How many ways can a podocyte die?

Podocytes are highly specialized epithelial cells that line the urinary surface of the glomerular capillary tuft. To maintain kidney filtration, podocytes oppose the high intraglomerular hydrostatic pressure, form a molecular sieve, secrete soluble factors to regulate other glomerular cell types, and provide synthesis and maintenance of the glomerular basement membrane. Impairment of any of these functions after podocyte injury results in proteinuria and possibly renal failure. Loss of glomerular podocytes is a key feature for the progression of renal diseases, and detached podocytes can be retrieved in the urine of patients with progressive glomerular diseases. Thus, the concept of podocyte loss as a hallmark of progressive glomerular disease has been widely accepted. However, the nature of events that promote podocyte detachment and whether detachment is preceded by any kind of podocyte cell death, such as apoptosis, necroptosis, or necrosis, still remains unclear and is discussed in this review.     

Increased tubular proliferation as an adaptive response to glomerular albuminuria

Renal tubular atrophy accompanies many proteinuric renal diseases, suggesting that glomerular proteinuria injures the tubules. However, local or systemic inflammation and filtration of abnormal proteins known to directly injure tubules are also present in many of these diseases and animal models; therefore, whether glomerular proteinuria directly causes tubular injury is unknown. Here, we examined the renal response to proteinuria induced by selective podocyte loss. We generated mice that express the diphtheria toxin receptor exclusively in podocytes, allowing reproducible dose-dependent, specific ablation of podocytes by administering diphtheria toxin. Ablation of <20% of podocytes resulted in profound albuminuria that resolved over 1-2 weeks after the re-establishment of normal podocyte morphology. Immediately after the onset of albuminuria, proximal tubule cells underwent a transient burst of proliferation without evidence of tubular damage or increased apoptosis, resulting in an increase in total tubular cell numbers. The proliferative response coincided with detection of the growth factor Gas6 in the urine and phosphorylation of the Gas6 receptor Axl in the apical membrane of renal tubular cells. In contrast, ablation of >40% of podocytes led to progressive glomerulosclerosis, profound tubular injury, and renal failure. These data suggest that glomerular proteinuria in the absence of severe structural glomerular injury activates tubular proliferation, potentially as an adaptive response to minimize the loss of filtered proteins.     

Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene

Glomerular injury and proteinuria in diabetes (types 1 and 2) and IgA nephropathy is related to the degree of podocyte depletion in humans. For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed. The rodent homologue does not act as a diphtheria toxin (DT) receptor, thereby making rodents resistant to DT. Injection of DT into transgenic rats but not wild-type rats resulted in dose-dependent podocyte depletion from glomeruli. Three stages of glomerular injury caused by podocyte depletion were identified: Stage 1, 0 to 20% depletion showed mesangial expansion, transient proteinuria and normal renal function; stage 2, 21 to 40% depletion showed mesangial expansion, capsular adhesions (synechiae), focal segmental glomerulosclerosis, mild persistent proteinuria, and normal renal function; and stage 3, >40% podocyte depletion showed segmental to global glomerulosclerosis with sustained high-grade proteinuria and reduced renal function. These pathophysiologic consequences of podocyte depletion parallel similar degrees of podocyte depletion, glomerulosclerosis, and proteinuria seen in diabetic glomerulosclerosis. This model system provides strong support for the concept that podocyte depletion could be a major mechanism driving glomerulosclerosis and progressive loss of renal function in human glomerular diseases.     

Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental nephrotic syndrome

Podocytes play an important role in maintaining normal glomerular function and structure, and podocyte injury leads to proteinuria and glomerulosclerosis. The family of mitogen-activated protein kinases (MAPK; extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase, and p38) may be implicated in the progression of various glomerulopathies, but the role of MAPK in podocyte injury remains elusive. This study examined phosphorylation of p38 MAPK in clinical glomerulopathies with podocyte injury, as well as in rat puromycin aminonucleoside (PAN) nephropathy and mouse adriamycin (ADR) nephropathy. The effect of treatment with FR167653, an inhibitor of p38 MAPK, was also investigated in rodent models. In human podocyte injury diseases, the increased phosphorylation of p38 MAPK was observed at podocytes. In PAN and ADR nephropathy, the phosphorylation of p38 MAPK and ERK was marked but transient, preceding overt proteinuria. Pretreatment with FR167653 (day -2 to day 14, subcutaneously) to PAN or ADR nephropathy completely inhibited p38 MAPK activation and attenuated ERK phosphorylation, with complete suppression of proteinuria. Electron microscopy and immunohistochemistry for nephrin and connexin43 revealed that podocyte injury was markedly ameliorated by FR167653. Furthermore, early treatment with FR167653 effectively prevented glomerulosclerosis and renal dysfunction in the chronic phase of ADR nephropathy. In cultured podocytes, PAN or oxidative stress induced the phosphorylation of p38 MAPK along with actin reorganization, and FR167653 inhibited such changes. These findings indicate that the activation of MAPK is necessary for podocyte injury, suggesting that p38 MAPK and, possibly, ERK should become a potential target for therapeutic intervention in proteinuric glomerulopathies.     

Darbepoetin alfa protects podocytes from apoptosis in vitro and in vivo

Detachment or apoptosis of podocytes leads to proteinuria and glomerulosclerosis. There are no current interventions for diabetic or non-diabetic glomerular diseases specifically preventing podocyte apoptosis. Binding of erythropoiesis stimulating proteins (ESPs) to receptors on non-hematopoietic cells has been shown to have anti-apoptotic effects in vitro, in vivo, and in preliminary human studies. Recently, erythropoietin receptors were identified on podocytes; therefore, we tested effects of darbepoetin alfa in preventing podocyte apoptosis. Cultured immortalized mouse podocytes were treated with low-dose ultraviolet-C (uv-C) irradiation to induce apoptosis in the absence or presence of darbepoetin alfa. Apoptosis was quantified by Hoechst staining and by caspase 3 cleavage assessed by Western blots. Pretreatment with darbepoetin alfa significantly reduced podocyte apoptosis with this effect involving intact Janus family protein kinase-2 (JAK2) and AKT signaling pathways. Additionally, darbepoetin alfa was found protective against transforming growth factor-beta1 but not puromycin aminonucleoside induced apoptosis. Mice with anti-glomerular antibody induced glomerulonephritis had significantly less proteinuria, glomerulosclerosis, and podocyte apoptosis when treated with darbepoetin alfa. Our studies show that treatment of progressive renal diseases characterized by podocyte apoptosis with ESPs may be beneficial in slowing progression of chronic kidney disease.     

Angiotensin II type 1 receptor blockade inhibits the development and progression of HIV-associated nephropathy in a mouse model

HIV-associated nephropathy (HIVAN) is characterized by a collapsed glomerular capillary tuft with hyperplasia and hypertrophy of podocytes. Recently generated were conditional transgenic mice (podocin/Vpr) that express one of the HIV-1 accessory genes, vpr, selectively in podocytes using podocin promoter and Tet-on system. These transgenic mice developed renal injury similar to HIVAN when treated with doxycycline for 8 to 12 wk. This study demonstrated that nephron reduction by heminephrectomy markedly enhanced phenotypic changes of podocytes and led to severe FSGS within 4 wk. Nephrotic-range proteinuria was observed already at 2 wk, together with dedifferentiation and dysregulation of podocytes, indicated by decreased expression of nephrin, synaptopodin, and Wilms' tumor 1 protein and increased expression of Ki-67. The acceleration of phenotypic changes of podocytes, proteinuria, and subsequent glomerulosclerosis by heminephrectomy was almost completely inhibited by angiotensin II type 1 receptor (AT1R) blocker olmesartan. In contrast, the renoprotective effect of the calcium channel antagonist azelnidipine was minimal, although it lowered systemic BP to the same level as olmesartan, demonstrating that the inhibitory effect of AT1R blocker was independent of systemic BP. Olmesartan also reduced proteinuria and prevented glomerulosclerosis even by the delayed treatment, which was initiated after the podocyte injury appeared. These data suggest that nephron reduction exaggerates podocyte injury and subsequent glomerulosclerosis, possibly through glomerular hypertension, in the mouse model of HIVAN. AT1R blockade could be beneficial in the treatment of HIVAN by ameliorating podocyte injury by avoiding the vicious cycle of nephron reduction and glomerular hypertension.     

