Pathophysiology of Acute Kidney Injury




This chapter focuses on the pathophysiological principles of Acute Kidney Injury (AKI), with special emphasis on the structural, cellular and molecular alterations occurring in ischemic, nephrotoxic and septic AKI. Changing concepts in the disease pathogenesis and approaches to treatment based on pathophysiological principles are also detailed.


Keywords


acute kidney injury, ischemia, acute tubular necrosis, sepsis, endothelial cell


Clinical Overview


Classifications and Definitions


Acute Kidney Injury (AKI) is a heterogenous syndrome defined by a rapid decline in glomerular filtration rate (GFR) which may lead to accumulation of metabolic waste products and disturbances in fluid, electrolyte and acid-base handling. In the last few years, the previous terminology of Acute Renal Failure (ARF) has largely been abandoned and the new term AKI has been widely adopted in the medical literature. In this chapter, the term AKI will be used to describe the entire spectrum of disorders that share the same physiologic characteristics but may be pathophysiologically distinct.


In 2002, based on the recommendations of the Acute Dialysis Quality Initiatives (ADQI) the staging of AKI was proposed. The RIFLE classification of AKI is divided into three levels of renal dysfunction namely, “ r isk”, “ i njury” and “ f ailure” based on either GFR or urine output criteria whichever is more severe. The GFR criteria assesses renal dysfunction by using either changes in serum creatinine or the percentage decline in GFR from baseline GFR. The parameters used have a high sensitivity, are prognostic based on severity of stage, but represent a retrospective analysis. Two other very important clinical outcomes are also incorporated into the RIFLE criteria: “ l oss” of kidney function as defined by persistent AKI with the need for renal replacement therapy (RRT) for more than four weeks, and “ e nd” stage kidney disease, defined as the need for RRT for greater than three months. These parameters are more strict and therefore have a higher specificity.


Recently, the Acute Kidney Injury Network (AKIN) proposed a modification of the RIFLE criteria with the addition of a>= 0.3 mg/dl increase in serum creatinine to the criteria that define Risk. Because of the linear relationship between GFR and 1/serum creatinine, it should be kept in mind that a small rise in serum creatinine, in the normal or near normal values, corresponds to relatively large decline in GFR. For example, in a steady state, a serum creatinine of 0.8 mg/dl in a young 50 kg female corresponds to a GFR of approximately 100 ml/min, while a serum creatinine of 1.6 mg/dl would reflect in a GFR of 50 ml/min, a decline of nearly 50% in GFR. In the non-steady state of AKI, the serum creatinine value should be considered very carefully, as this is not an equilibrium state. Thus an overestimation of the GFR is made if standard steady state calculations are made using formulas such as Cockroft-Gault or MDRD if the serum creatinine is rising. Hence during the evolution of acute renal dysfunction, serum creatinine underestimates the degree of renal dysfunction, and conversely it underestimates the degree of renal recovery as function recovers. Having said that, the change from baseline and perhaps the rate of rise of creatinine are still potentially useful in the clinical evaluation of GFR decline. Even creatinine clearance (when collection is accurate) suffers from the same limitation as mentioned above, and causes an overestimation of GFR. Determining an accurate GFR in clinical settings is rarely accomplished and perhaps more important would be the determination of whether the GFR is improving or not. Hence one should always remember that to detect AKI, it is essential to assess the change in creatinine , and that across all patients, a single creatinine valuenever corresponds to a given GFR .



Table 76.1

RIFLE and Acute Kidney Injury Network (AKIN) Definition and Staging of Acute Kidney Injury













RIFLE AKIN
Definition
An increase in serum creatinine of >50% developing over<7 days; or a urine output of <0.5 ml/kg/hr for>6 hours An increase in serum creatinine of>0.3 mg/dl or >50% developing over<48 hours; or a urine output of <0.5 ml/kg/hr for>6 hours





































Staging Criteria
RIFLE Stage Increase in Serum Creatinine Urine Output Criteria Increase in Serum Creatinine AKIN Stage
Risk ≥50% <0.5 ml/kg/hr for>6 hours ≥0.3 mg/dl; or ≥50% Stage 1
Injury ≥100% <0.5 ml/kg/hr for>12 hours ≥100% Stage 2
Failure ≥200% <0.5 ml/kg/hr for>24 hours or anuria for>12 hours ≥200% Stage 3
Loss Need for renal replacement therapy for>4 weeks
End-stage Need for renal replacement therapy for>3 months


The production rate and volume of distribution of creatinine also affect the plasma creatinine value, while other factors such as medications (e.g. cimetidine) can inhibit distal tubular creatinine secretion and cause a rise in serum creatinine without being secondary to AKI. Abnormalities in liver function, decreased muscle mass and aging decrease the production of creatinine, whereas fever, immobilization, glucocorticoids or muscle trauma increase its production.


Urine output is sensitive to changes in renal hemodynamics, but extremely insensitive to define or differentiate AKI. Oliguria has been defined as urine output of <400 ml/day or <0.3 ml/kg/hour, and has been found to be associated with a higher mortality as compared to non-oliguric ARF. However, severe AKI can exist in non-oliguric states as well, whereas obstructive causes could present with a fluctuating levels of urine output. The RIFLE criteria does employ urine output as markers of risk, injury or failure, though concordance between serum creatinine and urine output criteria has not been established, even with regards to mortality risk.


Acute Kidney Injury: Incidence and Risk Factors


Incidence


The exact incidence of AKI has varied in the past due to the absence, until recently, of a standard definition. It has been estimated that 3% to 20% of hospitalized patients and 25% to 67% of ICU patients develop AKI, with 5 to 6% of the ICU population requiring renal replacement therapy after developing AKI. Studies have indicated that there is an increasing incidence of AKI over the last twenty years. Amongst all Medicare patients, Eggers et al. noted that after adjusting for sex, age and ethnicity, AKI rates increased 10% per year from 14 cases per year in 1992 to 36 cases per year in 2001. The CDC noted >20 fold increase in the incidence of AKI for patients hospitalized between 1980–2005 using ICD-9 codes.


In another multinational, multicenter observational study of 29,269 critically ill patients, 5.7% developed severe AKI and 4.3% received renal replacement therapy. Although there is less confusion with regard to rates of AKI requiring renal replacement therapy, reported rates still vary due to differences in characteristics of patient populations and variability in criteria for the initiation of renal replacement therapy. Population based studies have shown a rate of 2147 cases of AKI per million population.


Risk Factors


An extremely important aspect in trying to prevent AKI is to identify high risk populations and clinical conditions, or high risk situations and procedures to devise strategies to minimize the incidence and impact of AKI. Numerous risk factors have been identifiedin different population groups, some of which are discussed here ( Table 76.2 ). Most studies have found age to be an independent risk factor for the development of AKI. In 2009 the URSDS, reported incidence rates of AKI in the US using three different datasets from 1995 to 2007 and found the largest increase in AKI incidence was seen among older individuals >85 years age. In a large observational cohort study across five academic centers in the United States, the mean age of patients was 59.5 years. Possible reasons why age could be an independent risk factor includes decreased residual renal function, presence of comorbidities and increased susceptibility to infections. Pre-existing renal insufficiency is a major risk factor for the development of AKI in the non-ICU and ICU setting. Even small decrements in systemic hemodynamics can lead to significant alterations in the renal hemodynamics, due to diminished capacity to auto-regulate the response to a decreased perfusion pressure, leading to a reduction in GFR. Increasing levels of risk are associated with more severe baseline chronic kidney disease (CKD). Compared to patients with baseline eGFR >60 ml/min/1.73 m 2 , those with eGFR values of 45–59 ml/min/1.73 m 2 had a nearly 2-fold increased risk of developing dialysis-requiring AKI. This risk increased to more than 40-fold among patients with baseline eGFR values <15 ml/min/1.73 m 2 . Underlying diabetes mellitus, hypertension, and the presence of proteinuria were also associated with increased the risk for hospital-acquired AKI. CKD patients are at high risk for development of AKI secondary to radio-contrast agents, aminoglycosides, atheroembolism, and cardiovascular surgery.



Table 76.2

Common Risk Factors for Developing Acute Kidney Injury














































Risk Factor
Age
Pre-existing Chronic Kidney Disease
Reduced Effective Arterial Volume
Volume Depletion
Nephrotic Syndrome
Congestive Heart Failure
Cirrhosis
Sepsis
Diabetes Mellitus
Drugs
Non-Steroidal Anti-Inflammatory Drugs
Aminoglycosides
Radio-Contrast Agents
Inflammatory States
Trauma
Burns
Sepsis
Post-Surgical State
Post Solid Organ/Allogenic Bone Marrow Transplant
Mechanical Ventilation


The presence of exo-and endo-toxins increases the risk for AKI. Antibiotics, non-steroidal anti-inflammatory agents, anesthetic agents, contrast media and diuretics are well defined risk factors for AKI. There is a synergestic increase in nephrotoxicity from such agents when there is renal hypoperfusion for any reason. Severe infections, especially in the setting of a surgical procedure are often associated with AKI. AKI complicating trauma is often multifactorial in origin, resulting from a combination of hypovolemia and myoglobin release from muscle tissue. AKI remains a frequent complication of surgery necessitating cardiopulmonary bypass despite off pump techniques.


Etiology


Frequently the cause for AKI is multifactorial, involving more than one insult. However, traditionally the etiology of AKI has been categorized anatomically into pre-renal, renal and post-renal ( Figure 76.1 ).




Figure 76.1


Etiology of AKI based on anatomical categories (pre/intrinsic/post).


