex. (D) Three-dimensional rendering of stellate-shaped CX3CR1 ⁺ GFP ⁺ cells within the interstitium demarcated by tubular segments in the medulla (red fluorescence). (E) Kidney sections from the medulla of CX3CR1 ⁺ /GFP ⁺ mice. GFP is expressed mainly on monocyte-macrophages and dendritic cells, and many CX3CR1 ⁺ GFP ⁺ green fluorescing cells are seen in the cortex; most IA ⁺ cells (red label) are seen in the medulla. Apparent in this image is the heterogeneity of CX3CR1 ⁺ /GFP ⁺ −only cells, IA ⁺ −only cells, and dual labeling with CX3CR1 ⁺ /GFP ⁺ and IA ⁺ , which in the latter case represents dendritic cells in the medulla. (F) Higher magnification, Z-stack projection image of five optical slides at 0.69-mm intervals. These results demonstrate the heterogeneity of mononuclear phagocytes within the kidney microenvironment.

A−D from Soos TJ, Sims TN, Barisoni L, et al. CX3CR1+ interstitial dendritic cells form a contiguous network throughout the entire kidney. Kidney Int. 2006;70[3]:591–596 and E, F from Li L, Huang L, Sung SS, et al. The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia-reperfusion injury. Kidney Int. 2008;74[12]:1526–1537.

Danger and stranger models.

Infections of pathogenic bacteria or viruses cause the release of pathogen-associated molecular patterns (PAMPs) that bind to pattern recognition receptors (PRRs) , such as Toll-like receptors (TLRs), on immune cells and stimulate an innate immune response that is accompanied by inflammation and activation of adaptive immunity, and eventually processes to resolve the infection and allow for tissue repair. The danger model recognizes that similar events occur when cells are stressed or injured and that necrotic cells release molecules normally hidden within the cell. In the extracellular space, these damage-associated molecular patterns (DAMPs) can bind to PRRs or to specialized DAMP receptors to elicit an immune response by promoting the release of proinflammatory mediators and recruiting immune cells to infiltrate the tissue. The immune cells that participate in these processes include, for example, antigen-presenting cells (APCs) , such as dendritic cells and macrophages, as well as T cells and neutrophils (polymorphonuclear leukocytes [PMNs] ). DAMPs may also stimulate adaptive immunity and participate in autoimmune responses and tissue repair. A wide variety of intracellular and extracellular molecules function as DAMPs when released from cells (see Table 27.1 ). The functions of such a diverse group of molecules may not yet be fully elucidated; it is unknown whether different DAMPs have specific roles, whether specific functions are elicited in different cell types or conditions, or even whether immune responses to DAMPs can be distinguished from those of PAMPs.

From Rosin DL, Okusa MD. Dangers within: DAMP responses to damage and cell death in kidney disease. J Am Soc Nephrol. 2011;22[3]:416–425.

Dendritic cells have enormous plasticity and can be antiinflammatory or proinflammatory. ^, They contribute early in the course of IRI-induced activation of NKT cells and the IL-17/IL-23 signaling pathway. Depletion of dendritic cells, using transgenic mice expressing the human diphtheria toxin receptor ([DTR]; human heparin-binding epidermal growth factor–like growth factor in CD11c ⁺ cells (CD11c-DTR mouse), demonstrates their importance in activating the innate immune response in AKI. Diphtheria toxin (DT)-treatment depleted CD11c-DTR mice had significantly less injury than CD11c-DTR mice in response to IRI compared with those mice pretreated with a catalytically inactive mutant DT (mDT), strongly supporting the concept that dendritic cells contribute to the early innate response in IRI. Dendritic cells are the earliest producers of IL-6, TNF-α, MCP-1, and RANTES (regulated on activation, normal T cell−expressed and secreted). In addition to initiating the recruitment of inflammatory cells, dendritic cells also participate in recovery via IL-10 production and can induce tolerance. Tolerogenic dendritic cells are functionally immature, express inadequate positive or enhanced negative costimulatory signals and reduced proinflammatory cytokines, and can generate immune tolerance by inducing T cell anergy or deletion or by induction or expansion of regulatory T (Treg) cells. ^, Adenosine 2A receptor (A ₂ AR)-induced tolerized dendritic cells suppressed NKT cell activation in vivo and attenuated kidney IRI. However, mature dendritic cells also can promote tolerance. ^, Both immature and mature dendritic cells can prime Treg cells thus preventing autoimmunity. In contrast to IRI, dendritic cell ablation in DTR mice increased injury in a cisplatin-induced model of AKI, which demonstrates the importance of dendritic cells for tissue protection in a distinct model of kidney injury. Thus these studies have revealed disparate roles of tissue-resident dendritic cells in AKI and suggest that the interstitial microenvironment created by different pathogenic circumstances, such as cisplatin toxicity, IRI, or endogenous molecules (e.g., PAMPS, DAMPS, and cytokines, autacoids such as adenosine), may differentially regulate dendritic function. Finally, dendritic cells migrate away from the kidney via the lymphatic system to present antigens and regulate adaptive lymphocytic responses. Thus dendritic cells serve at the crossroads of communication between the epithelium and endothelium, regulating both innate and adaptive immunity, self-tolerance, and tissue injury and repair.

Macrophages are phagocytic innate immune cells that contribute to host defenses and, based on surface markers, five distinct populations have been identified. ^, Tissue-resident macrophages are derived from a heterogeneous population of bone marrow-derived monocytes. ^, They are characterized by low surface expression of chemokine receptor 2 (CCR2), Gr-1, and Ly6C and high surface expression of the fractalkine receptor CX3CR1. ^, These monocytes migrate into normal tissue and differentiate into resident dendritic cells and macrophages. In contrast, macrophages that infiltrate inflamed tissue have a phenotype characterized by high surface expression of CCR2, Ly6C, and Gr-1 and low surface expression of CX3CR1. It is likely that the microenvironment in tissue determines macrophage phenotype. TNF-α, IL-4, and IL-15 skew monocyte differentiation toward a dendritic cell phenotype, whereas interferon-gamma (IFN-γ) and IL-6 direct monocyte differentiation toward a macrophage phenotype. ^,

Improving the ability to enhance clearance of debris (efferocytosis) by professional phagocytes, as well as tubular cells themselves, improves AKI outcomes. The junctional adhesion molecule-like protein (JAML) is upregulated in AKI kidneys and its deletion in macrophages, but not tubular cells, worsened AKI via regulation of efferocytosis through a macrophage-inducible C-type lectin-dependent mechanism. On the other hand, boosting the efferocytosis of tubular cells, but not myeloid cells, using overexpression of chimeric efferocytic receptors alleviated inflammation in models of AKI. Likewise, inhibition of retinoic acid receptors in PTECs protected against AKI that involved increased Kim-1 dependent efferocytosis, which enhanced dedifferentiation, proliferation, and metabolic reprogramming of PTECs. Treatment with geniposide, an herbal compound, also improved outcomes in experimental AKI by promoting the ability of macrophages to phagocytose NETs via AMPK-PI3K/Akt signaling.