Glomerular podocytes and adhesive interaction with glomerular basement membrane

Normal podocyte function requires attachment to the underlying glomerular basement membrane. Alteration or disruption of podocyte attachment occurs in many forms of glomerular injury, leading to the development of proteinuria and eventually progressive glomerulosclerosis. The inability of podocytes to proliferate and thereby recover denuded glomerular basement membrane areas may be central to the pathogenesis of certain forms of glomerular diseases.     

Urinary podocyte loss is a more specific marker of ongoing glomerular damage than proteinuria

Podocyte loss contributes to the development of glomerulosclerosis. Although podocyte detachment has been recognized as a new mechanism of podocyte loss in glomerular diseases, its time course and relationship to disease activity are not known. Urinary excretion of viable podocytes was quantified in two models of transient glomerular injury, i.e., rats with puromycin aminonucleoside-induced nephrosis (PAN) and mesangioproliferative nephropathy (anti-Thy 1.1 nephritis model), as well as in a model of continuous glomerular injury, i.e., hypertensive nephropathy (5/6-nephrectomy model), and in aging rats. The number of glomerular Wilm's tumor (WT)-1-positive podocytes and the glomerular expression of cell-cycle proteins in vivo were assessed. Urinary podocyte loss occurred in both primary (PAN) and secondary (anti-Thy 1.1 nephritis) in parallel to the onset of proteinuria. However, subsequently proteinuria persisted despite remission of podocyturia. In continuous glomerular injury, i.e., after 5/6-nephrectomy, podocyturia paralleled the course of proteinuria and of systemic hypertension, whereas no podocyturia became detectable during normal aging (up to 12 mo). Despite podocyte detachment of varying degrees, no decrease in glomerular podocyte counts (i.e., WT-1 positive nuclei) was noted in either disease model. Podocyturia in the PAN and anti-Thy 1.1 nephritis model was preceded by entry of glomerular podocytes into the cell cycle, i.e., cyclin D1, cdc2, and/or proliferating cell nuclear antigen (PCNA) expression. Podocyturia is a widespread phenomenon in glomerular disease and not simply a reflection of proteinuria because it is limited to phases of ongoing glomerular injury. The data suggest that podocyturia may become a more sensitive means to assess the activity of glomerular damage than proteinuria.     

Rosuvastatin protects against podocyte apoptosis in vitro

BACKGROUND: Clinical studies suggest that statins reduce proteinuria and slow the decline in kidney function in chronic kidney disease. Given a rich literature identifying podocyte apoptosis as an early step in the pathophysiological progression to proteinuria and glomerulosclerosis, we hypothesized that rosuvastatin protects podocytes from undergoing apoptosis. Regarding a potential mechanism, our lab has shown that the cell cycle protein, p21, has a prosurvial role in podocytes and there is literature showing statins upregulate p21 in other renal cells. Therefore, we queried whether rosuvastatin is prosurvival in podocytes through a p21-dependent pathway. METHODS: Two independent apoptotic triggers, puromycin aminonucleoside (PA) and adriamycin (ADR), were used to induce apoptosis in p21 +/+ and p21 -/- conditionally immortalized mouse podocytes with or without pre-exposure to rosuvastatin. Apoptosis was measured by two methods: Hoechst 33342 staining and fluorescence-activated cell sorting (FACS). To establish a role for p21, p21 levels were measured by western blotting following rosuvastatin exposure and p21 was stably transduced into p21 -/- mouse podocytes. RESULTS: Rosuvastatin protects against ADR- and PA-induced apoptosis in podocytes. Further, exposure to rosuvastatin increases p21 levels in podocytes in vitro. ADR induces apoptosis in p21 -/- mouse podocytes, but rosuvastatin's protective effect is not seen in the absence of p21. Reconstituting p21 in p21 -/- podocytes restores rosuvastatin's prosurvival effect. CONCLUSION: Rosuvastatin is prosurvival in injured podocytes. Rosuvastatin exerts its protective effect through a p21-dependent antiapoptotic pathway. These findings suggest that statins decrease proteinuria by protecting against podocyte apoptosis and subsequent podocyte depopulation.     