Prerenal Acute Kidney Injury


The most common cause of AKI is prerenal azotemia and it accounts for about 40–55% of the cases. It results from kidney hypoperfusion due to a reduced effective arterial volume (EAV). The effective arterial volme is the volume of blood effectively perfusing organs. Conditions causing hypovolemia resulting in a reduced EAV include hemorrhage (traumatic, gastrointestinal, surgical), GI losses (vomiting, diarrhea, nasogastric suction), kidney losses (over-diuresis, diabetes insipidus) and third spacing (pancreatitis, hypoalbuminemia). In addition cardiogenic shock, septic shock, cirrhosis, nephrotic syndrome and anaphylaxis all are pathophysiologic conditions that decrease effective circulating volume, independent of the volume status, resulting in reduced renal blood flow. Pre-renal AKI reverses rapidly once renal perfusion is restored because the renal parenchyma remains uninjured. However, when hypoperfusion is severe, it may result in ischemia leading to acute tubular necrosis.


Pre-renal azotemia has also been divided into volume responsive and volume non-responsive. In volume responsive pre-renal azotemia correction of the patients volume status results in increased kidney perfusion and resolution of the disorder. In volume non-responsive forms, additional intravenous volume is of no help in restoring kidney perfusion and function. Disease processes such as severe congestive heart failure and sepsis may not respond to intravenous fluids as markedly reduced cardiac output or a reduction in total vascular resistance, respectively, prevent volume mediated improvement in kidney perfusion. Therefore it is essential to understand the mechanism mediating pre-renal azotemia in order to correct it.


Hypovolemia causes a decrease in mean arterial pressure which activates baroreceptors and initiates a cascade of neural and humoral responses. This leads to the activation of sympathetic nervous system that causes increased production of catecholamines especially norepinephrine. The other major consequence is the activation of the renin-angiotgensin-aldosterone (RAAS) system that causes production of angiotensin II (ATII), a very potent vasoconstrictor. There is also an increased release of anti-diuretic hormone (ADH) mediated both by hypovolemia and a rise in extracellular osmolality, that retains water, as well as influencing urea back-diffusion into the papillary interstitium.


In response to volume depletion or states of decreased EAV there is increased intra-renal ATII activity. This increases proximal tubule Na + absorption through a complex effect in the glomerulus by preferentially increasing the efferent arterioral resistance. Thus the glomerular hydrostatic pressure is increased and preserves GFR. With severe volume depletion there is greater ATII activity leading to afferent arteriolar constriction, that reduces both renal plasma flow and the filtration fraction. ATII has also been shown to have direct effects on transport in proximal tubule through receptors located in the proximal tubule. It has also been postulated that the proximal tubule can locally produce ATII. Hence under conditions of volume depletion, ATII stimulates a larger fraction of the transport, whereas volume expansion will blunt this response.


There is also significantly increased renal sympathetic nerve activity in pre-renal azotemia. Studies have shown that in volume depleted states adrenergic activity independently constricts the afferent arteriole as well as changing the efferent arteriolar resistance through ATII. Renal nerve activity is linked to renin release through β-adrenergic receptors on renin-containing cells while α-a adrenergic influences primarily the vascular resistances within the kidney. In contrast α-2 adrenergic agonists primarily decrease the glomerular ultrafiltration coefficient via ATII. Although vasodilation might be expected as a result of acute removal of adrenergic activity, a transient increase in ATII is actually seen, along with constancy in GFR and renal blood flow. Even after sub-acute renal denervation renal vascular sensitivity increased to ATII as a result of major up-regulation of ATII receptors. Hence complex effects on the renin-angiotensin activity occur within the kidney secondary to renal adrenergic activity during pre-renal azotemia.


All these systems work together and stimulate vasoconstriction in musculocutaneous and splanchnic circulations, inhibit salt loss through sweat, stimulate thirst and retain salt and water to maintain blood pressure and preserve cardiac and cerebral perfusion. Various compensatory mechanisms preserve glomerular perfusion. Autoregulation is achieved by stretch receptors in afferent arterioles that cause vasodilation in arterioles in response to reduced perfusion pressure. Under physiologic conditions autoregulation works until a mean systemic arterial blood pressure of 75–80 mm Hg. Below this, the glomerular ultrafiltration pressure and GRF decline. Kidney production of prostaglandins, kallikrein and kinins as well as nitric oxide is increased contributing to the vasodilation. NSAIDs, by inhibiting prostaglandin production, worsen kidney perfusion in patients with hypoperfusion. Selective efferent arteriolar constriction, which is a result of ATII, helps preserve the intraglomerular pressure andGFR. ACE inhibitors inhibit synthesis of angiotensin II and so disturb this delicate balance in patients with severe reductions in EAV such as severe CHF or bilateral renal artery stenosis and can worsen prerenal azotemia. On the other hand, very high levels of angiotensin II seen in circulatory shock causes constriction of both afferent and efferent arterioles which negates its protective effect.


Although these compensatory mechanisms are protective against acute renal failure, they are overwhelmed in states of severe hypoperfusion. Renovascular disease, hypertensive nephrosclerosis, diabetic nephropathy as well as older age predispose patients to kidney hypoperfusion at lesser degrees of hypotension.


Post-Renal Acute Kidney Injury


Post-renal AKI occurs from either ureteric obstruction or bladder/urethral obstruction. AKI from ureteric obstruction requires that the blockage occur either bilaterally at any level of the ureters, or unilaterally in a patient with a solitary functioning kidney or CKD. Ureteric obstruction can be either intraluminal or external. Bilateral ureteric calculi, blood clots, and sloughed renal papillae can obstruct the lumen, while external compression from tumor or hemorrhage can block the ureters as well. Fibrosis of the ureters intrinsically or of the retroperitoneum can narrow the lumen to the point of complete luminal obstruction. The most common cause for post-renal azotemia is structural or functional obstruction of the bladder neck. Prostatic conditions, therapy with anti-cholinergic agents and a neurogenic bladder can all cause post-renal AKI. Relief of the obstruction usually causes prompt return of GFR if the duration of obstruction has not been excessive. The rate and magnitude of functional recovery is dependent on the extent and duration of the obstruction.


AKI resulting from obstruction usually accounts for less 5% of cases, although in certain settings, e.g., transplant, it can be as high as 6–10%. Clinically the patient can present with pain and oliguria, though these are non-specific. Because of the ease of ultrasonography, its diagnosis is usually straightforward, although a volume depleted patient or a patient with severe reduction in GFR may not show hydronephrosis on radiological assessment. Since initially during the course of the disease GFR is not affected, volume repletion can help with the diagnosis by increasing GFR and urine production into the ureter leading to dilation of the ureter proximal to the obstruction and enhancing ultrasound visualization. Early diagnosis and prompt relief of obstruction remain key goals in preventing long-term parenchymal damage as the shorter the period of obstruction the better the chances for recovery and long-term outcomes. The pathophysiology and treatment of obstructive uropathy are discussed extensively in another chapter.


Intrinsic or Intra-Renal Acute Kidney Injury


It is helpful to divide the causes of intrinsic renal azotemia among categories that delineate the site of the initiating injury. Thus the most useful classification is as follows: (1) Vascular—(a) large renal vessels and (b) renal microvasculature; (2) Glomerular; (3) Tubular and (4) Interstitial. AKI secondary to vasculitides and rapidly progressive glomerulonephritides are discussed elsewhere in the text. Below we shall focus on AKI pathophysiology secondary to tubular and interstitial diseases before going on to discuss ATN in detail.


Interstitial


Acute interstitial nephritis (AIN) represents a frequent cause of acute kidney injury, accounting for 15–27% of renal biopsies performed because of this condition. By and large, drug-induced AIN is currently the commonest etiology of AIN, with antimicrobials and nonsteroidal anti-inflammatory drugs being the most frequent offending agents. Other conditions such as leukemia, lymphoma, sarcoidosis, bacterial infections (e.g., E.coli) and viral infections (e.g., cytomegalovirus) can also cause acute interstitial disease leading to AKI. The inflammatory cellular infiltrates that characterize AIN, mainly composed of T lymphocytes and macrophages, are a powerful source of cytokines that increase the production of extracellular matrix and the number of interstitial fibroblasts, and induce an amplification process recruiting more inflammatory cells and eosinophils into the interstitium. These are often patchy and present most commonly in the deep cortex and outer medulla and mostly comprised of T-cell and monocytes/macrophages and eosinophils. These infiltrations are always associated with interstitial edema, and sometimes with patchy tubular necrosis that if present is usually in close proximity to areas with extensive inflammatory infiltrates. A few neutrophilic granulocytes may be present as well. The majority of cases of AIN are probably induced by extra-renal antigens being produced by drugs or infectious agents that may be able to induce AIN by: (1) binding to kidney structures, (2) modifying immunogenetics of native renal proteins, (3) mimicking renal antigens, or (4) precipitating as immune-complexes and hence serving as the site of antibody or cellular mediated injury. Medications and specific microbial antigens could elicit an immune reaction after their interstitial deposition (planted antigens). Conversely, tubular cells have the capacity to hydrolyze and process exogenous proteins. In this regard, medications can bind to a normal component of TBM, behaving as a hapten, or can mimic an antigen normally present within the TBM, inducing an immune response directed against this antigen. Immunofluorescence studies in renal biopsies of patients with AIN are generally negative, indicating the absence of antibody-mediated immunity that has a marginal, if any, pathogenic role.


Exogenous Nephrotoxins


The kidneys are vulnerable to toxicity as they are the major elimination/metabolizing route of many of these elements and also because epithelial cells reabsorb agents from the interstitium that is exposed to high concentrations of these agents.



Table 76.3

Classification of Various Common Drugs Based on Pathophysiological Categories of AKI







  • 1.

    Vasoconstriction/impaired microvasculature hemodynamics (pre-renal) – NSAID’s, ACE-inhibitors,angiotensin receptor blockers, norepinephrine, tacrolimus, cyclosporine,diuretics, cocaine, mitomycin C, estrogen, quinine, interleukin-2, COX-2 inhibitors


  • 2.

    Tubular cell toxicity– Antibiotics – Aminoglycosides, amphotericin B, vancomycin, rifampicin, foscarnet, pentamidine, cephaloridine, cephalothin. Radio-contrast agents, NSAID’s, acetaminophen, cyclosporine, cisplatin, mannitol, heavy metals, IVIG, ifosfamide, tenofovir.


