Chronic progressive nephropathies, independent of the type of the initial insult, have in common persistently high levels of urinary protein excretion, chronic tubulointerstitial lesions, and glomerular sclerosis. It has become clear that in otherwise comparable groups of patients, those with higher levels of proteinuria are at a greater risk of progression. It has been long known that the severity of tubular interstitial damage is highly correlated to the degree of deterioration of renal failure, even better than the glomerular lesions do . The recognition that proteinuria is an independent predictor and a possible contributor of progression rather than just a marker of the severity of glomerular damage represents a major change in our concepts of progressive kidney disease. Proteinuria is enlisted among modifiable risk factors of progressive renal insufficiency, and antiproteinuric treatments are consistently renoprotective. Proteinuria reduction must therefore be considered as a target of therapeutic intervention in managing the patient with a chronic proteinuric nephropathy.
Among the cellular mechanisms that may contribute to determine progression, proteins that gain access to the glomerular filtrate as a consequence of altered glomerular permeability reach the Bowman’s space and the tubular lumen and act as trigger of interstitial inflammatory and fibrotic reactions through the tubular synthesis of bioactive mediators. In vitro studies using polarized proximal tubular epithelial cells as a model system to mimic the effects of prolonged exposure to proteins have highlighted specific mechanisms underlying renal cell activation and renal interstitial injury.
The main focus of this chapter is on pathways of interstitial inflammation and fibrosis activated by ultrafiltered proteins that play major roles in the multifactorial process ultimately responsible for kidney scarring and loss of function ( Figure 87.1 ).
A relentless decline in glomerular filtration rate can follow initial insults to the kidney through a process termed as ‘renal disease progression’ by nephrologists since 1982. Studies in the early 19 th and 20 th centuries contributed to link a disparate body of knowledge about patients with albuminuria and pathological evidence of diseased kidneys. By summarizing his concept in term of renal ‘osmotic work’, Addis calculated how this work would vary with the amount of proteins in the diet and suggested that dietary protein restriction could possibly help patients with renal impairment.
When glomerular permeability is altered, as occurs in many glomerular diseases, considerable quantities of circulating proteins are abnormally filtered in the urinary space. Volhard and Fahr and subsequently Mollendorf and Stohr provided the early descriptions of hyalin droplets in the cytoplasm of proximal tubular cells and suggested that they reflected renal damage related to severe proteinuria. It was then proposed that such changes could represent the initial phase of activation of cellular pathways leading to cell necrosis. The appearance of droplets was also suggested to be associated with impairment of normal reabsorbption and degradation of plasma proteins by proximal tubular cells.
Hints about a possible association between proteinuria and renal function impairment formally emerged in the late 1970s. Cameron and colleagues showed that among patients with focal and segmental glomerulosclerosis those with nephrotic syndrome progressed more rapidly than those who had never been nephrotics. This was also true for mesangiocapillary glomerulonephritis and membranous nephropathy. Their speculation was that during the course of prolonged nephrotic syndrome intracapillary platelet thrombi induced segmental sclerosis, or that these glomerular lesions were a consequence of proteinuria. These observations were in agreement with previous findings that none of patients with focal and segmental glomerulosclerosis who responded to corticosteroid treatment with reduction of proteinuria developed renal failure.
In 1981 Brenner and associates introduced the concept that glomerular hemodynamic changes (glomerular hyperfiltration) ensuing as compensatory adaptation to nephron loss can cause progressive deterioration of remaining nephrons. Instrumental in clarifying the pathophysiology of renal adaptation to nephron loss was the model of renal ablation in the rat. After removal of a critical portion of renal mass, remnant nephrons undergo sudden hypertrophy with concomitant lowering of arteriolar resistance and increased glomerular plasma flow. Since the vascular tone drops more in afferent arterioles than in the efferent ones, glomerular capillary hydraulic pressure rises and more filtrate is formed per nephron. While such changes serve to enhance the filtration capacity of remaining nephron units, thus minimizing the functional consequences of nephron loss, they are ultimately detrimental. Therapies that attenuate such adaptive changes limit GFR decline and structural damage. In harmony with the data obtained in animal studies, clinical studies showed markedly reduced rates of GFR decline in patients with diabetes receiving antihypertensive agents. Elevated intraglomerular capillary pressure was suggested to act as a noxious stimulus to glomerular cells. Studies of mathematical models of the glomerular filtering membrane documented that the high glomerular capillary pressure acted to enlarge the radii of the pores perforating the glomerular membrane by a mechanism at least partly mediated by angiotensin II. This impairs the size-selective function of the barrier resulting in protein ultrafiltration. The association of protein ultrafiltration and progression of renal disease was underscored by Mathiesen and coworkers who reported that in diabetic patients the higher the microalbuminuria the faster the rate of GFR decline.
In 1986, it was suggested for the first time that proteins abnormally filtered through the glomerular capillary might have intrinsic renal toxicity that is relevant to the progression of renal damage. In the kidneys of proteinuric rats with adriamycin nephrosis, the accumulation of filtered proteins in the cytoplasm of proximal tubular cells and in luminal casts was associated with focal breaking of the tubular basement membrane and extravasation of the tubular content into the renal interstitium. Aging is associated with progressive proteinuria and glomerulosclerosis, which lead to the renal insufficiency of certain strains of rats. The severity of proteinuria predicted the development of glomerulosclerosis and tubulointerstitial changes. Implications of these studies were that the enhanced protein uptake may activate proximal tubular epithelial cells to promote the accumulation of inflammatory cells into the interstitium, in turn responsible for renal scarring. Studies using animal models and pharmacologic manipulations found that the dysfunction of the glomerular capillary barrier to proteins can precede the onset of structural lesions in the absence of glomerular hemodynamic changes, consistent with the possibility that—at least in certain settings—protein load of the nephron may contribute independently to progressive renal damage before maladaptive hemodynamic changes can take place or be measurable within the glomerulus.