Although controversial and challenged by recent studies, the original classification of M1 and M2 macrophages does provide an important functional framework. M1 macrophages, also referred to as “classic macrophages,” are activated by IFN-γ and LPS, and express high levels of inducible nitric oxide synthase (iNOS). M1 macrophages have high microbicidal activity through the production of proinflammatory cytokines, such as TNF-α, IL-6, and IL-12 via signal transducer and activator of transcription 1 (STAT1), NF-κB, and ROS. M2 macrophages, referred to as “alternatively activated macrophages,” are activated by IL-4 or IL-13 and participate in tissue repair and resolution of inflammation through insulin-like growth factor 1 (IGF-1), mannose receptor 1 (MRC1/CD206), and arginase-1 (Arg1) following STAT6 activation.

Evidence for these disparate macrophage roles can be assessed in vivo through the administration of liposomal clodronate. Liposomal clodronate is engulfed by macrophages and clodronate, which is released from the liposome through action of lysosomal phospholipases in the macrophage, becomes toxic and kills cells by apoptosis. The functional role of macrophages was determined by liposomal clodronate depletion studies that focused on the time course for macrophage depletion (early vs. late). Depletion of kidney and spleen macrophages using liposomal clodronate before renal IRI prevented AKI, and adoptive transfer of macrophages reconstituted AKI. However, depletion of macrophages 3 to 5 days after IRI slowed tubular cell proliferation and repair, suggesting that M1 macrophages exhibiting a proinflammatory phenotype are important for injury, whereas M2 macrophages exhibiting an antiinflammatory phenotype are important for tissue repair. ^{, ,}

Following IRI, the proinflammatory monocyte entering kidney tissue is activated to a proinflammatory macrophage through the infiltration of polymorphonuclear leukocytes, T cells, and NKT cells. ^{, ,} Resident dendritic cells activate NKT cells to produce INF-γ and other inflammatory mediators such as ROS, semaphorin-3A, DAMPs, and TNF-α, which conditions the microenvironment and favors the proinflammatory macrophage phenotype. These proinflammatory macrophages then produce proinflammatory cytokines, which leads to further tissue injury. Whether polarization of macrophages from M1 to M2 involves the recruitment of circulating cells or reprogramming is unclear. ^, Studies by Lee and colleagues have supported the concept that macrophage polarization occurs in situ. The investigators injected labeled M1 macrophages at the time of injury and examined labeled macrophages that infiltrated the injured kidneys. Most of the labeled cells maintained an M1 phenotype; however, when labeled M1 macrophages were injected 3 days after injury, most of the macrophages expressed an M2 phenotype. These studies support the concept that macrophages may be reprogrammed in situ ^{, ,} and that the kidney microenvironment provides important conditioning cues.

Macrophage phenotype is also regulated by mediators of inflammation. Treatment with a synthetic C-reactive protein (CRP) was shown to improve AKI and clear infection in the cecal-ligation and puncture (CLP)-induced septic AKI model that was associated with the M2 skewing of macrophages 24 hours after AKI. Similarly, stimulation of PGE ₂ receptor or E-type prostanoid receptor 4 (EP4) induced MafB expression in macrophages to induce an M2 phenotype, and deletion of Cox2 or EP4 following AKI led to increased renal fibrosis. Conversely, M2 phenotype macrophages are associated with fibrosis in a PTEN- and Irf4-dependent manner in late stages after AKI and deletion of Arg1, a marker of M2 macrophages, attenuated renal dysfunction, and reduced expression of IL-6 and IL-1.

Early inflammation is classically characterized by the margination of leukocytes to the activated vascular endothelium via interactions between selectins and ligands that allows rolling and later firm adhesion, followed by transmigration. ^, A number of potent mediators are generated by the injured proximal TEC, including proinflammatory cytokines such as TNF-α, IL-6, IL-1β, MCP-1, IL-8, transforming growth factor-β (TGF-β), and RANTES. ^, TLR2 is an important mediator of endothelial ischemic injury, and TLR4 plays a similar role in animal models of both ischemic and septic injury, especially in PTC. In septic AKI, F4/80 (EGF-like module-containing mucin-like hormone receptor-like 1 or EMR1) deletion in macrophages led to higher kidney IL-6 levels that were ameliorated by anti-IL-6 therapy. It was hypothesized that the F4/80 ^hi macrophages express IL-1R antagonist to restrict IL-6 production and limit septic AKI.

The neuroimmune axis regulates AKI; pulsed ultrasound ^, and vagus nerve stimulation ^, activate the cholinergic antiinflammatory pathway and are renoprotective. This stimulation activated α-7 nicotinic receptor (α7nAChR) expressed in splenocytes and peritoneal macrophages to attenuate AKI, and adoptive transfer of activated splenocytes or peritoneal macrophages reduced IRI in recipient mice. ^{, ,} This protection was abrogated in the absence of α7nAChR or upon splenectomy and was dependent on the expression of hairy and enhancer of split-1 (Hes1), a basic helix-loop-helix DNA-binding protein, in macrophages. ^{, ,} Administration of GTS-21, an α7nAChR-specific agonist, reduced the production of TNF-α in macrophages and in cisplatin-induced AKI.