  • 3.

    Acute Interstitial Nephritis – Antibiotics – Ampicillin, penicillin G, methicillin, oxacillin, rifampicin, ciprofloxacin, cephalothin, sulfonamides. NSAIDs, aspirin, fenoprofen, naproxen, piroxicam, phenybutazone, radio-contrast agents, thiazinde diuretics, phenytoin, furosemide, allopurinol, cimetidine, omeprazole


  • 4.

    Tubular Lumen obstruction – Sulfonamides, acyclovir, cidofovir, methotrexate, triamterene, methoxyflurane, protease inhibitors, ethylene glycol, indinivir, oral sodium phosphate bowel preparations.


  • 5.

    Thrombotic Microangiopathy – Clopidogrel, cocaine, ticlodipine, cyclosporine, tacrolimus, mitomycin C, oral contraceptives, gemcitibine, bevacizumab.


  • 6.

    Osmotic nephrosis – IVIG, Mannitol, dextrans, hetastarch



Radiocontrast Induced Nephropathy (CIN)


CIN is a common complication of radiological or angiographic procedures. The incidence varies from 3–7% in patients without any risk factors, but can be as high as 50% in patients with chronic kidney disease (CKD). Other risk factors include diabetes, intravascular volume depletion, high osmolar contrast, advanced age, proteinuria and anemia. The pathophysiology of CIN likely consists of combined hypoxic and toxic renal tubular damage associated with renal endothelial dysfunction and altered microcirculation. Initially, radiocontrast injection leads to an abrupt but transient increase in renal plasma flow, GFR and urinary output due to the hyperosmolar radio-contrast agent enhancing solute delivery to the distal nephron and leading to an increased oxygen consumption by enhanced tubular sodium reabsorption. A transient phase vasodilation is followed by a period of sustained vasoconstriction, resulting in hypoxic cell damage mainly to the outer medulla. Renal parenchymal oxygenation decreases especially in the outer medulla as documented in various studies where the cortical PO 2 declined from 40 to 25 mmHg, while the medullary PO 2 fell from 30–26mmHG to 9–15 mmHg. The renin-angiotensin system is thought to be activated by RC media, while there is also evidence that Ca 2+ as a second messenger is involved in the renal vasoconstriction. Perturbations in the local vasodilatory system are evident as suggested by aggravation of RCIN in the setting of concomitant NSAID presence, thus highlighting the role of altered renal prostaglandin production in its pathogenesis. Similarly, NO inhibition potentiates the renal damage while L-argninine, a precursor of NO, attenuates the damage implying that a disturbance in NO production likely worsens the decrease in RBF after RC infusion. Increased synthesis and release of endothelin (ET) and adenosine from endothelial cells, combined with suppression of NO production likely results in medullary hypoxia secondary to shunting of blood flow to the cortex. Although experimental animal studies have demonstrated beneficial effects of using ET-antagonists, their efficacy has not been reproduced in human clinical studies. Video microscopy studies have shown that radiocontrast agents markedly reduced inner medullary papillary blood flow, to the extent of near cessation of RBC movement in papillary vessels, associated with RBC aggregation within the papillary vasa recta. Lastly, mechanical factors such as the viscosity of the radiocontrast agent also plays a role, as the contrast agents increase blood viscosity in the inner medulla which already has hypertonic conditions.


Cell-culture studies indicate direct RC media toxicity to proximal tubule cells, which has been observed in human studies where biopsies have shown morphological features of proximal tubular vacuolization, tubular degeneration, and interstitial inflammation and edema. These effects are more pronounced under hypoxic or high-osmolarity conditions. Apoptosis is also induced by RC media in in vitro studies. RCIN typically manifests as an acute deline in GFR within 24 to 48 hours after administration with return to baseline by one to two weeks. Urinalysis in these patients can show either findings of pre-renal azotemia with low fractional excretion of sodium, but in severe cases, findings similar to ATN with tubular epithelial cells and coarse granular casts are seen. In human studies, volume expansion is key to prevention but possibly N-acetyl-cysteine or sodium bicarbonate therapy have been shown to be beneficial in reducing RCIN. The reno-protective effects of N-acetlycysteine may be related to improved NO dependent vasodilation and medullary oxygenation in addition to scavenging of free radicals.


Acute Phosphate Nephropathy


Oral sodium phosphate containing preparation solutions for colonscopic procedures have recently been identified as a cause for AKI. The pathogenesis is related to a transient and significant rise in serum phosphate concentration that occurs simultaneously in the setting of intravascular volume depletion due to the prep agent itself. Intra-tubular precipitation of calcium phosphate salts obstructs the tubular lumen and causes direct tubular damage. Although the complete mechanisms are not fully eludicated, risk factors for acute phosphate nephropathy include pre-existing volume depletion, use of ACE inhibitors and ARBs, CKD, older age, female sex, and higher doses of oral sodium phosphate.


Endogenous Nephrotoxins


Myoglobin and hemoglobin are endogenous toxinscommonly associated with ATN. Muscle injury due to insults such as trauma, excessive immobilization, ischemia, inflammatory myopathis, drugs and metabolic disorders, cause the rapid and excessive release of myoglobin. Myoglobin, a 17 kDa hemeproteinis highly filtered by the glomerulus, and enters the proximal tubule epithelial cells through endocytosis and is metabolized. It causes red-brown colored urine with a positive dipstick for heme, but relative absence of red cells. Intravascular hemolysis results in circulating free hemoglobin, which, when it exceeds haptoglobin-binding is filtered, resulting in hemoglobinuria, hemoglobin-cast formation and heme uptake by proximal tubule cells. AKI in rhabdomyolysis is due to a combination of factors including volume depletion, intra-renal vasoconstriction, direct heme-protein mediated cytotoxicity and intraluminal cast formation. The heme center of myoglobin may directly induce lipid peroxidation, generation of isoprostanes and liberation of free iron. Iron is an intermediate accelerator in the generation of free radicals. There is also evidence to suggest increased formation of H 2 0 2 in rat kidney model of myohemoglobinuria. The subsequent hydroxyl (OH ) radical plays a vital role in oxidative stress induced AKI through mechanisms discussed in detail later in the chapter. Iron chelators such as deferoxamine and scavengers of reactive oxygen species such as glutathione have been shown to provide protection against myo-hemoglobinuric AKI. Similarly, endothelin antagosists have also been shown to prevent hypofiltration and proteinuria in rats that underwent glycerol induced rhabdomyolysis. These studies implicate the important role of vascular mediators such as endothelin-1, thromboxane A, TNF-α, and F-isoprostance. Others have shown NO supplementation might be beneficial by preventing the heme induced renal vasoconstriction, as heme proteins scavenge nitric oxide.


Precipitation of myoglobin with Tamm-Horsfall protein and shed proximal tubule cells leads to cast formation and distal tubular obstruction which is enhanced in acidic urine. In human studies volume expansion and perhaps alkalinaztion of urine to limit cast formation are the preventive measures generally employed as none of the experimental agents used in animal studies have been convincingly beneficial.


Other endogenous nephrotoxins include uric acid and light chains. Excessive light chains, produced in diseases such as multiple myeloma, are filtered, absorbed and then catabolized in proximal tubule cells. The concentration of light chains leaving the proximal portion of the nephron depends on the capacity of the proximal tubule to reabsorb and catabolize them as well as the filtrate concentration. Certain light chains can be directly toxic to the proximal tubules themselves. Light chain-induced cytokine release has been associated with nuclear translocation of NF-κB suggesting that its endocytosis leads to production of inflammatory cytokines through activation of NF-κB. Once the capacity for proximal tubule uptake is overwhelmed, a light chain load is presented to the distal tubule where upon reaching a critical concentration the light chains aggregate and co-precipitate with Tamm-Horsfall protein and form characteristic light chain casts. Light chains, in the amount seen in plasma cell dyscrasiasts, are also capable of catalyzing the formation of H 2 O 2 in cultured HK-2 cells. H 2 O 2 stimulates the production of monocytes chemo-attractant protein (MCP-1), a key chemokine involved in monocytes/macrophage recruitment to proximal tubule cells.


Any process reducing GFR such as volume depletion, hypercalcemia or NSAID’s will accelerate and aggravate cast formation. It has been proposed that acutely reducing the presented light chain load by plasmapheresis might be beneficial in limiting cast formation and reducing the extent of the AKI in certain select patients. Tumor cell necrosis following chemotherapy can release large amounts of intracellular contents such as uric acid, phosphate and xanthine into the circulation that can potentially lead to AKI. Acute uric acid nephropathy with intratubular crystal obstruction and interstitial nephritis is not seen as commonly as it was in the past mainly due to prophylactic use of allopurinol prior to chemotherapy and or rasburicase to acutely lower the serum uric acid levels.


Other therapeutic agents such as amphotericin B, acyclovir, indinavir, cidofovir, foscarnet, pentamidine, and ifosfamide can all directly cause tubular injury.




Models of Acute Kidney Injury


Experimental Models of ARF


Despite a variety of animal and cell culture models of AKI, there remains a need to develop in vivo experimental models of ischemic AKI more closely mimicking clinical human AKI for the development of effective therapies. Some of the important principles in studying the pathophysiology of AKI in various models include the importance of measuring parameters at multiple appropriate time points and the ability to control physiological functions known to affect kidney function (e.g., temperature, blood pressure, anesthesia, fluid status etc.). A limitation in many experimental models is the lack of co-morbidities such as aged animals, chronic kidney disease, multi-organ failure, pre-existing vascular changes or multiple renal insults, which quite often co-exist in human AKI. We will briefly describe the pros and cons of using these experimental models ( Table 76.4 ).