Cause-and-effect relationships between enhanced transtubular protein passage and interstitial inflammation were investigated by Eddy in a rat model of protein overload. Repeated injections of albumin increased the permeability of the glomerular barrier leading to proteinuria and tubular changes, including the accumulation of injected albumin in intracellular droplets of tubular cells. These changes occurred in advance of heavy infiltration of macrophages and T lymphocytes into the renal interstitium. Excessive proteinuria was also induced in rats by transplanting a pituitary tumor (MtT SA5) that causes liver hyperplasia with both overproduction and abnormally high urinary excretion of albumin, followed by tubular damage and interstitial inflammation. Models of protein overload contributed to suggest that the pathogenic potential of ultrafiltered plasma proteins is not confined to pathways underlying tubular and interstitial damage. The availability of cultured cells with features of differentiated podocytes revived investigation on the effects of plasma proteins on the function of glomerular epithelial cells, currently recognized to play a key role in the progression of lesions toward glomerular sclerosis. Plasma proteins that are filtered accumulate as protein droplets within podocytes from the early stages of non-immunologically-induced glomerular damage . At the same stage, podocytes show signs of dedifferentiation and injury . These abnormalities are likely to play role in the development of glomerular sclerotic lesions. They will be discussed in the light of findings indicating another important pathway, i.e., the activation of complement upon excess protein ultrafiltration.
Tubular Handling of Proteins
Proteins that reach the tubular lumen are reabsorbed in the proximal segments by receptor-mediated endocytosis, via two multiligand-binding receptors, megalin and cubilin. Megalin, a 600-kDa transmembrane glycoprotein belonging to the low density-lipoprotein receptor family, represents the most abundant endocytic receptor in the proximal tubule, being concentrated in clathrin-coated pits (CCPs) and vesicles in the brush border region. It binds different ligands including albumin, hormones such as insulin, angiotensin II and prolactin, and vitamin-binding proteins. Megalin’s endocytic function is regulated by the G protein–mediated signaling pathway that includes Gαi3, GAIP and GIPC, the latter interacting with the cytoplasmic tail of megalin ( Figure 87.2 ). This portion contains recognition motifs for intracellular adaptor proteins and protein kinases involved in endocytosis, apical sorting of receptor, and signaling.
Among adaptor proteins, Disabled protein 2 (Dab2) colocalizes with megalin in CCPs and vesicles of renal proximal tubules where it binds the second endocytic motif of megalin, and, by interacting with the motor protein nonmuscle myosin heavy chain IIA, regulates trafficking of megalin through the endocytic/recycling pathway ( Figure 87.2 ). Suboptimal trafficking of megalin in Dab2 knockout mice leads to decreased megalin levels, possibly due to increased protein shedding and degradation, and impaired endocytosis as documented by urinary loss of megalin ligand. Association of motor proteins with adaptor proteins is now emerging to be instrumental for renal proximal tubular endocytosis. Myosin VI (Myo6) is highly expressed in the brush border where it associates with the CCP via its tail domain binding to Dab2 ( Figure 87.2 ). Myo6 functional null mice show increased urinary albumin excretion, similar to megalin- and cubilin-deficient animals, and reduced and delayed endocytic uptake and trafficking of horseradish peroxidase consistent with impaired endocytosis. This defect is associated with structural and phenotypic changes of tubular cells and fibrosis.
The expression and subcellular distribution of megalin is controlled by the chaperone receptor-associated protein RAP that prevents ligand–induced endoplasmic reticulum (ER) retention and degradation of the receptor and is possibly involved in its proper folding ( Figure 87.2 ). RAP knockout mice are characterized by both reduced expression of megalin and its preferential subcellular distribution to the ER. RAP deficiency is also associated with leakage of proteins in urine s due to impaired megalin – dependent tubular reabsorption. Pro-inflammatory and pro-fibrotic mediators such as transforming growth factor beta (TGF-β) and angiotensin II as well as nephrotoxicants also down-regulate megalin expression in proximal tubular cells.
Cubilin or intrinsic-factor B12 receptor, is a 460-kDa peripheral membrane protein that binds albumin, transferrin, IgG light chains and RAP, but lacks the sites for interaction with adaptor proteins or other mediators of clathrin-dependent endocytosis. Since megalin binds cubilin with high affinity, it has been hypothesized that megalin mediates endocytosis and intracellular trafficking of cubilin. In vitro , the uptake of the cubilin-specific ligands, including transferrin, was inhibited both by anti-megalin antibodies and by megalin anti-sense oligonucleotides. Furthermore, in megalin-deficient mice transferrin accumulates on the luminal membrane of the proximal tubule without being internalized. Cubilin binds amnionless (AMN), a 50-kD transmembrane protein that is required for membrane targeting and may permit internalization of cubilin. Patients with Imerslund-Grasbesk syndrome (IGS), a rare autosomal recessive disease caused by inheritable cubilin or AMN gene defect, show reduced protein reabsorption and proteinuria. Consistently, inappropriate apical membrane insertion of cubilin in the proximal tubule leads to proteinuria in a model of IGS in dog s with a mutation of the AMN gene. Kidney specific AMN knockout mice also show increased urinary excretion of transferrin.
The generation of mice with the genetic ablation of cubilin, with or without ablation of megalin, disclosed a mutual dependency of cubilin and AMN to form a functional membrane receptor complex. Cubilin is indispensable for albumin reabsorption in renal proximal tubule under physiological conditions, whereas megalin drives the internalization of cubilin-albumin complexes.
Megalin undergoes regulated intramembrane proteolysis with protease-mediated ectodomain shedding, producing a membrane–associated C-terminal fragment, which is in turn the substrate for the γ-secretase activity of a multimolecular complex of proteins, including the so-called presenilins, that release the C-terminal soluble cytosolic domain predicted to target the nucleus. In the rat kidney, the brush border exhibits both γ-secretase activity and presenilin-1 expression. In a cell line derived from opossum proximal tubule (OK cells), metalloprotease activity sheds the ectodomain of megalin, producing a membrane-bound megalin COOH-terminal fragment with the same size as a major fragment of megalin found in the kidney. This fragment is further cleaved by γ-secretase into a soluble intracellular domain leading to down-regulation of megalin and Na + /H + exchanger 3 (NHE-3) transcripts. This domain does not affect megalin expression and endocytic function in vivo at least under physiological conditions, although a regulatory effect on renal gene transcription under stress cannot be excluded.