Roflumilast enhances the renal protective effects of retinoids in an HIV-1 transgenic mouse model of rapidly progressive renal failure

Retinoic acid decreases proteinuria and glomerulosclerosis in several animal models of kidney disease by protecting podocytes from injury. Our recent in vitro studies suggest that all-trans retinoic acid induces podocyte differentiation by activating the retinoic acid receptor-α (RARα)/cAMP/PKA/CREB pathway. When used in combination with all-trans retinoic acid, an inhibitor of phosphodiesterase 4 further enhanced podocyte differentiation by increasing intracellular cAMP. Additionally, we found that Am580, a specific RARα agonist, has similar renal protective effects as all-trans retinoic acid in a rederived colony of HIV-1 transgenic mice with rapidly progressive renal failure (HIV-Tg) that mimics human HIV-associated nephropathy. Treatment with either the inhibitor of phosphodiesterase 4, roflumilast, or Am580 significantly reduced proteinuria, attenuated kidney injury, and improved podocyte differentiation in these HIV-Tg mice. Additional renal protective effects were found when roflumilast was combined with Am580. Consistent with the in vitro data, glomeruli from HIV-Tg mice treated with both Am580 and roflumilast had more active phosphorylated CREB than with either agent alone. Thus, phosphodiesterase 4 inhibitors could be used in combination with RARα agonists to provide additional renal protection.     

Glomerular Aging and Focal Global Glomerulosclerosis: A Podometric Perspective

Kidney aging is associated with an increasing proportion of globally scarred glomeruli, decreasing renal function, and exponentially increasing ESRD prevalence. In model systems, podocyte depletion causes glomerulosclerosis, suggesting age-associated glomerulosclerosis could be caused by a similar mechanism. We measured podocyte number, size, density, and glomerular volume in 89 normal kidney samples from living and deceased kidney donors and normal poles of nephrectomies. Podocyte nuclear density decreased with age due to a combination of decreased podocyte number per glomerulus and increased glomerular volume. Compensatory podocyte cell hypertrophy prevented a change in the proportion of tuft volume occupied by podocytes. Young kidneys had high podocyte reserve (podocyte density >300 per 10⁶ µm³), but by 70–80 years of age, average podocyte nuclear density decreased to, <100 per 10⁶ µm³, with corresponding podocyte hypertrophy. In older age podocyte detachment rate (urine podocin mRNA-to-creatinine ratio) was higher than at younger ages and podocytes were stressed (increased urine podocin-to-nephrin mRNA ratio). Moreover, in older kidneys, proteinaceous material accumulated in the Bowman space of glomeruli with low podocyte density. In a subset of these glomeruli, mass podocyte detachment events occurred in association with podocytes becoming binucleate (mitotic podocyte catastrophe) and subsequent wrinkling of glomerular capillaries, tuft collapse, and periglomerular fibrosis. In kidneys of young patients with underlying glomerular diseases similar pathologic events were identified in association with focal global glomerulosclerosis. Podocyte density reduction with age may therefore directly lead to focal global glomerulosclerosis, and all progressive glomerular diseases can be considered superimposed accelerators of this underlying process.     

PPAR-gamma agonist protects podocytes from injury

Podocyte injury and loss contribute to progressive glomerulosclerosis. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear hormone receptor, which we have found to be increased in podocytes in a variety of kidney diseases. It is not known if PPAR-gamma contributes to renal injury or if it serves as a countermeasure to limit renal injury during disease progression. We tested these possibilities utilizing the puromycin aminonucleoside (PAN) model of renal injury in immortalized mouse podocytes. The cultured podocytes expressed PPAR-gamma mRNA at baseline but this was decreased by PAN. Pioglitazone, a pharmacologic agonist of PPAR-gamma, increased both PPAR-gamma mRNA and activity in injured podocytes, as assessed by a reporter plasmid assay. Further, pioglitazone significantly decreased PAN-induced podocyte apoptosis and necrosis while restoring podocyte differentiation. The PPAR-gamma agonist significantly restored expression of the cyclin-dependent kinase inhibitor p27 and the antiapoptotic molecule Bcl-xL while significantly decreasing proapoptotic caspase-3 activity. Pioglitazone tended to decrease PAN-induced transforming growth factor-beta (TGF-beta) mRNA expression. Our study shows that PPAR-gamma is normally expressed by podocytes and its activation is protective against PAN-induced apoptosis and necrosis. We postulate that this protective effect may be mediated in part by effects on p27 and TGF-beta expression.     