Table 76.4

Comparison of Models of Studying Acute Kidney Injury “+” Minimally Applicable; “++++”Very Applicable












































































































































Humans Animals Cells
Ischemic Septic Toxic
Warm-Ischemia-reperfusion Cold-Ischemia-reperfusion Hypoperfusion/ Cardiac arrest Isolated Perfused Kidneys Endotoxin Cecal Ligation & Puncture Bacterial Infusion Contrast/ Pigment/ Glycerol/ Drug Isolated Proximal Tubule Cells Cultured Tubular Cells
Simplicity + ++++ ++ ++ ++ ++++ +++ +++ ++++ +++ ++++
Reproducibility ++ ++++ +++ +++ +++ +++ ++ +++ +++ +++ +++
Clinical Relevance ++++ ++ +++ ++++ ++ ++ ++++ +++ +++ + +
Therapeutic value +++ ++ +++ ++++ ++ ++ +++ +++ +++ + +
Studying Mechanisms ++ ++ ++ +++ ++ ++ +++ +++ +++ +++ +++
Controlling Extrinsic factors + + ++ ++ +++ ++ ++ ++ ++ ++++ ++++
Isolating single variables ++ + ++ + +++ +++ ++ ++ +++ ++++ ++++
Standardization Value + ++++ +++ ++ +++ +++ ++ +++ ++ ++ +++
Experimental Limitation ++++ +++ +++ +++ ++ ++ +++ ++ ++ + +


The warm ischemia-reperfusion renal clamp model is one of the most widely used experimental models in rats and mice because of its simplicity and reproducibility. In rats the inflammatory response, tubular injury and repair, and medullary congestion are similar and probably comparable to human ischemic ATN. However, in human AKI, isolated ischemia is seen rarely seen and neither is there usually complete cessation of blood flow to the kidneys. In this model, important mediators of injury suchreactive oxygen species (ROS) and perioxynitrite species may have a different or delayed role as compared to low oxygen states in hypoperfusion models. Total blood flow cessation also prevents the degradative products of the ischemic kidney from being eliminated. Other factors playing a role in the pathophysiology of AKI such as inflammatory mediators released from gut ischemia, endothelium, smooth vascular muscle cells need to be taken into consideration in any experimental model. Release of bowel proteins into the circulation can act as inflammatory mediators and increase the susceptibility to AKI. The S3 segment of the proximal tubule is almost completely necrosed in such models, a finding not seen very frequently in human ARF. In contrast to animal models, human AKI histological biopsy data are lacking at early time points from the onset of insult. This has made comparison between animal models and human AKI of limited value. Lastly, drug delivery is prevented in total occlusion models, which actually may be of significant value during the peak ischemic insult.


The cold ischemia-warm reperfusion model resembles AKI in human transplants but this model is inadequately studied and difficult experimentally. In the isolated perfused kidney model, the kidney is perfused in ex vivo using perfusates either with or without erythrocytes, and employs either ischemic (stopping perfusate) or hypoxic (reduced oxygen tension of erythrocytes) to induce functional impairment. The morphological patterns are different in erythrocyte free and erythrocyte rich perfusates. The latter system is more comparable with what is observed histologically in animal models. Additionally, limitations include exclusion of various inflammatory mediators, neuro-endocrine hemodynamic regulation, and systemic cytokine and growth factor interactions known to be present and play a pathophysiologic role in animal models.


Cardiac arrest is a common scenario leading to human ARF. Rabb et al. have described whole body ischemia reperfusion injury model induced by 10 minutes of cardiac arrest, followed by cardiac compression resuscitation, ventilation, epinephrine and fluids, which that lead to a significant rise in SCr and renal tubular injury at 24 hours. One of the unique advantages of this model is the cross talk between vital organs such as the brain, heart, lung and the renal hemodynamics. A hypoperfusion model of AKI using partial aortic clamping was first described by Zager et al. may be more representative of human AKI reflecting a state of reduced blood flow to the kidney with systolic blood pressure around 20 mm Hg, resulting in reproducible AKI. This was also recently adapted and refined in a study where a novel compound, soluble thrombomodulin, was used to minimize ischemic injury in a partial aortic clamp AKI model.


Toxic models of renal failure employ various known toxins, such as radiocontrast, gentamicin, cisplatin, glycerol and pigments including myoglobin and hemoglobin. Septic models to study AKI include cecal ligation and puncture, endotoxin infusion andbacterial infusion into the peritoneal cavity. The endotoxin model which is simple, inexpensive and suitable to study new pharmacological agents, has certain drawbacks as well. There is variability amongst sources and types of lipopolysaccharide (LPS) endotoxin, rate and method of administration, and it is usually of short duration due to the high mortality associated with the doses required to induce AKI. It also tends to be a vasoconstrictive model and does not recapitulate the hemodynamics nor inflammation of human sepsis. In the cecal ligation and puncture model (CLP), there is considerable similarity with sepsis in humans with acute lung injury, metabolic derangement and systemic vasodilation, accompanied by increased cardiac output initally. However there is some variability depending on the mode and size of cecal perforation. Star et al. have developed a new sepsis model keeping under consideration the following facts: (1) animals should received the same supportive therapy that is standard for ICU patients (i.e., fluid resuscitation and antibiotics); (2) age, chronic co-morbid conditions and genetic heterogeneity vary. Complex animal models of human sepsis that introduce these disease-modifying factors are likely more relevant and may be more pharmacologically relevantthan simple animal models. The zebra fish model developed by Bonventre et al. has the advantages of markedly improved accessibility of the kidney, feasibility of knock-down and upregulation of genes and a short phenotypic readout time, while at the same time possessing the complexity of an organism to study renal injury. These properties may make it a useful inexpensive tool to screen therapeutic agents in the future.


Experimental models of hypoxic acute kidney damage differ morphologically in the distribution of tubular cell injury and tubular segment types differ in their capacity to undergo anaerobic metabolism, mount hypoxia-adaptive responses mediated by hypoxia-inducible factors (HIFs). Hence it is important to keep them in mind the potential pitfalls when evaluating experimental studies or therapeutic interventions using these models. The lack of ability to demonstrate effectiveness of an agent in humans which has been shown to be efficacious in animal models, does not necessarily reflect a flaw with the model. Most often, the agent is administered very late in the course of the human disease, and the patient heterogeneity of the population makes it even more difficult to establish true efficacy.


Role of Biomarkers in AKI


Changes in serum creatinine and/or urine output to diagnose AKI may not be able to identify the early stages of intrinsic kidney injury. Early identification and subsequent early pharmacologic intervention may improve outcomes in AKI. In order to facilitate the early diagnosis of intrinsic injury, multiple biomarkers of tubular injury have been evaluated. Biomarkers for AKI include N-acetyl- B -D-glucosaminidase (NAG), kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and interlukin 18 (IL-18), among others. In addition, serum cystatin C has been proposed as more sensitive and timely than serum creatinine for detecting changes in GFR, and urinary cystatin C has been proposed as a marker of tubular injury. Although not utilized yet for routine clinical use, these biomarkers have the potential to provide an early diagnosis of intrinsic AKI and the ability to differentiate pre-renal AKI from intrinsic tubular damage, as well as to provide prognostic information of an episode of AKI. One biomarker or a panel of these biomarkers may eventually provide the necessary early diagnosis to allow therapies to limit kidney damage and promote recovery of kidney function. Please refer to Chapter 75 for a detailed explanation on the role of serum and urinary biomarkers in AKI.




Pathophysiology of Acute Kidney Injury


Morphological Changes of AKI


Acute Tubular Necrosis (ATN) is the most common form of AKI, and this process of renal tubular injury encompasses more than just cell death followed by repair. It is easier to understand the entire spectrum of injury if one looks at the different compartments involved and the phases they go through ( Fig. 76.2 ).




Figure 76.2


Overview of pathogenesis in acute kidney injury. The major pathways of impairment of glomerular filtration rate (GFR) in ischemic acute tubular necrosis as a result of vascular and tubular injury (see text for details).

(Source: unpublished figure from Encyclopedia of Intensive Care Medicine. Publisher, Springer. Eds, Vincent, Jean Louise Hall, Jesse B. Ch Epithelial Cell Injury by Asif Sharfuddin and Bruce Molitoris. First Edition due to published March 2012).


Tubular Epithelial Cell Injury


Although data regarding which nephron segments in humans with ATN are more severely affected is sparse due to lack of biopsies early in the course of ATN, experimental animal models provide sufficient information to help understand and delineate the mechanisms of ATN by histological analysis. In ATN the most severe tubular injury takes place within the outer medulla of the kidney, and involves the S3 segment of the proximal tubule (pars recta) and the medullary thick ascending limb (MTAL) of the distal nephron. The S3 segment has limited capacity to undergo anaerobic glycolysis. Secondly, due to its unique primarily venous capillary regional blood flow, there is marked hypoperfusion and congestion in this medullary region post injury that persists even though cortical blood flow may have returned to near normal levels after ischemic injury. Endothelial cell injury and dysfunction are primarily responsible for this phenomenon, known now as the “extension phase” of AKI. The proximal tubule S 1 and S 2 segments are most commonly involved in toxic nephropathy due to their high endocytic rates leading to increased cellular uptake of the toxin.


The apical brush border of proximal tubule cells (PTC) is damaged early resulting in microvilli disruption and detachment from the cell surface forming membrane bound “blebs” released into the tubular lumen. Loss of microvillar surface leads to ineffective enzymatic activity, endocytosis, channel and transporter density resulting in diminished effective transcellular absorption. Patchy detachment and subsequent loss of tubular cells exposing areas of denuded tubular basement and focal areas of proximal tubular dilatation along with the presence of distal tubular casts is also a major pathological findings in ATN. Because the surviving adjacent cells tend to spread out and become flattened, in an attempt to completely or partially cover the denuded epithelium, the appearance is often of a flattened and pauci-cellular epithelium.