Reduced expression of megalin is a common feature to settings of proteinuria and defective tubular uptake of filtered proteins, such as early stages of experimental diabet es or polycystic kidney disease, Fanconi syndrome and Dent’s disease in humans. Dent’s disease is caused by a mutation of the gene CLCN5 encoding the 2 chloride (Cl − )/proton (H + ) exchanger ClC-5. Inactivating mutations of ClC-5 in Dent’s disease and the deletion of CLCN5 in knockout mice lead to low – molecular – weight proteinuria due to reduced endocytic uptake of filtered proteins in the proximal tubule. This defect was initially attributed to impaired acidification of apical endosomes in these cells, but subsequent data indicate selective loss of expression of megalin and cubilin at the brush border as a major cause of defective protein endocytosis in this disease. Along this line, reduced megalin and cubilin levels have been found in tubules with the uncoupling E211A (unc) mutation that converts CLC-5 into a pure Cl − conductor. Despite maintaining active endosomal acidification, ClCN5 unc mice show impaired proximal tubular endocytosis and resultant low-molecular-weight proteinuria as found in CLC-5 knockout mice and in patients with Dent’s disease. These results indicate that endosomal chloride-proton exchange rather than chloride conductance is crucial for tubular endocytosis ( Figure 87.3 ).
During receptor-mediated endocytosis, ligand-receptor complexes are internalized and transported via clathrin-coated vesicles to early endosomes where they dissociate. The receptor is sorted to the plasma membrane (recycling pathway) and the endocytosed proteins are transported further into the degradative pathway. The endocytic pathway is regulated by a protein network. The inositol 5’-phosphatase OCRL -whose mutations are responsible for OculoCerebroRenal Syndrome of Lowe , an X-linked disorder characterized by cataract, mental retardation, and renal Fanconi syndrome- is present throughout the early endocytic pathway including CCPs and interacts with adaptor molecules involved in the early endocytic traffic of receptors in brain and kidney. On peripheral early endosomes OCRL binds the Rab5 effector APPL1, an adaptor/signaling protein that binds several transmembrane receptors and also the adaptor protein GIPC ( Figure 87.3 ). Since GIPC directly binds megalin (see above), loss of the interaction of OCRL with APPL1 due to disease mutation may have a causal role in the reabsorptive defect of kidney resulting in low-molecular-weight proteinuria in Lowe syndrome patients and in a subset of Dent’s disease patients.
Another protein, the transporter NHE – 3 contributes to the early phase of the apical endocytic pathway at least in part due to its involvement in endocytic vesicle fusion. NHE – 3 is associated with megalin and dipeptidyl IV in proximal tubule endosomes where it also participates in the acidification process ( Figure 87.3 ). Acidification of endosomal compartment is crucial not only for ligand-receptor dissociation and receptor recycling, but also for a correct protein endocytic trafficking from early to late endosomes, and finally lysosomes. The acidification of proximal tubule endosomes is driven by the vacuolar H + -ATPase (V-ATPase), an electrogenic pump that translocates protons from the cytoplasm to the endosomal lumen and in conjunction with a parallel chloride conductance generates the acidic milieu of the endosomal compartment ( Figure 87.3 ). V-ATPase acts as an endosomal pH sensor that recruits the small GTPase Arf6 (ADP-ribosylation factor) and its cognate GDP/GTP exchange factor ARNO (ADP-ribosylation factor nucleotide site opener) from the cytosol to the endosomal membranes by direct binding these proteins upon vesicle acidification ( Figure 87.3 ). Interaction of both Arf6 and ARNO with V-ATPase allows correct vesicular trafficking from early to late endosomes. By contrast, inhibition of V-ATPase or uncoupling of endosomal acidification impairs V-ATPase/ARNO/Arf6 interaction leading to accumulation of endocytosed proteins in early endosomes, and ultimately, to inhibition of endocytosis. An intact actin cytoskeleton is important for correct assembly and function of the V-ATPase. The disorganization of the actin cytoskeleton induced by statins through reduced prenylation of the small GTP-binding proteins result s in a defect of albumin endocytosis in proximal tubular cells.
Protein Overload Activates Phenotypic Changes in Cultured Proximal Tubular Cells
Insights into mechanisms linking the excess traffic of plasma proteins and interstitial injury have come from in vitro studies using polarized proximal tubular cells as a model to assess effects of apical exposure to proteins ( Figure 87.4 ). Challenge of cultured proximal tubular cells with high concentrations of proteins (delipidated or lipid-enriched albumin, IgG and transferrin) induced the synthesis of the vasoconstrictor peptide endothelin-1, a mediator of progressive renal injury due to its ability to stimulate renal cell proliferation and extracellular matrix production and to attract monocytes. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES or CCL5), potent chemoattractant s for monocytes/macrophages and T-lymphocytes, were upregulated in proximal tubular cells challenged with albumin and other plasma proteins. Albumin overload also induced tubular gene expression and production of the C-X-C chemokine interleukin-8 (IL-8), a potent chemoattractant for neutrophils and lymphocytes. Endothelin-1 and chemokines induced by plasma proteins were released mainly toward the basolateral compartment of the cell, which is indicative of a directional secretion potentially responsible for the tubulointerstitial inflammatory reaction as found in proteinuric nephropathies. Co-culture systems of proximal tubular epithelial cells and monocytes/T cells proved useful to document that the release of MCP-1 and RANTES upon apical exposure of tubular cells to albumin was further increased in the presence of monocytes or T cells, either through a cell-to-cell contact mechanism or mediated by soluble factors such as interleukin-1 and tumor necrosis factor. Moreover, the conditioned medium of tubular cells activated by albumin induced distinct patterns of chemokine receptor expression on T cells or monocytes, thereby implying that different arrays of chemokines may mediate the chemotactic activity of leukocytes in the tubulointerstitium during the proteinuric state.