Podocyte-specific deletion of integrin-linked kinase results in severe glomerular basement membrane alterations and progressive glomerulosclerosis

Alterations in glomerular podocyte cell-cell and cell-matrix contacts are key events in progressive glomerular failure. Integrin-linked kinase (ILK) has been implicated in podocyte cell-matrix interaction and is induced in proteinuria. For evaluation of ILK function in vivo, mice with a Cre-mediated podocyte-specific ILK inactivation were generated. These mice seemed normal at birth but developed progressive focal segmental glomerulosclerosis and died in terminal renal failure. The first ultrastructural lesions that are seen at onset of albuminuria are glomerular basement membrane (GBM) alterations with a significant increase in true harmonic mean GBM thickness. Podocyte foot process effacement and loss of slit diaphragm followed with progression to unselective proteinuria. No significant reduction of slit membrane molecules (podocin and nephrin), key GBM components (fibronectin, laminins, and collagen IV isoforms), or podocyte integrins could be observed at onset of proteinuria. However, alpha3-integrins were relocalized into a granular pattern along the GBM, consistent with altered integrin-mediated matrix assembly in ILK-deficient podocytes. As the increased GBM thickness precedes structural podocyte lesions and key components of the GBM were expressed at comparable levels to controls, these data suggest an essential role of ILK for the close interconnection of GBM structure and podocyte function.     

Podocyte injury in focal segmental glomerulosclerosis: Lessons from animal models (a play in five acts)

Genetic engineering in the mouse has ushered in a new era of disease modeling that has advanced our understanding of podocyte injury in the pathogenesis of focal segmental glomerulosclerosis. Historically, the major animal models of focal segmental glomerulosclerosis involve direct podocyte injury (exemplified by toxin models) and indirect podocyte injury due to adaptive responses (exemplified by renal ablation models). In both paradigms, recent evidence indicates that podocyte depletion is a major pathomechanism mediating proteinuria and glomerulosclerosis. Podocyte-specific toxin models support that podocyte loss is sufficient to cause focal segmental glomerulosclerosis in a dose-dependent manner. Knockout and transgenic models have provided proof of concept that mutations in specific podocyte proteins mediate genetic forms of focal segmental glomerulosclerosis. Transgenic models of HIV-associated nephropathy have helped to elucidate the role of direct viral infection and podocyte expression of viral gene products in the pathogenesis of this form of collapsing glomerulopathy. Taken together, emerging data support that injury directed to or inherent within the podocyte constitutes the critical event in diverse pathways to glomerulosclerosis.     

Angiotensin II-dependent persistent podocyte loss from destabilized glomeruli causes progression of end stage kidney disease

Podocyte depletion is a major mechanism driving glomerulosclerosis. Progression is the process by which progressive glomerulosclerosis leads to end stage kidney disease (ESKD). In order to determine mechanisms contributing to persistent podocyte loss, we used a human diphtheria toxin transgenic rat model. After initial diphtheria toxin-induced podocyte injury (over 30% loss in 4 weeks), glomeruli became destabilized, resulting in continued autonomous podocyte loss causing global podocyte depletion (ESKD) by 13 weeks. This was monitored by urine mRNA analysis and by quantitating podocytes in glomeruli. Similar patterns of podocyte depletion were found in the puromycin aminonucleoside and 5/6 nephrectomy rat models of progressive end-stage disease. Angiotensin II blockade (combined enalapril and losartan) restabilized the glomeruli, and prevented continuous podocyte loss and progression to ESKD. Discontinuing angiotensin II blockade resulted in recurrent glomerular destabilization, podocyte loss, and progression to ESKD. Reduction in blood pressure alone did not reduce proteinuria or prevent podocyte loss from destabilized glomeruli. The protective effect of angiotensin II blockade was entirely accounted for by reduced podocyte loss. Thus, an initiating event resulting in a critical degree of podocyte depletion can destabilize glomeruli and initiate a superimposed angiotensin II-dependent podocyte loss process that accelerates progression resulting in eventual global podocyte depletion and ESKD. These events can be monitored noninvasively in real-time through urine mRNA assays.     