Sloughed off tubular cells are also present in the tubular lumen, where they can be overtly necrotic or viable . These cells along with brush border vesicle remnants and cellular debris combine with Tamm-Hosrfall glycoprotein (THP) and form the classical “muddy-brown granular” casts, that have the potential to obstruct the tubular lumen. Cast formation may be potentiated by relative stasis of tubular fluid flow because of the reduction in GFR. On biopsy, these casts may not be captured since they exist within the medulla and actual site of obstruction may be a short segment. However, dilation of tubules proximal due to obstruction is often seen as long as the GFR and regional tubular epithelium are maintained. Eventually decompression of these tubules will occur because of decreased GFR, damage to proximal tubular cells, and persistent reabsorption of tubular fluid in uninjured areas. The injury to the proximal tubule cells in humans is also seen in experimental models of ischemic AKI ( Fig. 76.3 ).




Figure 76.3


A. Morphology of acute tubular necrosis in human biopsy specimen. Proximal tubules show loss of brush border, flattening of tubular cells with denuded basement membranes, blebbing of cytoplasm (arrowhead) and vacuolization in a patient with toluene induced acute tubular necrosis. There is also evidence of extensive interstitial edema and expansion with presence of inflammatory cells (arrow). ( Slide courtesy of Dr Carrie Phillips ). B. Morphology of acute tubular necrosis in rat kidney specimen subjected to 60 minutes of hypoperfusion. Areas of detachment of cell and intact cells within the tubular lumen. C. Cellular cast within the tubular lumen.


Apoptotic features are more commonly seen in both proximal and distal tubule cells as compared to necrosis which itself is inconspicuous and restricted to the highly susceptible outer medullary regions. Apart from the proximal tubular cells, the other major epithelial cells of the nephron are those of the medullary thick ascending limb located distally. Apoptotic changes have been detected in human AKI, as shown in distal nephron segments in nephrotoxic acute tubular necrosis. Distal tubular cell apoptosis also occurs in donor biopsies before engraftment, which was predictive of delayed graft function due to acute tubular necrosis. In an ex vivo model of hypoxic AKI, inhibition with FG-4497 (specific prolyl-hydroxylase inhibitor which leads to activation of HIF-Aplha) in the isolated perfused kidney led to decreased selective outer medullary distal tubular injury. The course of a tubular cell alterations may take different paths depending on the type and extent of injury as discussed later.


Glomerulus


The glomerular tuft collapses in ischemic injury, and some investigators have described the diameter of ischemic endothelial cell fenestrae on average to be larger than that of untreated kidneys. Other human biopsy studies have documented enlargement of juxtaglomerular apparatus during the oligoanuric phase, and thickening and coarsening of foot processes. But these findings have not been confirmed and there still exists a paucity of data on glomerular changes in human ATN in different stages. Glomerular epithelial cell injury in ischemic, septic or nephrotoxic injury is not classically seen although some studies have shown thickening and coarsening of foot processes and recently Wagner et al. have shown podocyte specific molecular and cellular changes.


Epithelial Cytoskeletal Abnormalities


Cytoskeletal Alterations


Cellular structure and function are mediated by an interactive and dynamic role of the actin cytoskeleton including but not limited to proximal tubule brush border microvilli structure and function, cell polarity, endocytosis, signal transduction, cell motility, movement of organelles, exocytosis, cellular division and migration, barrier function of the junctional complex, cell-matrix adhesion and signal transduction. Actin is present in globular form (G-actin) that can self-assemble into filamentous (F-actin) to form helical microfilaments. In conjunction with actin-binding proteins, guanosine triphosphatases (GTPases) and adenosine triphophate (ATP), the dynamic process of actin assembly and disassembly is accomplished. The actin cytoskeleton is present as a layer of microfilaments below the apical plasma membrane, forming the terminal web. The architectural integrity of brush border microvilli is dependent upon extensions of actin filaments from the terminal web to the tip of the individual microvilli.


Under physiologic conditions most actin monomers are ATP-bound, while the bulk of actin found within actin filaments is ADP-bound. The unique binding sites of actin for nucleotides and divalent cations allow conformational changes, apart from its hydrolytic properties that are activated by polymerization. The G-actin ADP complex released on depolymerization (disassembly) undergoes nucleotide exchange with abundant cytosolic ATP, and is then stored as a high energy G-actin ATP intermediate by thymosin-sequestering proteins until needed for polymerization. Four groups of actin-binding protein mediate different effects on the actin cytoskeleton including actin sequestering, capping, severing and nucleation. These effects in turn coordinate the continual remodeling of the cytoskeleton and give it the ability to respond to various internal and external stimuli.


Ischemic insults to proximal tubule cells induce a rapid and severe degeneration of the microvillar F-actin core, which in turn mediates the plasma membrane finger-like microvillar structural morphology changes including loss of the apical membrane through blebbing. The degeneration of this F-actin core occurs as a result of ATP depletion leading to depolymerization of microvillar actin. In addition ezrin, an actin binding phosphorylated protein, becomes dephosphorylated during ischemia and the attachment between the microvillar F-actin core and the overlying plasma membrane is lost ( Fig. 76.4 ). The apical membrane is then either exfoliated as “blebs” into the tubule lumen or internalized with the capability of being recycled during cellular recovery. Furthermore, the concentration of F-actin in the cell increases with the formation of large cytosolic aggregates in the perinuclear region and also near the junctional complexes and basolateral membrane. ATP-G-actin levels decrease rapidly during ischemia. Since ADP G-actin cannot be sequestered by thymosin its concentration exceeds a critical threshold resulting in the polymerization in an unregulated fashion.




Figure 76.4


Cytoskeletal and tight junctions alterations in AKI. Ischemic insult to a proximal tubule cell disrupts actin cytoskeleton and junctional complexes. The orderly arrangement of the actin microfilaments extends from terminal web (TW) into microvilli (MV) as well as interacting with tight junction proteins zonula adherens (ZO), and adherens junction proteins zonula adherens (ZA). Occludin (OC) is transmembrance integral protein of the tight junction forming a multiprotein complex with ZO, controlling paracellular permeability. Severe ATP depletion results in occludin translocating to the cytoplasm, compromising adhesion and permeability. Similarly adherens junction proteins such E-cadherin (EC) and catenins (C) that interact with actin and other junctional components are compromised. ADF or cofilin is activated with ischemia that translocates and gets recruited to apical microvilli and binds to F-actin structures, resulting in severing and depolymerization of F-actin. This leads to subsequent apical membrane disruption and bleb formation.


The actin binding protein family of cofilin, also known as actin depolymerizing factor (ADF), has been shown by Molitoris and colleagues to be a critical mediator in F-actin severing during ischemic injury. In proximal tubule cells (PTC) ADF/cofilin, which when phosphorylated is inactive and does not bind actin, gets rapidly dephosphorylated and therefore activated by renal ischemia. This leads to relocalization from the cytoplasm to the surface membrane, as well as in shed membrane-bound vesicles seen in PTC lumen. The mechanism by which disruption of F-actin structure occurs also involves the role of another family of actin binding protein called tropomyosin. Under physiologic conditions tropomyosin binds to and stabilizes the F-actin microfilament core in the terminal web, and protects the filaments from ADF/cofilin induced severing and depolymerization. Ashworth et al. have demonstrated that after ischemic injury there is dissociation of tropomyosin from the microfilament core providing access to microfilaments in the terminal web for F-actin binding, severing and depolymerizing actions of ADF/cofilin proteins.


Alterations in the activity of Rho family of GTPases also contributes to changes in actin cytoskeleton associated with ischemia. First, chemical ATP depletion was shown to cause Rho GTPase inactivation. Secondly, GTP depletion during ischemia could also inactivate Rho GTPase function. These two findings, coupled with the finding that cells transfected with a constitutively active form of RhoA during chemical ATP depletion are protected against actin depolymerization, provide evidence of ischemia-induced RhoGTPase inactivation. An additional cytoskeletal component important for cellular polarity and protein trafficking is microtubules. Wald et al. have shown that in reperfused rat proximal tubules non-centrosomal microtubule organizing centers (MTOCs) were fully detached from the cytoskeleton and scattered throughout the cytoplasm at three days after reperfusion when brush borders membranes were mostly reassembled with normal F-actin distribution. At that time microtubules were also fully reassembled but lacked their normal apicobasal orientation, hence demonstrating that the reestablishment of the submembrane F-actin does not seem to be sufficient for a full polarization of the cells. Microtubule formation also occurs by continuous assembly and disassembly of α and β tubulin heterodimer with an intricate polymerization process. Studies indicate that during ischemia α and β tubulin do not participate in microtubule polymerization and their localizations are also different. The fact that GTP levels are depleted by 90% after 30 minutes kidney ischemia in rats supports this assumption of impaired microtubule polymerization.


Epithelial cells are characterized by an asymmetrical distribution of proteins and lipids in the apical and basolateral membrane resulting in surface membrane polarity of these cells. In ischemic ATN this polarity is abolished, but has the potential for re-establishment during recovery. Molitoris et al. have shown evidence to suggest the Na + K + -ATPase pump that normally resides in the basolateral membrane of proximal tubule cells, under conditions of chemical anoxia, is redistributed to the apical membrane. This redistribution, which can occur as early as 10 min after ischemia, is another consequence of the disruption of the actin cytoskeleton, which normally maintains the attachment of the Na + K + -ATPase to the basolateral membrane. Furthermore, both ankyrin and fodrin dissociated from F-actin and each other during ATP depletion. These data were confirmed and indicate wide spread actin cytoskeleton alterations during ATP depletion lead to altered protein-protein interactions. Other nephron segments such as the distal tubule cells, and TAL do not show similar apical redistribution of the Na + K + -ATPase. This redistribution results in functional consequences reflected in the loss of unidirectional transport of salt and water across the epithelial cell, resulting in one mechanism of the high fractional excretion of Na + seen in patients with ATN ( Fig. 76.5 ).