Protein overload promotes the tubular expression of fractalkine/CX3CL1, a cell-membrane anchored chemokine that serves as an adhesive molecule to promote firm adhesion of mononuclear cells expressing the specific receptor CX3CR1, in addition to functioning in its cleaved soluble form as a chemoattractant. Upregulation of fractalkine mRNA and increased synthesis of both membrane-bound and soluble forms of the protein occurred upon stimulation of human proximal tubular cells with albumin. In a murine model of protein overload proteinuria, fractalkine mRNA was overexpressed in the kidney, and fractalkine staining was detected in tubular epithelial cells in a focal distribution. Treatment of mice with an antibody against CX3CR1 limited the accumulation of monocytes/macrophages in the renal interstitium. These data suggest that in proteinuric conditions, fractalkine overproduction might contribute to direct mononuclear cells into the peritubular interstitium and possibly enhance their adhesive property.
TGFβ is a profibrogenic cytokine capable of directly stimulating the proliferation of fibroblasts and the synthesis of matrix proteins, in addition to exerting indirect stimulatory effects via inflammatory infiltrating cells. TGFβ acts as a key stimulus for epithelial-to-mesenchymal transition (EMT), by which tubular cells acquire features of fibroblasts. High concentrations of albumin induced in cultured proximal tubular cells the transcription of the TGFβ gene resulting in the enhanced release of the cytokine in the cell supernatant . Albumin upregulated TGFβ receptor type II expression in proximal tubular cells, which became more susceptible to the matrix-stimulatory actions of TGFβ. Albumin stimulated the accumulation of extracellular collagen type IV, laminin, and fibronectin by proximal tubular cells through a post-transcriptional mechanism. A reduced degradation could be responsible for the increased accumulation of extracellular matrix protein components, as indicated by induction of tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2, in response to albumin.
Advances have been made in clarifying the mechanisms of albumin-induced TGFβ production in proximal tubular cells. In vitro , albumin-induced secretion of TGFβ1 by proximal tubular cells could occur in the absence of albumin endocytosis. Thus two cell lines, opossum kidney (OK) proximal tubular cells and HKC-8 proximal tubular cells, which display different degrees of endocytosis, produced an equivalent amount of TGFβ1 when exposed to albumin. Moreover, inhibiting albumin endocytosis in OK cells with two agents with different mechanisms of action as EIPA (a sodium/hydrogen exchanger-3 inhibitor) or simvastatin (a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor) did not reduce albumin-induced TGF-β1 secretion. On the other hand, RAP, a known inhibitor of binding of albumin to megalin, inhibited albumin endocytosis but it did not affect TGFβ1 production, suggesting that albumin-induced secretion of TGFβ1 is not via megalin signaling . Along this line, further studies showed that primary cultures of mouse proximal tubular cells exhibited substantial albumin endocytosis that could be similarly inhibited by both statins and thiazolinediones, but only statins had the ability to limit albumin-stimulated MCP-1 production. It was concluded that inhibition of albumin endocytosis alone was insufficient to attenuate chemokine production and therefore to protect proximal tubular cells from the toxic effects of albumin.
The contact between tubular cells and interstitial matrix is operated by specialized adhesion molecules, the integrins. They are α/β transmembrane heterodimers that mediate adhesion of the basal surface of cells to the underlying substratum by recognizing extracellular matrix components and by interacting with different cytoskeletal molecules. In vitro these molecules are clustered in specialized structures, called focal contacts. In tubular cells the expression of integrins can be regulated by cytokines, including TGFβ, that either directly influence mRNA transcription of integrins or modulate the synthesis of interstitial matrix and the consequent rearrangement of integrins at focal contacts. On the other hand, matrix proteins can modulate integrin expression and via integrin-generated signals, regulate the synthesis, degradation and organization of the matrix itself. Therefore, alterations in the expression and/or functional status of tubular cell integrins may play roles in the events leading to tubulointerstitial fibrosis. In cultured tubular cells the addition of albumin increased membrane expression of αvβ5 integrin which became organized in typical focal contacts dose-fashion related.
The Role of Proteinuria in Tubular Apoptosis
Protein overload is a stimulus for apoptosis. A dose- and time-dependent induction of apoptosis by albumin was demonstrated in cultured proximal tubular cells as revealed by internucleosomal DNA fragmentation, morphological changes including cell shrinkage and nuclear condensation, and plasma membrane alterations. Apoptosis in this setting was associated with activation of Fas-FADD-caspase 8 pathway, suggesting activation via the extrinsic pathway of apoptosis. Peroxisome proliferator activated receptor (PPAR)-γ is also implicated in molecular mechanisms underlying albumin-induced apoptosis. A lbumin-bound fatty acids stimulated PPAR-γ in primary cultures of human proximal tubular cells and caused apoptosis that could be blocked by PPAR response element decoy oligonucleotides. Moreover, transfection experiments revealed that the transient overexpression of PPAR-γ in tubular cells resulted in enhanced apoptosis. In HKC-8 human proximal tubular cells, albumin-induced apoptosis was mainly mediated by the intrinsic pathway of apoptosis, characterized by Bax translocation to mitochondria and cytochrome c release from the organelles. This pathway was recently confirmed in rat proximal tubular cells by inhibition with Bcl-2 transfection and was found to be mediated by protein kinase C-delta (PKC-δ), a novel subfamily member of PKC serine/threonine protein kinases.
