Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats

Angiotensin II (AngII) is a critical determinant of glomerular function involving both hemodynamic and pressure-independent effects that are insufficiently understood. A novel transgenic rat (TGR) model with overexpression of the human AngII type 1 receptor (hAT1) in podocytes was developed to study the consequences of an increased AT1 signaling on the structure and function of the glomerular filter. Use of the nephrin promoter to target the podocytes resulted in an expression of the hAT1 at a level roughly two times higher than the endogenous AT1 throughout life. All male TGR developed significant albuminuria starting at 8 to 15 wk of age; systolic BP was not elevated. More or less concurrently, structural changes at the glomerulus were encountered, starting with ubiquitous formation of pseudocysts at podocytes, followed by foot process effacement and local detachments. This damage progressed to nephron loss via the well known pathway typical for classic focal segmental glomerulosclerosis. The structural changes significantly correlated with age (r(2) = 0.76) and urinary albumin excretion (r(2) = 0.70). The data provide direct evidence that increased AT1 signaling in podocytes leads to protein leakage and structural podocyte damage progressing to focal segmental glomerulosclerosis.     

Inducible podocyte injury and proteinuria in transgenic zebrafish

Damage or loss of podocytes causes glomerulosclerosis in murine models, and mutations in podocyte-specific genes cause nephrotic syndrome in humans. Zebrafish provide a valuable model for kidney research, but disruption of pronephroi leads to death within a few days, thereby preventing the study of CKD. In this study, we generated an inducible model of podocyte injury in zebrafish (pod::NTR-mCherry) by expressing a bacterial nitroreductase, which converts metronidazole to a cytotoxin, specifically in podocytes under the control of the zebrafish nphs2/podocin promoter. Application of the prodrug metronidazole to the transgenic fish induces acute damage to the podocytes in pronephroi of larval zebrafish and the mesonephroi of adult zebrafish, resulting in foot-process effacement and podocyte loss. We also developed a functional assay of the glomerular filtration barrier by creating transgenic zebrafish expressing green fluorescent protein (GFP)-tagged vitamin D-binding protein (VDBP) as a tracer for proteinuria. In the VDBP-GFP and pod::NTR-mCherry double-transgenic fish, induction of podocyte damage led to whole-body edema, and the proximal tubules reabsorbed and accumulated VDBP-GFP that leaked through the glomeruli, mimicking the phenotype of human nephrotic syndrome. Moreover, expression of wt1b::GFP, a marker for the developing nephron, extended into the Bowman capsule in response to podocyte injury, suggesting that zebrafish have a podocyte-specific repair process known to occur in mammalian metanephros. These data support the use of these transgenic zebrafish as a model system for studies of glomerular pathogenesis and podocyte regeneration.     

Growth-dependent podocyte failure causes glomerulosclerosis

Podocyte depletion leads to glomerulosclerosis, but whether an impaired capacity of podocytes to respond to hypertrophic stress also causes glomerulosclerosis is unknown. We generated transgenic Fischer 344 rats that express a dominant negative AA-4E-BP1 transgene driven by the podocin promoter; a member of the mammalian target of rapamycin complex 1 (mTORC1) pathway, 4E-BP1 modulates cap-dependent translation, which is a key determinant of a cell's hypertrophic response to nutrients and growth factors. AA-4E-BP1 rat podocytes expressed the transgene and had normal kidney histology and protein excretion at 100 g of body weight but developed ESRD by 12 months. Proteinuria and glomerulosclerosis were linearly related to both increasing body weight and transgene dose. Uni-nephrectomy reduced the body weight at which proteinuria first developed by 40%-50%. The initial histologic manifestation of disease was the appearance of bare areas of glomerular basement membrane from the pulling apart of podocyte foot processes, followed by adhesions to the Bowman capsule. Morphometric analysis confirmed the mismatch between glomerular tuft volume and total podocyte volume (number × size) per tuft in relation to weight gain and nephrectomy. Proteinuria and glomerulosclerosis did not develop if dietary calorie restriction prevented weight gain and glomerular enlargement. In summary, failure of podocytes to match glomerular tuft growth in response to growth signaling through the mTORC1 pathway can trigger proteinuria, glomerulosclerosis, and progression to ESRD. Reducing body weight and glomerular growth may be useful adjunctive therapies to slow or prevent progression to ESRD.     