Figure 76.5


An overview of sublethal injury to tubular cells. An overview of sublethally injured tubular cells. Na/K/ATPase pumps are normally located at the basolateral membrane. In sublethal ischemia the pumps redistribute to the apical membrane of the proximal tubule. Upon reperfusion, the pumps reverse back to their basolateral location.

(Source: unpublished figure from Encyclopedia of Intensive Care Medicine. Publisher Springer. Eds, Vincent, Jean Louise; Hall, Jesse B. Ch Epithelial Cell Injury by Asif Sharfuddin and Bruce Molitoris. First Edition due to published March 2012.)


Junctional Defects and Permeability Alterations


Cell-cell junctional complexes actively participate in the establishment and maintenance of cell polarity, paracellular transport, cytoskeletal interactions, and rearrangements in cellular shape. Ischemia also induces functional changes in the epithelial junctional complexes, which are comprised of at least three structures: adherens junctions (also known as zonula adherens (ZA)), tight junctions (zonula occludens (ZO)) and desmosomes. Tight junctions are located directly apical to the adherens junction, are composed of a growing list of proteins such as occludin, claudin, protein kinase C (PKC), ZO-1 etc., with multiple functions such as adhesion, permeability, structural integrity and paracellular transport of solutes. The actin present in the cortical belt, and in the terminal web, is also linked to the tight junction. Adherens junction, which are located directly below the tight junction, form strong cell–cell adhesion complexes and are composed of proteins such as cadherins and catenins are associated with numerous other junctional and cytoplasmic proteins. They are responsible for adhesion of adjacent cells, regulation of adhesion, and are also linked to the actin cytoskeleton.


In vivo and in vitro studies indicate that in early ischemic injury there is an “opening” of the tight junctions as the proteins such as ZO-1 and cingulin become insoluble during ATP depletion, and associate into macromolecular intracellular complexes. This leads to increased permeability of the tight junctions in sub-lethal injury resulting in back-leakage of glomerular filtrate, which is an important factor in the reduction of GFR as discussed later. If ATP is repleted before lethal injury the permeability defect resolves. PKC signaling regulates both tight junction and adherens junction assembly. Evidence suggest that tight and adherens junction proteins including ZO-1, ZO-2, ZO-3, occludin, vinculin, and p100–p120 are affected by this kinase signaling pathway. It is also likely that disruption of the actin cytoskeleton and reduction in activity of Rho-GTPases also contribute to the changes in the tight junction during ischemia. The loss of adherens integrity is in part caused by activation of c-Src which translocates to the adherens junction and tyrosine phosphorylates components such as β-catenin. Nigam et al. have demonstrated the role of tyrosine kinases and phosphatases in the disassociation of adherens junction. The importance of these animal model findings is highlighted by findings in human allografts with ATN where Kwon et al. have shown the same features noted in experimental models of loss of cell polarity and tight junctions.


Epithelial cells also lose their attachment to the underlying extracellular matrix, the mechanism for which has been elucidated as at least being partially due to loss of polarity and redistribution from the basal membrane to the apical membrane of β1-integrins. Integrins are transmembrane proteins normally responsible for the anchoring of epithelial cell to the matrix through actin cytoskeleton and actin-binding proteins. In vitro studies of MDCK cells have shown that adherence of these cells to a collagen I substratum is mediated by peripheral actin filaments and adhesion complexes regulated by myosin light chain kinases and adhesion complexes controlled by RhoA. The detachment and loss of tubular cells into the lumen also contributes to the back-leakage of the glomerular filtrate, and at the same time the β1-integrins and E-cadherins might even play a role in mediating the aggregation of these exfoliated cells worsening intraluminal cast formation.


Although the glomerular injury is not as prominent in AKI, Wagner et al. have demonstrated in an in vivo rat model that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria owing to rapid loss of interactions between the tight junction proteins Neph1 and ZO-1. Cell culture models using human podocytes further showed that ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding, and redistribution of Neph1 and ZO-1 proteins from the cell membrane to cytoplasm; ATP recovery increased phosphorylation of Neph1 and restored Neph1 and ZO-1 binding and their localization at the cell membrane.


Tubular Obstruction


Tubular obstruction has been noted in ischemic as well as toxic models of injury. Renal tubular epithelial cells can be seen in the urine of patients with AKI, and can be either alive, apoptotic or necrotic. Micropuncture studies, done over 20 years ago, demonstrated elevation of intratubular pressure early after reperfusion following renal artery occlusion. This is characteristically evident as tubular dilatation, with cast deposition in the distal nephron causing luminal obstruction and back pressure. Although intratubular pressures tend to fall towards normal after 24 hours, the presence of persistent obstruction can be revealed by extracellular volume expansion, which again elevates intratubule pressures. An obstructing cast in the collecting duct could potentially impair the function of multiple nephron units, as many nephrons drain into a single collecting duct.


The term “back-leak” generally implies the passive movement of GFR into the interstitium from the tubular lumen, eventually being recirculated to the systemic vasculature through the venous network. Studies have revealed that if radiolabeled compounds are microinjected into renal tubules after ischemic injury, they can be detected in the contra-lateral kidney. Human studies by Myers et al. provided evidence of transtubular leakage of GFR after ischemic renal failure as well as tubular obstruction, leading to a reduction in measured or effective GFR. The presence of areas of open PTC tight junction or denuded basement membrane in electron-microscopy biopsy specimens provides a logical morphological explanation of back-leakage. However, understanding the mechanism responsible for tight junction dysfunction or detachment of tubular cells is key to defining the event of cast formation and tubular obstruction. The integrin superfamily of proteins, located on the basal aspect of the cell, is responsible for the complex cell-matrix adhesion events. The β chains coupled with α chains form β1 integrins, which interact with the actin cytoskeleton and actin-binding proteins such as α-actinin, vinculin and talin. The extracellular domain of the β1 integrins attaches to receptors of proteins such as collagen and fibronectin, which are abundant and constitute the tubular basement membrane. The tri-peptide sequence of arginine-glycine-asparagine (abbreviated as RGD), is a well define receptor for β1 integrin on the extracellular matrix. The loss of polarity causes redistribution of β1 integrins, which become expressed in the apical domain of sublethally injured cell. It was hence hypothesized that administraton of an excess of soluble RGD containing molecules would saturate the extracellular binding site of all exposed β1 integrins within the lumen of the nephron, and thus prevent luminal renal tubular cell-cell adhesion, and this prevent intratubular obstruction. Both intravenous and direct intrarenal infusion of RGD peptides resulted in amelioration of ischemic AKI. It is also possible that the detached tubular cells adhere to Tamm-Horsfall protein (THP) in the distal tubule by RGD sequence peptides. By using dual labeled RGD peptide sequences, it was also discovered that RGD peptides also mapped to intimal surface of vessels in ischemic kidneys. Further studies utilizing RGD peptides in ARF could provide key answers to questions of vascular and epithelial injury in ATN. Recent studies also show a role of the sphingosine-1 phosphate receptor (S1PR) in maintaining structural integrity after AKI. Okusa et al. have shown that S1PRs in the proximal tubule are necessary for stress-induced cell survival, and S1P 1 R agonists are renoprotective via direct effects on tubular cells.




Microvascular Insult in AKI—Functional Basis and Morphological Changes


Microvasculature and Interstitium


A prominent feature of AKI is interstitial edema, which is in part due to altered endothelial permeability, as well as increased tubular pressure, perhaps via backleak through the wall of injured or distended tubular cells. Solez et al. in 1974 demonstrated there was intravascular leukocyte accumulation following ischemic injury, a finding which is still commonly seen in peritubular capillaries, particularly in the ascending vasa recta in the outer and inner medulla. The pathogenesis and significance of this finding is discussed in detailed later. The peritubular capillaries of the corticomedullary junction and outer medulla are most often affected in ischemic human AKI, exhibiting vascular congestion, accumulation of inflammatory cells and either compression or dilatation of vessels. Subtle and few changes may be seen in larger vessels such as arterioles and arteries. The pathologic profile of interlobular and afferent arteriolar vessels in renal artery clamp experiments shows vacuolization in the muscular layer as early as four hours post-ischemic insult, followed by focal necrosis in the smooth muscle.


Medullary Ischemia


Renal Blood flow (RBF) approximates 20–25% of the total cardiac output, and various forces regulate glomerular filtration as a result of autoregulation of renal blood flow. A small fraction of RBF is delivered to the medulla, while the cortex receives the majority. A relatively hypoxic region thus exists in the medulla with partial pressures of oxygen as low as 20–30 mm Hg. In contrast the partial pressure of oxygen in the cortex is about 80–90 mmHg. It has been known for years that restoration of total RBF to near normal, shortly after an ischemic insult does not prevent the extension or maintainence phase of AKI. Thus a sequence of endothelial and epithelial cell processes is triggered that are independent of re-establishing total RBF.


The principal determinant of the medullary oxygen requirement is the rate of active Na + reabsorption along the mTAL. Therefore, not only the reduction of oxygen delivery, but also the increment of oxygen demand can cause an imbalance. Dehydration, volume depletion and renal hypoperfusion are major stimuli of urine concentration through active sodium reabsorption, which may further exacerbate hypoxic tubular damage. By volume repletion and salt loading, this workload is decreased, obviating the need for urine concentration, and hence able to tilt the balance back to match the oxygen supply. The kidney does have its own protective mechanism known as tubuloglomerular feedback (TGF), the stimulus for which appears to be the sodium concentration of the tubular fluid as sensed by the macula densa of the juxtaglomerular apparatus. Increased sodium sensed in this nephron segment, will in turn activate TGF to reduce the GFR, resulting in reduced metabolic demand placed on the tubule, giving the nephron an optimal oxygen supply versus demand balance. Clinically this results in oliguria, which could be termed as an appropriate physiological response to an insult. When this response system is overwhelmed, due to continued insults such as sever hypoxia, or hypoperfusion, this balance is lost, leading to cell death or necrosis.