Albumin-dependent signaling and albumin endocytosis appear to act as interrelated processes regulating the fate of proximal tubular cells in vitro . Megalin may behave as a sensor molecule that determines whether the cells will be protected from or injured by albumin, pending on the protein concentration. On one hand, low concentrations of albumin lead to activation of serine/threonine kinase PKB and phosphorylation of Bad protein, which inhibits apoptosis. On the other hand, albumin overload decreased the expression of megalin on the plasma membrane that was associated with a reduction of PKB activity and Bad phosphorylation, favoring apoptosis. As well as albumin load of tubular cells, a balance has also been suggested between the induction of a NF-kB dependent, Bcl-xL mediated antiapoptotic pathway and the induction of AP-1 mediated clusterin overexpression that instead, by inhibiting the pathway, would favor a switch from inflammatory phenotype to apoptotic injury.
Multiple pathways of apoptosis can be activated in renal tubular cells during proteinuric kidney diseases. Apoptotic responses to protein load were documented in the rat model of albumin overload proteinuria, showing increased numbers of terminal dUTP nick-end labeling positive apoptotic cells both in the tubulointerstitial compartment and in glomeruli. In tubuli, most of the positive cells were found in profiles expressing angiotensin II type 2 (AT2) receptor. Findings of reduced phosphorylation of ERK and Bcl-2 were suggested to reflect an AT2 receptor-mediated mechanism underlying tubular cell apoptosis. Proximal tubular cell apoptosis may contribute to glomerular-tubule disconnection and atrophy in response to proteinuria in rats with accelerated passive Heymann nephritis. Another study, in agreement with results obtained using cultured proximal tubular cells, showed PKC-δ overexpression and tubular apoptosis in kidneys of mice upon albumin overload, whereas PKC-δ knockout mice were protected . Levels of proteinuria remained comparable, indicating lower susceptibility to apoptosis rather than differences in protein exposure. Mechanistically, PKC-δ could promote apoptosis by activation of apoptotic genes, phosphorylation of caspases, interaction with apoptotic regulators, remodeling of cell membranes, or interference with mitochondrial function possibly including ER-mitochondrial crosstalk during ER-stress-induced apoptosis. ER stress has been demonstrated both in proximal tubular cells in vitro and in kidneys of rats with albumin-overload or puromycin aminonucleoside-nephrosis.
Apoptotic cells were also detected both in proximal and distal tubular profiles in biopsy specimens of patients with primary focal segmental glomerulosclerosis. A positive correlation was found between proteinuria and incidence of tubular cell apoptosis, which was identified as a strong predictor of outcome in these patients. Albumin exposure caused the activation of the Fas pathway and apoptosis in cultured Madin-Darby canine kidney epithelial (distal/collecting) cells, hence extending to the distal nephron the pro-apoptotic potential of enhanced protein load. Besides promoting tubulo-glomerular disconnection at proximal level, tubular apoptosis could create and sustain a local proinflammatory microenvironment via release of molecules that serve as danger signals by dying cells. Danger-associated molecular patterns (DAMPs) trigger inflammation by engaging Toll-like receptors (TLR) and nucleotides-binding domains, leucin-rich, repeat-containing proteins (NLRs). Engaged NLR form complexes with apoptosis-associated proteins to produce macromolecular complexes termed inflammasomes that cleave proinflammatory cytokines to their mature forms. A role for inflammasomes in progressive renal disease has been recently shown in NLRP3 -/- mice that were protected from injury and fibrosis of unilateral ureteral obstruction. Increased expression of NLRP3 was also detected in kidney biopsy specimens of patients with progressive kidney diseases.
Intracellular Signaling of Protein Overload
In vitro studies have documented that protein overload activates signal transduction cascades in proximal tubular cells resulting in transcriptional upregulation of proinflammatory and fibrogenic molecules.
Molecular mechanisms leading to chemokine gene induction as a consequence of enhanced protein uptake have been identified. Nuclear transcription factors such as NF-kB have attracted attention as candidate pathways and are seen as a potential target of therapeutic intervention against proteinuria-induced tubulointerstitial injury. The NF-kB/Rel family includes homodimeric or heterodimeric complexes designated as p50, p52, p65, c-rRel and RelB. The prototype NF-kB is composed of p50-p65 subunits. NF-kB proteins normally exist in the cytoplasm bound to the inhibitory protein IkBα. Upon cell activation by different stimuli, such as cytokines, viruses and oxidants, IkBα is phosphorylated by the IkB kinase (IKK) complex, ubiquitinated and degraded, allowing NF-kB translocation into the nucleus for binding to DNA motifs in gene promoters. In cultured proximal tubular cells albumin dose-dependently enhanced NF-kB activity resulting in upregulation of RANTES, MCP-1 and IL-8. The role of NF-kB activation in chemokine mRNA induction by protein overload was supported by experiments showing that adenovirus-mediated gene transfer of IkBα or the dominant negative mutant of IkB kinase-2 (IKK-2) which fails to phosphorylate IkBα, reduced upregulation of fractalkine mRNA in proximal tubular cells exposed to albumin. Reactive oxygen species (ROS) served as second messengers in protein overload-induced NF-kB activation. Albumin and IgG caused a rapid and sustained generation of hydrogen peroxide (H 2 O 2 ) in human proximal tubular cells, and antioxidants while preventing hydrogen peroxide production, almost abolished the enhanced NF-kB activity induced by both proteins. Oxidant generation is upstream regulated by protein kinase C (PKC) which, once activated, translocates from the cytoplasm to cell membrane to mediate ROS production and NF-kB activation. Inhibitors of PKC prevented hydrogen peroxide generation, NF-kB activation, and MCP-1 and IL-8 gene upregulation induced by albumin, suggesting a cascade of signals from PKC-dependent oxygen radical generation to nuclear translocation of NF-kB and consequent gene upregulation.
NF-kB transcriptional activity also depends on mitogen-activated protein kinase (MAPK) cascade pathways that transduce external stress stimuli into intracellular responses. In human proximal tubular cells albumin caused rapid phosphorylation of p38 MAPK. Treatment with a specific p38 inhibitor resulted in inhibition of the transcription of NF-kB promoter/luciferase reporter gene construct, consistent with a role of p38 as regulator in this pathway. The p38 blockade limited the overexpression of fractalkine mRNA and protein induced by albumin. Moreover, activation of the MAPK signaling pathway was involved in albumin-stimulated TGFβ-1 gene expression and protein secretion in rat proximal tubular cells. Strong and rapid activation of p44/42 MAPK was detected following cell exposure to albumin. A specific MAPK inhibitor abolished the increase of TGFβ-1 mRNA induced by albumin.