Thus a diverse set of data indicate that the kinetics and localization of macrophages during and after AKI may dictate the role of macrophage phenotype. While the early infiltration of monocyte-derived M1 phenotype macrophages may be proinflammatory, their later skewing to an M2 phenotype or the kidney-resident macrophages may play a protective role in AKI. An atlas of the murine kidney-resident macrophage system in the normal and injured kidney identifies several clusters of macrophages; the largest clusters in the papilla near the collecting ducts may drive fibrosis and those in the cortex around the PTC are enriched for iron-handling transcripts. Induction of AKI disrupted localization of all the clusters, which was not fully restored even 28 days after the injury; persistent macrophage-disrupted localization as a result of severe injury may dictate increased susceptibility to AKI-to-CKD transition.

Neutrophils accumulate early in ischemic injury in animal models. Both IL-17A and IFN-γ are produced by neutrophils and may positively regulate neutrophil transmigration to the injured kidney following kidney IRI ( eFig. 27.4 ). Although neutrophils are seen early in rodent models of AKI, whether they play a pathogenic role remains controversial because inhibition or depletion studies of neutrophils variably leads to either protection ^{, ,} or lack of protection. Blockade of neutrophil function or neutrophil depletion provides only partial protection against injury. Vascular adhesion protein-1 (VAP-1) is an adhesion molecule ^, associated with inflammatory conditions. VAP-1 is expressed primarily in pericytes, and a specific VAP-1 inhibitor, RTU-1096, attenuated renal IRI and decreased neutrophil infiltration. The protective effect of VAP-1 inhibition was absent in neutrophil-depleted rats, suggesting an important role of neutrophil infiltration.

Localization of neutrophils to interstitial and marginated compartments in the kidney.

Immunofluorescence staining of kidney outer medulla using antibodies to 7/4 ( green , neutrophils) and CD31 ( red, vascular endothelium). Nuclei are depicted by DAPI labeling (blue). Neutrophils in both sides of the vascular endothelial wall. Inset, Z-stack image (7.0 μm) of 12 optical slices of the kidney at 0.6-μm intervals. Shown are kidney neutrophils in the interstitium and peritubular capillaries (arrows) ; neutrophils that have transmigrated into the lumen of the tubule (∗) are shown in the inset.

From Awad AS, Rouse M, Huang L, et al. Compartmentalization of neutrophils in the kidney and lung following acute ischemic kidney injury. Kidney Int. 2009;75[7]:689–698.

NETosis is a specific form of regulated neutrophil death in which neutrophils release neutrophil extracellular traps (NETs), which are extracellular structures composed of chromatin and histones that enable immobilization and killing of bacteria. Infiltrating neutrophils undergoing NETosis contribute to organ damage in ischemic AKI. In the IRI model, infiltrating neutrophils undergo NETosis, leading to the release of cytotoxic DAMPs, such as histones, which exacerbate TEC injury and interstitial inflammation. Neutrophil peptidyl arginine deiminase-4 (PAD4) plays an important role in the formation of NETs. In CKD patients, SARS-CoV-2 led to the induction of vasculopathy leading to AKI via greater NETosis and microthromboses. ^, In sepsis-induced AKI in mice and humans, colocalization of NETs and fibroblast growth factor–inducible molecule 14 (Fn14) was seen. Simultaneous PAD4 deletion and Fn14 inhibition alleviated AKI. These findings suggest that NETosis is an important early target for intervention in diverse forms of AKI.

Early work by Burne-Taney and coworkers ^{, ,} demonstrated that T lymphocytes contribute importantly to renal IRI. However, conventional CD4 ⁺ T cells are thought to play an obligatory role in antigen-specific, cognate immunity that requires 2 to 4 days for naïve T cell processing, a time course that cannot explain the rapid, innate immune response following IRI. Studies have shown that RTEC may directly stimulate the proliferation of TCR double negative (DN) T cells and not peripheral CD4 and CD8 T cells are stimulated via this mechanism. Infiltration of T cells and the presence of T cell cytokines (IL-2, IL-10, and TNF-α) was markedly increased in the urine of patients who developed AKI associated with immune checkpoint inhibitors (AKI-ICI). T cells can be identified in kidneys several weeks after AKI despite recovery of renal function. In a unilateral IRI model, without contralateral nephrectomy, there was a second wave of T cell and neutrophil infiltration around day 14 that was accompanied by higher expression of tubular injury genes and decreased proportion of tubules when compared with mice that had contralateral nephrectomy.

Natural killer T (NKT) cells are a T cell sublineage and, in mice, NKT cells express an invariant T cell receptor TCRα chain, Vα14-J18. In contrast to conventional T cells, the NKT cell TCR does not interact with peptide antigen presented by classic major histocompatibility complex (MHC) class I or II; rather, it recognizes glycolipids presented by the class I–like molecule, CD1d. Upon activation of NKT cells, vigorous cytokine secretion occurs within 1 to 2 hours, including both Th1-type (IFN-γ, TNF) and Th2-type (IL-4, IL-13) cytokines. The rapid response by NKT cells following activation can amplify and regulate the function of dendritic cells, Treg cells, NK and B cells, and conventional T cells and thus link innate and adaptive immunity. In AKI, NKT cells participate in the early innate immune response to kidney IRI. NKT-produced IFN-γ was found as early as 3 hours following IRI, supporting previous work demonstrating that CD4 ⁺ T cell IFN-γ production is responsible for kidney injury. ^, These data demonstrate the central role of IFN-γ from CD4 ⁺ T and/or NKT cells in the pathogenesis of renal IRI.

T cell receptor ⁺ CD4 ^– CD8 ^– DN T cells may be protective post AKI. DN T cells are present in both mouse and human kidneys and are enriched for the expression of Kcnq5, Klrb1c, Fcer1g, and Klre1 . Mouse kidneys are also enriched for CD4 ⁺ T cells expressing the checkpoint molecule T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) after IR injury, but the population was restricted to T/natural killer cell subsets in patients with AKI. Kidney Treg cells also expressed TIGIT at baseline that was reduced post-IRI. In another study, deletion of Kelch-like ECH-associated protein 1 (Keap1) in CD4 ⁺ T cells increased the expression of Nuclear factor erythroid2-like 2 (Nrf2) target genes, and adoptive transfer of Keap1-KO CD4 ⁺ T cells before IRI was protective in IRI in T cell–deficient nu/nu mice.