Although tubular injury is a major mechanism initiating the decrease in GFR with AKI, the concomitant vascular changes are now being recognized as an important pathophysiologic variable. The glomerular capillary hydraulic pressure is maintained by variations in the preglomerular and postglomerular arteriolar resistances. GFR remains relatively constant despite variations in renal perfusion pressure through the process of autoregulation which includes tubuloglomerular feedback (TF) and myogenic alterations of arteriolar tone. Structurally, resting or basal tone is determined by intrinsic smooth muscle tone, and endothelial cells. Endothelial cells can detect changes in shear stress and mediating responses to altered flow. Basal nitric oxide (NO) activity is an important determinant of resting vascular tone. Responses to extrinsic stimuli are used to measure vascular function, also termed as vascular reactivity. These stimuli can be systemically generated, e.g., ANP, catecholamines, angiotensin II, or locally (paracrine), e.g., thromboxane A2, PGH2, endothelin-1 (ET-1), platelet activating factor (PAF), NO, amongst others. The kidney of all organs has the greatest vasoconstrictor sensitivity to ET-1. Characteristics of tubular fluid also modulate autoregulation of GFR to maintain fluid and electrolyte balance.


It is very important to note that abnormalities in RBF in AKI lead to persistent hypoxia in certain areas of the kidney. Severe hypoperfusion of the outer medulla persists long after the insult which initiated ATN has resolved. As cortical blood flow improves after reperfusion and cortical tubule cells demonstrate repair and regeneration. However, the S3 and MTAL segments of the outer medulla experience ongoing ischemia, thought to be due to “shunting” of oxygen between descending and ascending vasa recta, and vascular congestion in the peritubular capillaries. This contributes to ongoing injury to the S3 PTC and mTALC causing the extension phase of ATN. Studies have also emphasized the role of tubular cell swelling as a cause for vascular congestion, as well as a possibility of compression of capillaries by swollen tubules causing mechanical impediment, limiting reperfusion to the cortico-medullary junction of the kidney. Finally, WBC attachment, especially to the outer stripe venous capillaries and RBC rouleaux formation, leads to reduced and even stagnant flow to that area.


Nitric Oxide in ATN


A variety of vasoactive substances are vasconstrictive mediators of the microcirculation and thought to be major determinants of decreased RBF in AKI. Endothelial cell damage contributes to intra-renal vasoconstriction, by an imbalance of vasodilators and vascoconstrictors. The role of NO in the kidney ranges from homeostatic regulation and integration of tubular, vascular and glomerular functions, to integral cellular functions including energy metabolism, cellular respiration, proliferation and transcription. Specifically NO helps regulate local renal circulation, renal afferent and efferent nerve activity and direct fluid and electrolyte reabsorption in tubules. It is produced in both renal and non-renal vasculaturefrom L-arginine by the Nitric Oxide Synthase (NOS) isoforms, of which the three principal forms are neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). Intrarenal NO is responsible for up to one third of the normal renal blood flow and helps maintain the low renal vascular resistance under physiological conditions. NO also plays a key role in regulating perfusion of the renal medulla and local infusion of NOS inhibitors into animals reduces medullary blood flow and promotes salt retention. Conversely L-arginine infusion increases NO levels and enhances medullary blood flow.


During AKI the production of NO increases in tubular cells as a result of cytokine induced increase in expression of iNOS. Studies by Ling et al. have shown that proximal tubule cells isolated from mice with iNOS deficiency were resistant to damage by hypoxia, while mice lacking eNOS or nNOS were damaged by hypoxia. Inhibition of eNOS is also known to occur as endothelial dysfunction develops. Furthermore high output NO production by iNOS may suppress the activity of eNOS without changing its abundance. Hence in ischemic AKI, there is an imbalance of eNOS and iNOS . Goligorsky et al. have proposed that due to a relative decrease in eNOS, secondary to endothelial dysfunction and damage, there is a loss of anti-thrombogenic properties of the endothelium, hence leading to increased susceptibility to microvascular thrombosis. The decrease also leads to enhanced PMN adhesion, and vasoconstriction. On the other hand, the relative increase in iNOS leads to enhanced PMN motility, induction of tubular epithelial cell injury, loss of vasomotor response and suppression of eNOS. Generation of superoxide and NO in ischemia/reperfusion injury results in the formation of peroxynitrite anion (ONOO ). This metabolite is cytotoxic and is capable of causing lipid peroxidation and DNA damage. Effective scavenging of peroxynitrite by ebselen resulted in amelioration of renal dysfunction and a decrease in nitrotyrosine formation. Hence, apart from oxidative stress, there is a role for nitrosative stress in ensuing loss of kidney function (See section on Reactive Oxygen Species). Selective inhibition, depletion or deletion of iNOS have clearly shown renoprotective effects during ischemia. This effect is in part due to rescue of tubular cells from injury by iNOS or its reactive oxidized by products. Administration of L-arginine, NO-donor molsidomine, or the eNOS cofactor tetrahydrobiopterin can preserve medullary perfusion and attenuate acute kidney injury (AKI) induced by ischemia/reperfusion (I/R); conversely the administration of N <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='ω’>𝜔ω
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-nitro-L-arginine methyl ester, an NO blocker, has been reported to aggravate the course of AKI following I/R injury.


Certain other forms of AKI, such as rhabdomyolysis, also have their own unique effect on NO balance. This is due to the fact that heme proteins are potent scavengers of NO, and hence when there is an excess of heme proteins, the basal vasodilatory effect of NO is abolished. Since NO has an inhibitory effect of endothelin-1 synthesis (ET-1), there is a surge in ET production thus aggravating the vasoconstriction. It should also be emphasized that both myoglobin and free hemoglobin themselves can induce iNOS thus acting as another pathogen in the AKI associated with rhabdomyolysis.




Role of Endothelin


The role of endothelin-1 (ET-1) in AKI has been extensively studied. Ischemic and toxic injury increase the expression of pro-ET-1 gene, and the release of mature ET-1 from endothelial cells. ET-1 is the one the most potent vasoconstrictor known, and is produced from a precursor of 38–39 amino acids by the enzyme endothelin-converting enzyme in endothelial cells. It has been shown that ET-1 is detectable in the plasma of human and animals and also in a variety of tissues. The kidney is a major site of ET-1 production and its effect. The major actions of ET-1 are: (1) Hemodynamic-renal vasoconstriction and mesangial cell contraction. (2) Transport: low doses cause diuresis and natriuresis. High doses cause profound anti-natriuretic and anti-diuretic effects. (3) Proliferation: mitogenesis and proliferation of mesangial cells through ET-A receptor stimulation. (4) Inflammation: recruitment and activation of leukocytes.


The renal vasconstrictive effects are produced by vascular smooth muscle cell contraction following activation of ET-A receptor causing a flux of intracellular calcium. The end result is reduction in both RBF and GFR. With the development of various specific ET receptor antagonists there is compelling evidence of its role in ARF. Most ET receptor anatagonists are able to ameliorate the renal injury after ischemic and toxic injury. Jerkic et al. demonstrated that administration of bosentan, a dual ET receptor antagonist, in experimental ischemic AKI resulted in decreased tubular cell injury and increased RBF and GFR. Other toxic injury models such as endotoxin, CsA and myoglobinuric-induced renal failure have all shown to be associated with elevated plasma ET levels.




Endothelial Cell Injury in AKI


It has been known for over thirty years that endothelial cells in the renal vasculature undergo an early swelling during ischemia leading to a narrowing of the lumen. Evidence of endothelial dysfunction also comes from experiments that have found over expression of ICAM-1 by vascular endothelial cells and enhanced expression of the Arg-Gly-Asp (RGD) peptide binding integrins in ischemic AKI . Using minimally invasive intravital microscopy of the glomerular and peritubular capillaries, Goligorsky et al. have shown endothelial dysfunction andthe no-reflow phenomenon manifested by reversal, deceleration and cessation of blood flow, occurring in a sporadic fashion in pre- and postglomerular capillaries in post-ischemic kidneys.


The integrity of endothelial barrier is also impaired in AKI as shown by in vitro studies demonstrating endothelial cell desquamation and formation of gaps between confluent endothelial cells treated with thrombin, while in vivo studies in inflammatory states have provided direct evidence for increased gaps between endothelial cells with increased permeability. The cytoskeletal structure of endothelial cells includes actin filament bundles that form a supportive ring around the periphery, along with the adhesion complexes that provide the integrity of the endothelial layer. Alteration of the normal actin cytoskeleton of endothelial cells in vitro has been demonstrated with ATP depletion as a model of ischemic injury and with H 2 O 2 as a model of oxidant-mediated reperfusion injury. ATP depletion has been demonstrated to rapidly and reversibly disrupt the normal cortical and basal F-actin structures in endothelial cells resulting in F-actin aggregation and polymerization. Oxidant-mediated endothelial cell injury also has been demonstrated to disrupt the cortical actin band in cultured endothelial cells. The assembly and disassembly of actin filaments is regulated by a large family of actin binding proteins including actin depolymerizing factor (ADF)/cofilin. With ischemic injury, the normal architecture of the actin cytoskeleton is markedly changed along with endothelial cell swelling, impaired cell-cell and cell-substrate adhesion and loss of tight junction barrier functions. ATP depletion of cultured endothelial cells has been shown to induce dephosphorylation/activation of ADF/cofilin in a direct and concentration-dependant fashion. This results in depolymerized and severed actin filaments, seen as filamentous (F) actin aggregates at the basolateral aspects of the cell ( Fig. 76.6 ).




Figure 76.6


Endothelial injry in AKI. Key events in endothelial cell activation and injury. Ischemia causes upregulation and expression of genes coding for various cell surface proteins such as E-(endothelial) and P-(platelet) selectin, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and reduced thrombomodulin (TM). Activated leukocytes adhere to endothelial cells through these adhesion molecules. Endothelial injury increases the production of endothelin-1 and decreases endothelial derived NOS (eNOS) which serve to induce vasoconstriction and platelet aggregation. The combination of leukocyte adhesion and activation, platelet aggregation and endothelial injury serves as the platform for vascular congestion of the medullary microvasculature. There are permeability defects between endothelial cells as a result of tight and adherens junctional altrerations.