There is evidence in mouse proximal tubular cells that albumin activated extracellular signal-regulated kinase (ERK) and that blockade of ERK activation by a specific inhibitor of the mitogen-activated protein kinase kinase (MEK), the immediate upstream regulator of ERK, partially reduced albumin-induced MCP-1 expression, inhibited the increase in AP-1 and NF-kB DNA-binding activity, and prevented the degradation of IkB in albumin treated cells. It has been shown that in a proximal tubular epithelial cell line engineered to constitutively express heme oxygenase-1 (HO-1) MCP-1 production in response to albumin was reduced and that this inhibitory effect involved mechanisms that are distal to the activation of ERK and are associated with a suppressive effect on NF-kB activation. Another study showed that high concentrations of albumin stimulated ERK via an EGF-receptor-dependent pathway. The mechanism underlying albumin-induced stimulation of EGF-R is undefined. Based on the observations that EGF-like activators of EGF-R exist as transmembrane precursors that must undergo ectodomain cleavage to release the soluble EGF-activating fragment, and that the extracellular domain of megalin has 17 EGF-type repeats, it has been suggested that abnormal EGF activation could result from ectodomain shedding of megalin occurring from proximal tubular cells in the pathological setting of proteinuria. In addition, it has also been proposed that transactivation of EGF-R by G-protein-coupled receptors could in part be regulated by PKC and c-Src, that activate ERK and are stimulated by albumin. Moreover, in primary cultured proximal tubular cells albumin stimulated DNA synthesis by Ca 2+ /PKC as well as the EGFR-dependent p44/42 MAPK and NK-kB signal pathways.
The activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is an important mechanism converting cytokine and growth factor signals into gene expression programs that regulate cell proliferation and survival. The evidence that albumin activated JAK/STAT in murine proximal tubular cells led to the suggestion that albumin may stimulate proximal tubular cells in the manner of a cytokine. The activation of STAT was also observed after treatment of the cells with apotransferrin, one of the major components of plasma proteins consisting of transferrin not saturated with iron. Findings that albumin and apotransferrin induced in tubular cells the upregulation of intracellular ROS generation and that antioxidants prevented albumin-induced phosphorylation of STAT suggested that STAT activation could occur by way of the ROS generating system.
Among genes differentially regulated by excess proteins in proximal tubular cells of mice with protein overload proteinuria, glia maturation factor-B (GMF-B), a 17-kD intracellular protein originally purified from the brain, that regulates the life/death signaling by activating p38 was identified. Proximal tubular cells engineered to overexpress GMF-B acquired susceptibility to cell death under sustained oxidative stress through p38 pathway activation. H 2 O 2 stimulation persisted in GMF-B overexpressing cells in which the H 2 O 2 -generating enzyme, CuZn-SOD, was upregulated, and the H 2 O 2 -reducing enzymes glutathione peroxidase and catalase were down-regulated.
In Vivo Evidence for Proinflammatory and Profibrogenic Signaling in Tubular Cells Activated by Proteinuria
Proinflammatory signals . Studies of experimental models of kidney disease or injury, followed by further investigation in human nephropathies, have shown that activation of transcription factors and overexpression of chemokines can indeed be elicited by the proteinuric condition contributing to the development of progressive renal damage. In rats with protein-overload proteinuria the upregulation of MCP-1 and osteopontin in tubular epithelial cells was closely associated with an interstitial inflammatory reaction. In this model, enhanced renal NF-kB activity was localized preferentially to tubular epithelial cells. In rats with 5/6 nephrectomy, the increased urinary protein excretion over time was attended by a remarkable increase in NF-kB activity in the remnant kidney. Strong nuclear staining for the p50 NF-kB subunit was visualized in proximal tubular cells and in sparse cells within the renal interstitium. A progressive increase in renal expression of MCP-1 gene occurred over time concomitant to the activation of NF-kB. Tubular MCP-1 mRNA upregulation became detectable at stages that preceded the accumulation of monocytes/macrophages and T lymphocytes in the remnant kidney interstitium, suggesting MCP-1 dependent recruitment of the cells. In other animal models of proteinuric nephropathies, renal MCP-1 overexpression preceded or coincided with the interstitial infiltration of mononuclear cells. The administration of a neutralizing anti-MCP-1 antibody to rats with tubulointerstitial nephritis significantly decreased macrophage infiltration, strenghtening the possibility that MCP-1 is functionally important in eliciting an interstitial inflammatory response. The possibility that excess protein load and reabsorption by proximal tubular cells could indeed play a role in the development of interstitial inflammation and fibrosis was supported by results of analysis of time course and sites of protein accumulation and interstitial infiltration by macrophages and MHC-II-positive cells in the rat models of 5/6 nephrectomy and passive Heymann nephritis. A link was also observed between excess plasma protein reabsorption by proximal tubuli and the expression of osteopontin, a cytokine responsible for the attraction of mononuclear cells. Osteopontin was detected in cells of proximal tubuli congested with ultrafiltered proteins, and the sites of colocalization revealed a strict relationship with adjacent infiltrates. If the interstitial inflammatory reaction ensued as a consequence of excessive ultrafiltration and proximal tubular reabsorption of proteins that also promoted NF-kB activation and chemokine synthesis, limiting the enhanced protein traffic should also limit the biological effect of excessive tubular protein reabsorption and should slow renal disease progression. The best strategy to reduce protein traffic both in experimental animals and humans relies on ACE inhibitors effectively employed to slow the pace of progression of renal disease. ACE inhibitor given to rats with a remnant kidney reduced urinary protein excretion and at the same time almost suppressed NF-kB DNA-binding activity and reduced MCP-1 mRNA expression and interstitial inflammatory cell infiltrates. Similar effects were observed in the immune model of passive Heymann nephritis. The decrease of NF-kB activation was associated with down-regulation of MCP-1 expression and reduction of interstitial inflammation.