The gut microbiota influences T cell phenotype and its consequence for AKI, as colonization of mice with post-AKI microbiota worsened AKI outcomes and microbiota depletion with antibiotics protected against IRI or accelerated recovery, along with lower kidney Th1, Th17 cells, and TNF-α expressing double negative T cells and higher Treg, Foxp3 ⁺ CD8 ⁺ T cells.

Treg cells ^, also play a role in ischemic AKI. ^, In a murine model of ischemic AKI, there is significant trafficking of Treg cells into the kidneys after 3 and 10 days. Anti-CD25 antibodies increased IRI-induced tubule damage, tubular proliferation, immune cell infiltration, and cytokine production. ^, FoxP3 (forkhead box P3) ⁺ Treg cell–deficient mice accumulated more inflammatory leukocytes after renal IRI than mice containing Treg cells, and cotransfer of isolated Treg cells and Scurfy lymph node cells (Treg deficient lymph node cells) significantly attenuated IRI-induced renal injury and leukocyte accumulation. These studies demonstrate that Treg cells infiltrate kidneys after IRI and promote repair, likely through modulation of proinflammatory cytokine production of other T cell subsets.

The capacity of Treg cells to protect mice from IRI depends on IL-10 production, adenosine production through CD73, expression of the adenosine 2A receptor, and programmed cell death 1 (PD-1) on the cell surface. PD-1, a negative costimulatory molecule expressed on T lymphocytes, monocytes, dendritic cells, and B cells, ^, is indispensable for Treg function. Administration of monoclonal antibodies to PD-L1 or a genetic deficiency of PD-1 on Tregs exacerbated impaired kidney function and ATN after subthreshold ischemia. Stremska and coworkers synthesized a hybrid cytokine, IL233, which combines IL-2 and IL-33. Tregs require IL-2 for homeostasis and upregulate IL-33R, which promotes their recruitment and activation of innate lymphoid cells (ILC2s) in response to IL-33. Collectively, ILCs are the latest family of innate immune cells to be discovered, originating from common lymphoid progenitors (CLPs). In response to pathogenic tissue damage, ILCs play a crucial role in immunity by secreting signaling molecules and regulating both innate and adaptive immune cells. Primarily residing in tissues, ILCs are found in both lymphoid (immune-associated) and nonlymphoid tissues and are rarely present in the blood. ILC2 cells, or type 2 innate lymphoid cells, are a subset of innate lymphoid cells. Unlike ILCs as a broader category, ILC2 cells specifically belong to the lymphoid lineage and are derived from common lymphoid progenitors. These cells do not possess antigen-specific B or T cell receptors due to the absence of recombination-activating genes. Administration of the novel hybrid cytokine, IL233, increased endogenous Tregs in blood and spleen and prevented IRI more efficiently than a mixture of IL-2 and IL-33 administered separately. IL233 also increased the proportion of ILC2s in blood and kidneys, and adoptive transfer of ILC2s protected mice from IRI. Thus the many barriers to cell-based therapy may be overcome through this hybrid cytokine, which increases endogenous Tregs. We need a greater understanding of the role of ILCs, especially the ILC2 subtype, in AKI. We argue that the rapid translation to human studies would be a major advancement in the AKI field.

Studies to evaluate the role of B cells and the B1 subset on ischemia-induced AKI have remained controversial, with some studies showing that B cells may be deleterious. Burne-Taney and associates showed that μMT mice, which lack B cells and all immunoglobulins, are protected from AKI despite similar levels of infiltrating granulocytes and macrophages in the postischemic kidneys of wild-type (WT) mice. Sensitivity to ischemia-induced AKI was restored after replenishing these mice with WT serum, but not B cells, thus indicating that a serum factor enhanced the cytotoxic effect of infiltrating granulocytes and macrophages on the ischemic tubular cells. However, Renner and colleagues, using the same μMT mice, could not show that these mice were protected from ischemia-induced AKI. In fact, they had more severe injuries than control WT mice. Additionally, Lobo and associates failed to show protection from ischemia-induced AKI when WT mice were acutely depleted of B cells (with anti-CD20) and not immunoglobulins. More studies are needed to study the role of B cells and immunoglobulins in AKI; it may be preferable to deplete B cells or subsets of B cells acutely to study their role. μMT mice have other immune deficiencies, including low Tregs and lack of T cell receptor (TCR) diversification, which may have contributed to more severe injury in the experiments by Thurman and colleagues. ^, Deletion of CD157/Bst1 (a modulator of immune response), mainly found in B cells and neutrophils, attenuated migration of neutrophils and renal IRI. BAFF (B cell activating factor) also plays a proinflammatory role in AKI, and deletion of BAFF can protect kidneys from IRI. Surprisingly, BAFF-receptor KO worsened AKI. The basis for this paradox is not fully understood.

The role of the B1 subset of B cells in murine models of ischemia has also been controversial. Natural IgM produced by B1 cells is deleterious in ischemia-induced injury of murine skeletal muscle, cardiac muscle, and bowel. ^{, ,} Conversely, Lobo and colleagues showed that natural IgM protects WT kidneys from ischemic AKI by inhibiting the innate inflammation that occurs after ischemic injury. Such observations demonstrate that the mechanisms that cause ischemic injury in the kidney may be different from those of other organs.

Conger and associates were among the first to demonstrate that postischemic rat kidneys manifest vasoconstriction in response to decreased renal perfusion pressure and hence cannot autoregulate blood flow, even when total renal blood flow had returned to baseline values up to 1 week after injury. ^, Single-fiber, laser Doppler flow cytometry revealed that blood flow is reduced to 60% and 16% of preischemic values in cortex and medulla, respectively, and increased 125% in inner medulla following ischemia. Selective inhibition, depletion, or deletion of iNOS has renoprotective effects during ischemia. ^, NO production from the endothelium (eNOS) may be impaired at the level of enzyme activity or modified by ROS to impair normal vasodilatory activity. In ischemic AKI, there is an imbalance of eNOS and iNOS and a relative decrease in eNOS, secondary to endothelial dysfunction and damage, which leads to a loss of the antithrombogenic properties of the endothelium, as well as increased susceptibility to microvascular thrombosis. Administration of l -arginine, the NO donor molsidomine, or the eNOS cofactor tetrahydrobiopterin can preserve medullary perfusion and attenuate AKI induced by IRI. Conversely, the administration of N ^ω -nitro- l -arginine methyl ester, a NOS enzymatic blocker, aggravates the course of AKI following IRI. ^,