(Source: unpublished figure from Encyclopedia of Intensive Care Medicine. Publisher Springer. Eds, Vincent, Jean Louise; Hall, Jesse B. Ch Endothelial Cell Injury by Asif Sharfuddin and Bruce Molitoris. First Edition March 2012.)


Endothelial Permeability Defects


The endothelial barrier serves to separate the inner space of the blood vessel from the surrounding tissue and to control the exchange of cells and fluids between the two. It is defined by a combination of transcellular and paracellular pathways, the latter being a major contributor to the inflammation-induced barrier dysfunction.


Sutton et al. have studied the role of endothelial cells in acute kidney injury by a series of experiments utilizing florescent dextrans and two-photon intra-vital imaging. The increased microvascular permeability observed in acute kidney injury is likely a combination of numerous factors such as: loss of endothelial monolayer, breakdown of perivascular matrix, alterations of endothelial cell contacts and upregulated leukocyte-endothelial interactions. They have shown that 24h after ischemic injury there was loss of localization in 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 two 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 known to be substrates of matrix metalloproteinase (MMP)-2 and MMP-9, which are collectively known as gelatinases. Breakdown of barrier function may also be due to matrix metalloproteinase -2 or -9 activation and this up-regulation is temporally correlated with an increase in microvascular permeability. 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 findings indicate 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 that can ultimately impact microvascular density ( Fig. 76.8 ).




Endothelium and Coagulation Abnormalities In AKI


Endothelial cells have a central role in coagulation through their interaction with protein C mediated by the endothelial cell protein C receptor (EPCR) and thrombomodulin. Protein C is activated by thrombin-mediated cleavage and the rate of this reaction is augmented 1000-fold when thrombin binds to the endothelial cell-surface receptor 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. Activated protein C acquires antithrombotic and profibrinolytic properties, and participates in numerous anti-inflammatory and cytoprotective pathways to restore normal homeostasis. Activated protein C is also an agonist of protease activated receptor-1. Animal studies have shown pre-treatment with aPC to be beneficial in ameliorating AKI from ischemic or septic injury in rats by inhibiting leukocyte activation through TNF-α, and not by inhibiting coagulation abnormalities. It has also shown that both pre -treatment and post-injury treatment with soluble thrombomodulin attenuates renal injury with minimization of vascular permeabilility defects with improvement in capillary renal blood flow.


Injury to endothelial cells could have a role in chronic disease, Basile et al . documented a significant decrease in the density of blood vessels following acute ischemic injury, which led to the phenomenon of “vascular dropout.” This phenomenon was verified by Horbelt et al. who found that vascular density was reduced by almost 45% at four weeks after an ischemic insult. This observation indicates that, unlike renal epithelial tubular cells, the renal vascular system lacks comparable regenerative potential. Ischemia has been shown to inhibit VEGF, while inducing the VEGF inhibitor ADAM-TS 1. The lack of vascular repair was postulated to be due to the reduction in VEGF expression, as administration of VEGF to postischemic rats preserved microvascular density. Vascular dropout might mediate increases in the expression of hypoxia inducible factor (HIF), increase fibrosis, and alters proper hemodynamics, leading to hypertension. Basile et al. have also shown that the poor regenerative potential of endothelial cells and transformation into fibroblasts is in large part owing to the lack of VEGF expression. This may have a critical role in accelerating progression of CKD following initial recovery from ischemia or reperfusion-induced AKI. Vascular dropout could predispose individuals to recurrent ischemic events and AKI.




Inflammation in Acute Kidney Injury


Inflammatory Response, Adhesion Molecules and the Role of Leukocytes


Inflammation and recruitment of leukocytes during epithelial injury are now recognized as major mediators of all phases of endothelial and tubular cell injury. Human AKI/ATN biopsies seldom have accumulated neutrophils, as compared to the abundance in experimental animal ischemic studies. Neutrophils likely play a modest role as an effector cell in the initiation and extension phases, while T-cells and B-cells and macrophages probably have major modulatory roles in the extension and repair phases. A complex series of events involving several classes of adhesion molecules including selectins, mucins, integrins, and the Ig superfamily occurs.


Leukocyte recruitment into most organs occurs in a cascade-like fashion. Adherence to the vascular endothelium is a dynamic multifaceted process involving both the leukocyte as well as the endothelial cell. For the leukocyte to be activated to release cytokines it has to receive 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. Upon activation, leukocyte integrins change their confirmation and bind to endothelial ligands to promote firm adhesion. For neutrophil recruitment β2-intgerin (CD18) seems to be most important. These interactions with the endothelium are mediated through endothelial adhesion molecules that are upregulated during ischemic conditions.


Initially there is slow neutrophil migration mediated by tethering interactions between selectins and their endothelial cell ligands. Singbartl et al. found that platelet P-selectin and not endothelial P-selectin was the main determinant in neutrophil mediated ischemic renal 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 gene-deficient for E-selectin or P-selectin or both were completely protected. Selectin-deficient mice revealed unchanged intraperitoneal leukocyte recruitment but altered cytokine levels when compared to wild-type mice. Therefore, it is possible that selectins exert their effects through modulation of systemic cytokine profiles rather than through engagement in leukocyte-endothelial cell interactions.


After the initial rolling, firm adhesion is imparted by interaction between endothelial cell integrins and intercellular adhesion molecule-1 (ICAM-1). Blockade of integrin CD11/CD18, ICAM-1, or deficiency of ICAM-1, were all found to protect from ischemic renal injury. Treatment of humans with anti-ICAM-1 antibodies however did not reduce the rate of delayed graft function or acute rejection following renal transplantation. Alpha-melanocyte-stimulating hormone (α-MSH), a known inhibitor of interleukin-8 (IL-8) and ICAM-1 induction, initially thought to be protective through these mechanisms, was found to be protective independent of inhibition of neutrophil recruitment.


Biopsies of human AKI have demonstrated lymphocytes in ATN, and only recently has the modulatory role of T-cells in the mediation of ischemic injury been established. Rabb et al. in a series of recent experiments showed that T-cell deficient nu/nu mice are protected against ischemic injury, along with attenuation of ICAM-1 expression. An early transient increase in T-cells might explain how T-cells could still have a role without being present in histological analysis. Additional evidence that suggests a role of T-cells is derived from studies where blockade of the CD28-B7 co-stimulatory pathway reduced injury, as well as data from STAT-4 deficient mice, which have impaired Th1 phenotype of T-cells, revealing the deleterious role of Th1 phenotypes. There is also now evidence that B-cell deficient mice are protected from ischemic renal injury, while transfer of serum from wild type to B-cell deficient mice restored injury. Macrophage chemoattractants are up-regulated during ischemia, resulting inmigration of macrophages into the outer medulla of the rat kidney. Data from osteopontin knockout mice revealed there was less macrophage infiltration and less fibrosis, even though the course of renal dysfunction was similar to wild type mice. Okusa et al. have also shown recently that although macrophages are required for the full extent of the ischemic renal injury, activation of their adenosine 2A receptors reduces neutrophil accumulation and provides protection against injury, as seen in experiments with macrophage depleted and adenosine 2A receptors deficient mice. The protective effect of adenosine 2A receptor activation is independent of IL-6 and TGF-β mRNA induction.


Macrophages produce proinflammatory cytokines that can stimulate the activity of other leukocytes. Day et al . showed that depletion of macrophages in the kidney and spleen using liposomal clodronate prior to renal ischemia reperfusion injury prevented AKI, whereas adoptive transfer of macrophages reconstituted AKI. This group also showed that agonists of sphingosine-1-phosphate induced lymphopenia, which had a protective effect. However, studies have also shown a lymphocyte independent role of the sphingosine-1 phosphate receptor (S1PR) in maintaining structural integrity after AKI as S1PRs in the proximal tubule are necessary for stress-induced cell survival, and agonists of this receptor are renoprotective via direct effects on tubular cells. Dendritic cells are also thought to have a role in AKI; Dong et al. demonstrated that after AKI, renal dendritic cells produce the proinflammatory cytokines TNF, IL-6, C-C motif chemokine 2, and C-C motif chemokine 5, and that depletion of dendritic cells prior to ischemia substantially reduced the levels of TNF produced in the kidney.


Cytokines in AKI


There is increased renal expression of many proinflammatory cytokines in response to acute ischemic or toxic injury. These include TNF-α, interferon-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukins 1,2,18, as well as chemokines such as monocytes chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), and RANTES. It is also possible that AKI is associated with up-regulation of IL-10, an anti-inflammatory cytokine supported by studies showing protection against AKI in ischemic and cisplatin models of injury by IL-10 administration.


Recent human data suggest the role of both pro-inflammatory as well as anti-inflammatory cytokines in predicting mortality, with higher IL-6, IL-8 and IL-10 plasma levels in non-survivors amongst critically ill patients with AKI. Interestingly, IL-1 and TNF-α were not predictive. Critically ill patients with AKI also have decreased and impaired monocyte cytokine production and elevated plasma cytokine levels in a pattern that closely resembles critically ill patients without AKI, suggesting the very complex role of these cytokines. It is also important to remember that the maximal capacity to produce cytokines in response to stimulation can have considerable inter-individual variation due to genetic pre-determination for their expression. Single nucleotide polymorphisms (SNPs) within the promoter region of these cytokines genes with stable allelic variants have been identified, and in a recent prospective evaluation of these polymorphisms, Jaber et al. found TNF-α and IL-10 gene polymorphismsrelated to the risk of death among patients with ARF who require dialysis. As the list of these cytokine gene polymorphisms grows it is possible that we may be better able to identify patients at higher risk of organ injury.

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Jun 6, 2019 | Posted by in NEPHROLOGY | Comments Off on Pathophysiology of Acute Kidney Injury
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