Urinary and glomerular podocytes in patients with chronic kidney diseases

Backgrounds

Podocytes are highly differentiated epithelial cells involved in glomerular filtration. This study determines the clinical and histological significance of podocyte detachment and excretion in urine in patients with chronic kidney diseases.

Methods

Renal biopsy was performed in 59 patients (30 males, 29 females; mean age 48 ± 2 years), including 24 patients with immunoglobulin (Ig)A nephropathy, six each with focal segmental glomerulosclerosis, membranous nephropathy, and minimal change nephrotic syndrome, and 17 with other renal disorders. The number of glomerular podocytes and severity of morphological damage were evaluated in renal biopsy samples. Urinary podocytes were detected by anti-human podocalyxin antibody. The urinary IgG/albumin ratio and urinary peroxide products were assessed by gel electrophoresis and the 2′,7′-dichlorodihydrofluorescein-diacetate method, respectively.

Results

A decrease in glomerular podocytes was associated with age (r = ?0.33; P < 0.05), glomerulosclerosis (r = ?0.43; P < 0.01), tubulointerstitial lesions (r = ?0.46; P < 0.01), and low estimated glomerular filtration rates (r = 0.32; P < 0.05). Increased urinary podocyte excretion correlated with proteinuria (r = 0.36; P < 0.01), and was observed more frequently in patients with active histological lesions. Podocyte loss correlated with lower selectivity of proteinuria in patients with minimal change nephrotic syndrome and focal segmental glomerulosclerosis (r = ?0.90; P < 0.001). Moreover, urinary peroxide products increased in association with glomerulosclerosis (r = 0.39; P < 0.05).

Conclusions

Urinary podocyte excretion reflects ongoing glomerular injury in various kidney diseases, and podocyte loss correlated with glomerulosclerosis and impaired selectivity of proteinuria.     

Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: role in podocyte proliferation and maturation

BACKGROUND: Normal human podocytes are terminally differentiated and quiescent cells. It is not known why podocytes fail to proliferate in response to most forms of injury. Proliferation is regulated by cell cycle proteins and their inhibitors. The Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) in general prevent proliferation by inhibiting cyclin-CDK complexes. In the current study, we determined the expression and possible role of specific CDK inhibitors in podocyte proliferation in human disease characterized by podocyte injury. METHODS: Immunostaining was performed for the CDK inhibitors p21, p27, and p57 and the proliferation marker Ki-67 on renal biopsies from patients with minimal change disease (MCD; N = 6), membranous glomerulopathy (MGN; N = 19), cellular variant of focal segmental glomerulosclerosis (FSGS; N = 12), collapsing glomerulopathy (CG; N = 9), and HIV-associated nephropathy (HIVAN; N = 16). Adult nephrectomy specimens without evidence of glomerular disease served as controls (N = 9). RESULTS: Normal quiescent podocytes express p27 and p57, but not p21. In diseases without podocyte proliferation (MCD, MGN), p21, p27, and p57 expression did not change. In contrast, there was a uniform decrease in p27 and p57 immunostaining in diseases with podocyte proliferation (cellular FSGS, CG, and HIVAN). This was accompanied by the de novo expression of p21 in podocytes. CONCLUSIONS: Our results show that podocyte quiescence may require the presence of the CDK inhibitors p27 and p57. In human glomerular diseases, a decrease in p27 and p57 may be permissive for the altered proliferative podocyte phenotype. p21 may have a multifactorial role in podocyte cell cycle regulation.