The pattern of renal blood flow in experimental sepsis is inconsistent. Renal blood flow was decreased in 62% of studies and unchanged or increased in 38%. Administering Escherichia coli to sheep was shown to induce hyperdynamic sepsis associated with increased cardiac output, decreased mean arterial pressure, and AKI, as evidenced by a rise in creatinine levels. Furthermore, there was marked increase in renal blood flow, decrease in urine output, and decrease in creatinine clearance. When angiotensin was dose titrated to restore blood pressure to presepsis levels, these effects were reversed; there was a significant decrease in renal blood flow, increase in urine output, and increase in creatinine clearance. These studies suggest that improvement of function may be more than simply an improvement in mean arterial pressure and that the increase in glomerular filtration pressure may be through selective constriction of efferent arterioles and/or increase in mesangial cell tone. On the basis of these principles, patients with vasodilatory shock who were receiving another vasopressor were randomized in a clinical trial to Ang II or placebo. The group that received Ang II had higher mean blood pressure and, in a subgroup of patients with AKI who required renal replacement therapy, 28-day survival, mean arterial pressure, and rate of discontinuation of renal replacement were higher in the angiotensin group. These results together show the potential benefit of Ang II in hyperdynamic sepsis, which may in part be due to improved glomerular hemodynamics.

The glycocalyx, which is located on the blood side of the endothelium and has a depth of 1 to 3 μm, is composed of cell-bound proteoglycans, glycosaminoglycan side chains, and sialoproteins. Sandwiched between blood components and the endothelium, the glycocalyx is situated to play a key role in microvascular and endothelial homeostasis and endothelial physiology. The specific functions served by the endothelial glycocalyx include shear stress mechanotransduction to endothelial cells, regulation of vascular permeability, inhibition of intravascular thrombosis, and protection of the endothelium from platelet and leukocyte adhesion. ^, The glycocalyx may be damaged by local changes in shear stress, ROS, altered oncotic pressure, altered fluid composition, and inflammatory molecules. Shedding of the glycocalyx occurs, exposing the endothelial adhesion molecules to circulating immune cells and leading to immune cell activation, inflammation, and lipoprotein leakage to the subendothelial space. Coronary ischemia-reperfusion leads to shedding of the glycocalyx and an increase in neutrophil adhesion with subsequent myocardial injury. Similar events are likely to occur in septic AKI and kidney IRI ( Fig. 27.8 ).

Microvascular dysfunction in septic acute kidney injury.

Multiple mechanisms are involved in the development of sepsis-related microvascular dysfunction, among which endothelial dysfunction (related partly to circulating host-derived and pathogen-derived mediators, as well as to reactive oxygen species) and an altered glycocalyx have major roles. The glycocalyx is a thin layer of glycosaminoglycans that covers the endothelial surface, facilitating the flow of red blood cells and limiting the adhesion of leukocytes and platelets to the endothelium. The glycocalyx may be substantially altered during sepsis, so interactions between the vascular endothelium and circulating cells (e.g., leukocytes and platelets) are impaired, and leukocyte rolling and adhesion to the endothelium may occur. Activation of coagulation and the generation of microthrombi might also participate in sepsis-induced microvascular alterations, as well as alterations in erythrocyte deformability and/or their adhesion to the endothelium. All these phenomena might cause heterogeneity in microvascular blood flow, with a decrease in vascular density and nonperfused capillaries, resulting in an increased diffusion distance for oxygen and in alterations in oxygen extraction. vWF, von Willebrand factor.

From Lelubre C, Vincent JL. Mechanisms and treatment of organ failure in sepsis. Nat Rev Nephrol. 2018;14[7]:417–427.

Within 24 hours after ischemic injury, there is loss of vascular endothelial cadherin immunostaining, suggesting severe alterations in the integrity of the adherens junctions of the renal microvasculature. In vivo two-photon imaging demonstrated a loss of capillary barrier function within 2 hours of reperfusion, as evidenced by leakiness of high-molecular-weight dextrans (>300,000 Da) into the interstitial space.

Critical constituents of the perivascular matrix, including collagen IV, are substrates for matrix metalloproteinase-2 (MMP-2), MMP-7, and MMP-9, which are collectively known as gelatinases. Breakdown of barrier function may also be due to MMP-2 or MMP-9 activation, and this upregulation is temporally correlated with an increase in microvascular permeability. ^{, ,}

MMP-7 has a renoprotective effect by degrading Fas ligand (FasL) and E-cadherin. It mobilizes β-catenin, activates AKT protein kinase, suppresses P53 expression, and proapoptotic protein Bax, and attenuates renal tubule apoptosis. ^, MMP-9 gene deletion stabilizes microvascular density following ischemic AKI, in part by preserving tissue VEGF levels. MMP-9 also attenuates renal apoptosis via the soluble stem cell factor (SCF)-c-kit pathway. In contrast, MMP-2 contributes to AKI thought to be due to degradation of peritubular capillary basement membrane leading to hemorrhage and tubule necrosis.

In addition, minocycline, a broad-based MMP inhibitor, and the gelatinase-specific inhibitor ABT-518 both ameliorated the increase in microvascular permeability in this model. Taken together, many studies have indicated that the loss of endothelial cells following ischemic injury is not a major contributor to altered microvascular permeability, although renal microvascular endothelial cells are vulnerable to the initiation of apoptotic mechanisms following ischemic injury, which can ultimately reduce microvascular density.

Krüppel-like factors (KLF) are a family of zinc-finger transcription factors involved in a number of fundamental cellular processes (cell cycling, differentiation, regeneration, and human disease). ^, KLF2, KLF4, and KLF11 are expressed in kidney endothelial cells, and KLF2 and KLF4 modulate antithrombotic and antiinflammatory processes. Less studied is the endothelial-specific KLF11. KLF11 may be a novel renal protective member of the KLF family. KLF11-deficient mice were found to be more susceptible to bilateral IRI. They demonstrated more vascular congestion and induction of endothelin 1 and IL-6, suggesting the importance of KLF11’s effect on endothelial function.