In line with the possibility that NF-kB activation has a role in tubulointerstitial injury in proteinuric rats, there are data showing that in rats with adriamycin-induced nephropathy, chronic treatment with the putative NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC), initiated at the time of overt proteinuria, suppressed cortical NF-kB activation and markedly reduced interstitial monocyte infiltration. Chronic inhibition of NF-kB with PDTC also attenuated renal inflammation and injury in rats with 5/6 nephrectomy. The mechanism by which PDTC inhibits NF-kB is unclear. It can directly impede the degradation of IkB or it may act through its antioxidant properties to inhibit the stimulatory effect of oxidative stress on NF-kB system. Although in the above models no major side effects were observed, studies on the clinical toxicity and safety of PDTC and other NF-kB inhibitors are needed before considering their potential use against renal disease progression. Systemic inhibition of NF-kB might have unwanted effects on the induction of genes critically regulating inflammatory and immunologic responses. Several conventional therapies in clinical use, including ACE inhibitors, AT1 receptor antagonists, glucocorticoids and hydroxymethyl glutaryl-CoA reductase inhibitors, are able to modulate NF-kB activation, but data on specific NF-kB inhibition in human disease are still lacking.
To more specifically inhibit NF-kB activation, a recombinant adenovirus vector expressing the truncated form of IkBα that lacks the phosphorylation sites essential for the activation of NF-kB, was injected into renal arteries of rats with protein overload proteinuria. This maneuver prevented NF-kB activation in tubular cells and attenuated the interstitial infiltration of mononuclear cells, interstitial edema, and fibrosis. These data suggest the possibility of using gene therapy targeting NF-kB as a means of interrupting the process of tubulointerstitial injury.
Specific blockade of the MCP-1/CCR2 signaling pathways attenuated interstitial nephritis induced by protein overload proteinuria. A hydrodynamic-based gene transfer technique was used to introduce naked plasmid encoding 7ND (a MCP-1 antagonist) into the left kidney of rats subsequently given repeated injections of bovine serum albumin. Anti-MCP-1 gene therapy reduced interstitial inflammation and fibrosis and tubular damage, and limited the number of apoptotic cells in the treated kidney but not in the contralateral one. Finding that 7ND acted locally would envision a potential therapeutic application for this strategy against tubulointerstitial injury in the clinical setting.
Analysis of renal biopsy specimens from patients with severe proteinuria revealed NF-kB activation in tubular epithelial cells, which significantly correlated with the magnitude of proteinuria. There was a concomitant upregulation of proinflammatory chemokines, MCP-1, RANTES and osteopontin, found mainly in tubular epithelial cells, with the strongest expression in patients with progressive nephropathy. NF-kB activation and MCP-1 upregulation in proximal tubular cells were also shown in patients with diabetic nephropathy. A relationship between proteinuria and MCP-1 mediated interstitial damage was documented in a prospective study of patients who underwent renal biopsy for chronic kidney disease. Furthermore, transcriptome analysis by complementary DNA microarray of renal proximal tubular epithelial cells isolated by laser capture microdissection from patients with proteinuric nephropathies revealed more than 160 differentially expressed genes, including those encoding for signal transduction, cell cycle control, intracellular transport and metabolism .
Profibrogenic signal. Interstitial fibrosis represents the final common pathway of any form of progressive renal disease. There is general consensus that fibroblasts within fibrotic tubulointerstitium proliferate and that fibrosis-generating myofibroblasts – i.e, activated matrix secreting cells with features and gene patterns of smooth muscle cells – are an hallmark of the process . In proteinuric settings, protein load and reabsorption by proximal tubular cells initiate or enhance fibrogenesis by at least two mechanisms. First, proximal tubular epithelial cells have the potential to interact directly with the adjacent interstitial fibroblasts via paracrine mechanisms. Indeed, proximal tubular cells by their ability to synthesize platelet derived growth factor (PDGF) and TGFβ1 stimulated renal cortical fibroblasts in coculture to grow and synthesize collagen. On the other hand, the proinflammatory activation of tubular cells fosters local recruitment of macrophages and lymphocytes that by releasing TGFβ, PDGF and other cytokines stimulate interstitial cells to produce excess matrix. That both the tubular paracrine pathway and the inflammatory cell-mediated pathway are activated after the onset of proteinuria was suggested by findings that in remnant kidneys of rats, cells expressing the myofibroblast-associated marker α-smooth muscle actin (α-SMA) were first detectable in the interstitial areas and colocalized with macrophages surrounding proximal tubular cells that were engaged in excess protein reabsorption. TGFβ mRNA was upregulated in proximal tubular cells in parallel with the nearby accumulation of inflammatory cells and α-SMA positive cells. Treatment of rats with remnant kidney with an ACE inhibitor limited excess protein accumulation and interstitial inflammatory cell infiltration and also abrogated abnormal TGFβ1 gene expression in tubular cells, and myofibroblast formation.
In addition to the activation of interstitial cells, the fibrogenic reaction involved a phenotypic reversal of tubular epithelial cells termed as EMT. The epithelial cells can be induced by several stimuli to become α-SMA expressing myofibroblasts. A huge number of studies have documented the abnormal expression of α-SMA and other myofibroblast markers in the renal tubule both in human and experimental nephropathies (for review, see ). Urinary proteins from nephrotic patients with focal segmental sclerosis, or to a lesser extent from patients with minimal change disease, induced cultured proximal tubular cells to express EMT-related patterns including α-SMA and vimentin via ERK1/2 and p38 pathway. In remnant kidneys, α-SMA expression was also found in focal areas both in the context of the tubular epithelium and in peritubular microvascular cells with features of endothelium following the onset of proteinuria and TGFβ overexpression. However, the primary source(s) of activated fibroblasts or myofibroblasts even in experimental settings are not clear . Evidence was provided both for EMT in tubular epithelium and endothelial-mesenchymal-transition. Complete EMT appears difficult to detect as compared to intermediate phenotypic EMT stages. Fate-tracing studies of epithelial cells in non-proteinuric mouse models failed to identify myofibroblasts originating from the epithelium. A more predominant role is emerging for mesenchyme-derived stromal cells of the developing kidney that mature into perivascular pericytes in the adult kidney.