The angiopoietin (Angpt)/Tie2 system consists of the transmembrane endothelial tyrosine kinase Tie2 and circulating ligands (Angpt-1 and 2). ^, In endothelial cells, Tie 2 phosphorylation governs by the mutually antagonistic properties of Angpt-1 and Angpt-2. In inflammatory states, Angpt-1 attenuates vascular inflammation, leakage, and apoptosis, whereas Angpt-2 has the opposite effect. In septic individuals, Angpt-2 increased up to 50 times, whereas Angpt-1 was unchanged. ^, Although seemingly harmful, somewhat surprising results were obtained from global Angpt-2 deficient mice in which Angpt-2 ^–/– mice were not protected from sepsis-associated AKI. Additional studies are necessary to determine the role of Angpt/Tie2 in sepsis-associated AKI.

Endothelial cells play a central role in coagulation via interactions with protein C through the EPCR and thrombomodulin. The protein C pathway, which helps maintain normal homeostasis and limits inflammatory responses, is activated by thrombin-mediated cleavage, and the rate of this reaction is further augmented (by 1000-fold) when thrombin binds to the endothelial cell surface receptor protein thrombomodulin. The activation rate of protein C is further increased by approximately 10-fold when EPCR binds protein C and presents it to the thrombin-thrombomodulin complex. Essentially, activated protein C then has antithrombotic actions and profibrinolytic properties and participates in numerous antiinflammatory and cytoprotective pathways to restore normal homeostasis. On the basis of these properties, the endothelial cell plays an absolutely essential and critical role in maintaining a normal and healthy vasculature and endothelial bed. In AKI, microvascular function is ultimately compromised, resulting in disseminated intravascular coagulation and microvascular thrombosis, decreased tissue perfusion, and hypoxemia, leading to organ dysfunction and failure. Both pretreatment and postinjury treatment with soluble thrombomodulin attenuate renal injury, with minimization of vascular permeability defects and improvement in capillary renal blood flow.

Leukocyte and endothelial cells are dynamically involved in the process of adherence of leukocytes to the vascular endothelium. Leukocyte activation and cytokine release require signals through chemokines circulating in the bloodstream or through direct contact with the endothelium. Rolling leukocytes can be activated by chemoattractants such as complement C5a and platelet-activating factor. Once activated, leukocyte integrins bind to endothelial ligands to promote firm adhesion. β ₂ -Integrin (CD18) seems to be the most important neutrophil ligand for endothelial adherence. These interactions with the endothelium are mediated through endothelial adhesion molecules that are upregulated during ischemic conditions. The initial phase starts with slow neutrophil migration mediated by tethering interactions between adhesion molecules and their endothelial cell ligands. Within 2 to 4 hours of IRI or sepsis, endothelial P-selectin and intercellular adhesion molecule 1 (ICAM1) are expressed on endothelial cells associated with leukocyte trapping in peritubular capillaries. Platelet P-selectin and not endothelial P-selectin is the main determinant in neutrophil-mediated ischemic kidney injury. There is also significant protection from both ischemic injury and mortality by blockade of the shared ligand to all three selectins (E-, P-, and L-selectin), which seems to be dependent on the presence of a key fucosyl sugar on the selectin ligand. In a CLP model of septic azotemia, mice deficient for E-selectin, P-selectin, or both were completely protected. Furthermore, selectin-deficient mice demonstrated similar intraperitoneal leukocyte recruitment but altered cytokine levels when compared with WT mice, in addition to engagement in leukocyte-endothelial cell interactions. Other adhesion molecules such as VAP-1 appear to play a role in AKI.

The critical role that leukocyte integrin CD11b−CD18 contributes to both AKI and CKD was demonstrated in nonhuman primates. In an IRI model, mAb107, a CD11b−CD18 inhibitor, markedly reduced plasma creatinine and ATN, indicating that the leukocyte integrin CD11b−C18 contributes to AKI. Moreover, subsequent assessment up to 9 months after AKI revealed improvement in microvascular perfusion and histology.

Microparticles (MPs) are cell membrane–derived particles of 0.2 to 2 μM in diameter that can promote coagulation and inflammation and may be involved in sepsis-related AKI. In the cecal ligation model of sepsis, overexpression of calpastatin, which limits procoagulant microparticle release, improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. Increased calpastatin expression decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. Transferring microparticles from septic WT to septic calpastatin transgenic mice worsened survival and coagulopathy, thus demonstrating the deleterious effect of microparticles in this model. The development of therapeutics aimed to prevent the release of MPs from endothelium or other inflammatory cells may be a reasonable target in microvascular dysfunction during sepsis-induced AKI.

Kidney pericytes are extensively branched cells that are scattered on the outer wall of capillaries and embedded in the capillary basement membrane; they serve a homeostatic role in angiogenesis and vessel maturation. Following kidney injury, they detach and migrate into the kidney interstitium and, in some cases, transform into myofibroblasts, leading to progressive kidney fibrosis. Thus the endothelial-pericyte crosstalk, involving VEGF receptors and platelet-derived growth factor receptors, contributes to normal microvascular health and disease. Selective pericyte ablation caused an abrupt decline in renal function and an increase in albuminuria within 96 hours. These results demonstrate that pericytes are essential for normal kidney microvascular function.

During kidney recovery, renal and extrarenal cells participate in the wound-healing response and can initiate fibrosis. Immune cells of the mononuclear phagocyte system, including macrophages and dendritic cells, contribute to injury and have also emerged as important cells in the recovery of kidney function during adaptive repair or fibrosis during maladaptive repair. The balance between wound healing and progressive fibrosis dictates the final outcome. The intrinsic plasticity of monocytes-macrophages and dendritic cells, as well as attempts to relate in vitro studies to in vivo findings, makes the functional definition and phenotype of this myeloid population in kidney pathophysiology complex. In vitro studies have led to two well-defined mononuclear phagocytes. Classically activated macrophages—M1 mononuclear phagocytes consisting of macrophages and dendritic cells—are produced by exposure to LPS or INF-γ and are largely thought to be proinflammatory and contribute to initial kidney injury. Alternatively activated macrophages (M2 mononuclear phagocytes) are induced by IL-4 and IL-10 and appear later, after AKI has been established, and have a genetic signature associated with wound healing and/or fibrosis. These mononuclear phagocyte phenotypes depend on the complex local tissue microenvironment, which may induce phenotype switching.

A key feature of fibrosis is the activation of extracellular matrix−producing myofibroblasts. ^, Other factors relevant in CKD progression include endothelial cell damage and vascular damage in AKI, hypoxia-inducible factor (HIF), innate and adaptive immunity, cell cycle arrest, and epigenetic mechanisms. ^,

Although some injured tubules undergo repair and regeneration, injury may also be accompanied by inflammation, maturation, and proliferation of fibroblasts, and extracellular matrix deposition as part of the process of fibrosis. Tissue fibrosis is a common component of inflammatory diseases, including various forms of CKD. Collagen deposition, characteristically formed during normal wound healing, is reversible, but progressive irreversible fibrosis may occur through repeated injury due to dysregulated inflammation. Some studies have focused on the role of pericytes or resident fibroblasts that contribute to normal homeostatic microvascular function but transform into myofibroblasts on activation. Myofibroblasts are the primary collagen-producing cells.

The source of fibroblasts in the injured kidney has been controversial. ^, Although the myofibroblast is the cell type responsible for depositing collagen, investigators have been searching for their precursors. Other than interstitial fibroblasts that are known to transition, it has been suggested that pericytes, dendritic, endothelial, and epithelial cells may also represent potential contributors. In vivo and in vitro studies have demonstrated that endothelial cells can develop a myofibroblast phenotype. ^, Epithelial cells grown in culture can express genes that are typically expressed in myofibroblasts, suggesting EMT. Fate-mapping studies indicate that although epithelial cells can express mesenchymal markers in vitro, they appear not to penetrate the basement membrane to enter into the interstitial space and differentiate into myofibroblasts in vivo. Furthermore, lineage analysis demonstrated that platelet-derived growth factor receptor (PDGFR)-β–positive pericytes differentiate into myofibroblasts in a UUO model. Following AKI, a potential step in the AKI-CKD transition is the dissociation of pericytes from endothelial cells. This was revealed by using bigenic Gli1-CreERt2; R26tdTomato reporter mice. Pericyte ablation resulted in endothelial damage, and pericyte loss led to a significantly reduced capillary number, a hallmark of CKD.

Two studies have shed light on this controversy. Snai1 (encodes snail family zinc finger 1, Snail1) expressed in mouse renal epithelial cells is required for kidney fibrosis. The Snail family of transcription factors plays a key role in repressing the adhesion molecule E-cadherin, thereby regulating the EMT during embryonic development. Moreover, Twist (encodes twist family bHLH transcription factor 1) genes are also essential regulators of EMT. Inhibition or deletion of these genes or overexpression includes or inhibits EMT, respectively. ^, Lineage tracing studies have demonstrated that during activation, labeled epithelial cells do not contribute to myofibroblasts or interstitial cells; however, there was downregulation of epithelial markers and loss of epithelial polarity and cell differentiation. Thus partial EMT leads to cell cycle arrest, blocks proliferation and repair, and blocks secretion of growth factors, such as TGF-β, that drive proliferation of myofibroblasts ( Fig. 27.10 ). Genes and pathways that are associated with successful or failed repair are shown in Fig 27.11 .

Critical cells and signaling pathways activated in progressive chronic kidney disease (CKD) .

CKD is initiated by cellular injury, either to the epithelial or endothelial compartment. In the tubulointerstitium, injury leads to epithelial dedifferentiation that may be induced by transforming growth factor-β (TGF-β) and Notch pathway upregulation. Dedifferentiated epithelial cells secrete paracrine signaling factors, such as hedgehog and Wnt ligands, that act on interstitial pericytes and mesenchymal stem cell−like cells to activate myofibroblast differentiation, proliferation, and matrix secretion. This in turn causes peritubular capillary rarefaction and ongoing hypoxia. Chronic tubular injury leads to epithelial cell cycle arrest and senescence, with accompanying secretion of proinflammatory cytokines that amplify inflammation. These interrelated events ultimately drive nephron loss, ongoing interstitial fibrosis, and kidney failure.

From Humphreys BD. Mechanisms of renal fibrosis. Annu Rev Physiol. 2018;80:309–326.

Successful and failed-repair postacute kidney injury.

Multiomics studies have revealed the productive and deleterious genes and pathways that regulate successful and maladaptive (failed) repair, respectively. The injured tubular cells release several mediators that activate tubular cell-intrinsic and extrinsic pathways. Activation of uncontrolled cell death pathways, such as necroptosis, ferroptosis, inflammasome activation, and release of ATP lead to recruitment of proinflammatory cells such as neutrophils, which further cause NETosis, as well as NK/NKT cells and M1 macrophages. Early activation of pathways, such as apoptosis, which promotes mitophagy, autophagy, and efferocytosis, promotes clearance of cell debris. Recruitment of antiinflammatory cells such as Tregs and M2 macrophages, which may be mobilized from the spleen by vagus nerve stimulation (VNS) and Choline-acetyl transferase (ChAT) pathway, induce anti-inflammatory mediators including adenosine. Transcriptomic and metabolomic studies showed that the tubular and phagocytic cells that engulf apoptotic debris and upregulate the pathways regulating the uptake and oxidative metabolism of lipid cargo (repairing cell) transition toward successful repair as indicated by enhanced expression of genes related to cell cycle, dedifferentiation, tubular progenitors, and solute transporters related to function. On the other hand, cells that are enriched for expression of proinflammatory genes (failing repair) and genes related to dysregulation of glucose, lactate, and amino acid metabolism eventually activate profibrotic genes.

Created in BioRender. Sharma R. 2025. https://BioRender.com/idu28is.