TGFβ remains the most important cytokine for renal fibrogenesis. It has also been identified as the best characterized stimulus for EMT in renal tubular cells. Studies have focused on the signaling pathways which are activated during TGFβ-induced EMT. TGFβ caused Smad2 phosphorylation in a tubular epithelial cell line, and overexpression of the inhibitory Smad protein, Smad7, inhibited TGFβ−induced Smad2 activation, thereby preventing EMT and collagen synthesis. An endogenous antagonist of TGFβ1–induced EMT has been identified as bone morphogenic protein-7 (BMP-7), a member of TGFβ superfamily whose genetic deletion in mice leads to severe impairment of kidney development. BMP-7 reversed TGFβ1–induced EMT through a Smad-dependent reinduction of E-cadherin, an adhesive junction protein serving to maintain the structural integrity and polarity of epithelial cells. Systemic administration of recombinant BMP-7 repaired severely damaged renal tubular epithelial cells and reversed renal injury in mice with nephrotoxic serum nephritis. Investigation of a candidate mechanism downstream of BMP-7 suggested a role for TRPS1 (whose mutations cause tricho-rhino-pharyngeal syndrome) acting as an essential regulator of nephron development. Trps1 was found in proximal tubular epithelial cells of mice and its expression was reduced by ureteral obstruction. Trps haploinsufficiency promoted interstitial fibrosis via increased phosphorylation of Smad3 and decreased Smad7 protein. In vitro studies using proximal tubular cells suggested that the mechanism s underlying both TGFβ1–induced EMT and fibrosis were related to reduction of amount of Smad7 protein through ubiquitin-mediated degradation. Other mediators that may critically contribute to fibrogenesis include PDGF and endothelin-1 able to activate α-SMA gene expression in renal fibroblasts and vascular smooth muscle cells, respectively. IL18 and parathyroid hormone-related protein were found to promote EMT in vitro and to contribute to interstitial fibrosis in murine models of ureteral obstruction.
Growth factors in the ultrafiltrate, such as hepatocyte growth factor (HGF) and TGFβ1 itself, may contribute to the induction of fibrosis in vivo . In rats with glomerular proteinuria due to diabetic nephropathy, HGF and TGFβ were both detected in proximal tubular fluid and their receptors were upregulated in apical tubular membranes. In cultured proximal tubular cells, the exposure to ultrafiltrate rich in HGF and TGFβ induced the expression of fibronectin and PDGF as well as the basolateral secretion of MCP-1 and RANTES. The latter by interacting with macrophages in the renal interstitium caused them to secrete TGFβ which in turn, stimulated the expression of collagen type I and II, and fibronectin by interstitial myofibroblasts. Proximal tubular fluid from proteinuric, diabetic rats also increased the expression of connective tissue growth factor (CTGF) in tubular cells. Interestingly, CTGF induces moderate pro-fibrogenic activity and raises the expression of fibronectin in proximal tubular cells as well as extracellular matrix proteins in renal fibroblasts.
Thus, a multitude of profibrotic cytokines are generated as a consequence of glomerular inflammation and are conveyed with proteins into the tubular urine to amplify interstitial injury. Evidence indicates that glomerular proinflammatory cytokines combined with massive proteinuria are major determinants of subsequent tubulo-interstitial injury and progressive kidney failure in experimental and human glomerulonephritis. An elegant experiment, however, yielded data to dissect the role of glomerular inflammation from that of protein toxicity. Besides closed nephrons the kidney of a primitive amphibium, the axolotl, contains nephrons with ciliated peritoneal funnels (nephrostomes) that have free access to the peritoneal fluid. Injection of albumin into the peritoneal cavity caused selective uptake of proteins in tubular epithelial cells of nephrons with nephrostomes. As a consequence, protein and lipid droplets massively accumulated in the tubular cells. Furthermore, tubular lumen dilatation occurred and progressive focal accumulation of fibrous tissue was noted around protein-storing tubules, with the presence of fibronectin and TGFβ both in the tubular epithelial cells and in interstitial cells. Thus, protein loading in vivo directly induces tubulointerstitial activation and interstitial fibrosis, in the absence of glomerular inflammatory lesions. In this respect, myosin 1e (myo1e) knockout mice are a novel interesting model of glomerular disease, due to the disruption of a podocyte associated molecular motor protein that evolves to progressive tubulointerstitial injury. Early ultrastructural glomerular changes consisting of increased thickness of glomerular basement membrane and effacement of podocyte foot processes were detected in 1–3 week old mice and were followed by albuminuria, formation of protein droplets, and accumulation of immunoglobulin and C3 in proximal tubular cells. Tubular changes were associated with excess deposition of collagen V and fibronectin in the periglomerular and peritubular interstitium, that could be related to proteinuria-associated signaling, in the absence of any evidence of immune or inflammatory glomerular disease. Another report basing on an albumin-tracing analysis on serial sections of kidneys of OVE26 diabetic mice showed significant inter-nephron heterogeneicity with sharp localization of albumin-staining to proximal tubuli showing various degrees of injury from minimal protein droplet formation to ultrastructural damage and dilatation. Similar findings were obtained in biopsy samples of proteinuric patients. The intensity of positive staining was dependent on the level of albuminuria and immunoglobulin and C3 stained in albumin positive tubules. Connections were visualized between albumin-leaking glomeruli and tubuli accumulating albumin, either prior to or in association with the onset of fibrosis. Collectively these findings favor the possibility that both progressive injury within individual nephrons and consequent scarring were directly related to worsening of proteinuria of glomerular origin.