Signaling pathways Wnt, hedgehog (Hh), and Notch play important roles during renal fibrosis. In CKD, numerous Wnt ligands are upregulated, resulting in prolonged activation of the Wnt/β-catenin pathway. ^, Epithelial β-catenin activation leads to epithelial dedifferentiation and interstitial fibrosis. Wnt derived from tubules is necessary for myofibroblast activation and interstitial fibrosis. The sphingolipid pathway is important in progressive fibrosis. ^{, ,} Sphingosine 1-phosphate (S1P), a pleiotropic lysophospholipid that is involved in diverse functions such as cell growth and survival, lymphocyte trafficking, and vascular stability, is the product of sphingosine phosphorylation by two sphingosine kinase isoforms (SphK1 and SphK2). SphK2 is localized to the nucleus and produces nuclear S1P, which acts as a histone deacetylase (HDAC) inhibitor, thereby allowing gene expression to be induced. IFN-γ may mediate reduced fibrosis in Sphk2 ^–/– mice or mice treated with SpHK2 inhibitor at day 14. ^, Perivascular cell S1P transported via spinster homolog 2 (Spns2) bound to S1P1 in an autocrine fashion to enhance production of proinflammatory cytokines/chemokines upon injury, leading to immune cell infiltration and subsequent fibrosis. Inhibition of S1P transport through perivascular deficient Spns2 ^–/– or the use of small molecule inhibitors ameliorated kidney fibrosis in mice. These studies demonstrated that targeting the perivascular SPHK2–SPNS2–S1P1 axis may serve as a novel therapeutic approach for attenuating kidney fibrosis. ^{, ,}

Activation of myofibroblasts is also mediated through paracrine signals from immune cells (lymphocytes and antigen-presenting cells), including cytokines, chemokines, angiogenic factors, growth factors, peroxisome proliferator-activated receptors (PPARs), and other molecules. Although Th2 cytokines may induce fibrosis, Th1 cytokines such as IL-12 or IFN-γ inhibit pulmonary ^, and experimental renal fibrosis.

An unbiased approach to identify upregulated and downregulated pericyte genes in injury revealed increased activation and expression of ADAMTS1 (a disintegrin-like metalloprotease with thrombospondin type 1) and downregulation of MMP-3 inhibitor TIMP Metallopeptidase Inhibitor 3 (TIMP-3). TIMP-3−stabilized pericytes maintained collagen capillary tube networks, whereas ADAMTS1-treated pericytes led to enhanced destabilization. TIMP-3 has many functions including regulating VEGF signaling, pericyte migration, and MMP-2 and MMP-9 activity. Furthermore, TGF-β1 activates the pericyte-myofibroblast transition, thus adding to the stimuli for pericyte involvement in fibrosis. Injured epithelial cells reduce production of VEGF, a trophic factor for endothelial maintenance, and increase production of TGF-β and PDGFR, which enhance pericyte dedifferentiation into myofibroblasts. Finally, PDGFR blockade on pericytes or VEGF receptor 2 (VEGFR-2) on endothelial cells led to reduced fibrosis and stabilized the microvasculature in the UUO model. Thus numerous factors favor microvascular maladaptation after injury, including TGF-β production and lack of VEGF production by epithelial cells, reductions in PDGF production by endothelial cells, and increased ADAMTS1 expression, with reduced TIMP-3 production by pericytes. These events also suggest several therapeutic targets that could limit microvascular dropout and loss of kidney function secondary to the fibrotic process. Normal pericyte function therefore becomes deranged following transformation to activated myofibroblasts, which leads to loss of microvascular stability, ineffective angiogenesis, increased permeability, and capillary rarefaction, which are all processes leading to fibrosis. ^,

As discussed earlier, proximal tubules are highly metabolically active, relying on aerobic metabolism. Mitochondrial FAO is a major source of energy, and mitochondrial dysfunction leads to lipid accumulation, ATP depletion, and fibrosis in proximal tubule cells. A number of therapeutic approaches are in development, including some agents in clinical trials for diabetic nephropathy and other kidney diseases, ^, but as yet there are no approved treatments to prevent the development of kidney fibrosis or accelerate repair. The development of effective treatments requires a better understanding of the inflammatory, injury, wound healing, matrix deposition, and cellular repair processes that accompany fibrosis. ^{, , ,} For example, mitochondria may be an important therapeutic target in AKI-to-CKD progression. One approach is a novel mitoprotective drug, SS31. SS peptides bind selectively to cardiolipin in the mitochondrial inner membrane to protect mitochondrial cristae. SS31 allows better function of the respiratory chain complexes, prevents peroxidase activity, and enhances electron transfer through the cytochrome complex to improve oxidative phosphorylation and ATP production. SS31 administered 1 month after renal IRI attenuated glomerulosclerosis and senescence and reduced parietal epithelial cell activation and changes in podocyte and endothelial structure. Dynamin-1-like protein (DRP1) is a GTPase enzyme responsible for regulating mitochondrial fission. Proximal tubule–specific deletion of DRP1 prevented renal IRI, inflammation, and programmed cell death and promoted epithelial recovery, which was associated with activation of the renoprotective β-hydroxybutyrate signaling pathway. Importantly, delayed deletion of proximal tubule Drp1 using a tamoxifen-inducible system after IRI attenuated kidney fibrosis. These results highlight DRP1 and mitochondrial dynamics as an important mediator of AKI and progression to fibrosis and suggest that DRP1 may serve as a therapeutic target for AKI.

AKI has been described as a complication following the administration of oral sodium phosphate solution as a bowel cathartic in preparation for colonoscopy and bowel surgery. Although the mechanism linking oral sodium phosphate administration with AKI remains incompletely understood, the pathogenesis likely relates to a transient and significant rise in the serum phosphate concentration that occurs simultaneously with intravascular volume depletion.

When the urine is oversaturated and buffering factors such as pH, citrate, and pyrophosphate are overwhelmed, renal phosphorus excretion becomes compromised, especially when flow rates are reduced. This may lead to the intratubular precipitation of calcium phosphate salts when the solubility coefficient is exceeded, leading to obstruction of the tubular lumen and direct tubular damage. ROS generated by the binding of calcium phosphate crystals further promotes tubular damage. Risk factors for acute phosphate nephropathy include preexisting volume depletion, the use of ACE inhibitors and angiotensin receptor blockers (ARBs), NSAIDs, CKD, older age, female gender, and higher dosage of oral sodium phosphate. ^, Patients who develop acute phosphate nephropathy typically present with elevated serum creatinine concentrations days to months after the administration of oral sodium phosphate solution and can experience progression to CKD and ESKD. As in other conditions associated with hypercalcemia, hyperphosphatemia, or hyperphosphaturia, calcium phosphate precipitation in renal tubules is seen on renal biopsy as bluish-purple crystals that are nonpolarizable.