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.     

HIV-1 genes vpr and nef synergistically damage podocytes, leading to glomerulosclerosis

This study aimed to identify the causative gene for HIV-1 associated nephropathy, a paradigmatic podocytopathy. A previous study demonstrated that transgenic expression of nonstructural HIV-1 genes selectively in podocytes in mice with FVB/N genetic background resulted in podocyte injury and glomerulosclerosis. In this study, transgenic mice that expressed individual HIV-1 genes in podocytes were generated. Five of six transgenic mice that expressed vpr developed podocyte damage and glomerulosclerosis. Analysis of an established vpr transgenic line revealed that transgenic mice on FVB/N but not on C57BL/6 genetic background developed podocyte injury by 8 wk of age, with later glomerulosclerosis. Four of 11 transgenic mice that expressed nef also developed podocyte injury. One transgenic line was established from the nef founder mouse with the mildest phenotype. Transgenic mice in this line developed mesangial expansion at 3 wk of age and mild focal podocyte damage at 10 wk of age. Mating with FVB/N mice did not augment nephropathy. None of the transgenic mice that expressed vif, tat, rev, or vpu in podocytes, even with the FVB/N genetic background, developed podocyte injury. For testing effects of simultaneous expression of vpr and nef, these two lines were mated. All nef:vpr double-transgenic mice showed severe podocyte injury and glomerulosclerosis by 4 wk of age. In contrast, all vpr or nef single-transgenic mice in the same litter uniformly showed no or much milder podocyte injury. These findings indicate that vpr and nef each can induce podocyte injury with a prominent synergistic interaction.     

Thiazolidinedione attenuate proteinuria and glomerulosclerosis in Adriamycin-induced nephropathy rats via slit diaphragm protection

Aim:   The slit diaphragm (SD) of podocyte impairment contributes to massive proteinuria and progressive glomerulosclerosis in many human glomerular diseases. The aim of the study was to determine if thiazolidinedione (TZD) reduce proteinuria and glomerulosclerosis in focal segmental glomerulosclerosis (FSGS) by preserving the structure and function of SD.
Methods:   Adriamycin-induced FSGS rat models were employed. Urinary protein content was measured dynamically during the experiment. Additional biochemical parameters in serum samples were measured after the animals were killed. Glomerular sclerosis index (SI) and podocyte foot processes fusion rate (PFR) were evaluated. The protein and mRNA expressing levels of nephrin, podocin and CD2-associated protein (CD2AP) in glomeruli were assessed by immunohistochemistry and real-time quantitative polymerase chain reaction, respectively. The density of podocytes was also evaluated after anti-Wilms' tumour-1 immunohistochemical staining.
Results:   Rosiglitazone treatment partially reduced proteinuria, but did not significantly affect the serum levels of triglyceride, cholesterol, albumin, glucose, urea nitrogen and creatinine in Adriamycin-induced FSGS rats. Glomerular SI and podocyte foot PFR were significantly attenuated by rosiglitazone treatment. Rosiglitazone prevented the reduction of nephrin, podocin and CD2AP protein expression induced by Adriamycin, however, the mRNA expression levels of these SD-related markers did not change significantly. Rosiglitazone therapy did not reverse Adriamycin-mediated reduction of the density of podocytes.
Conclusions:   The study data suggest that TZD are promising therapeutic agents on FSGS, and the mechanism may be mediated in part by directly protecting the structure and function of SD.     

Essential role of integrin-linked kinase in podocyte biology: Bridging the integrin and slit diaphragm signaling

Integrin-linked kinase (ILK) has been implicated in the pathogenesis of proteinuria and congenital nephrotic syndrome. However, the function of ILK in glomerular podocyte in a physiologic setting remains unknown. In this study, a mouse model was generated in which ILK gene was selectively disrupted in podocytes by using the Cre-LoxP system. Podocyte-specific ablation of ILK resulted in heavy albuminuria, glomerulosclerosis, and kidney failure, which led to animal death beginning at 10 wk of age. Podocyte detachment and apoptosis were not observed at 4 wk of age, when albuminuria became prominent, indicating that they are not the initial cause of proteinuria. Electron microscopy revealed an early foot process effacement, as well as morphologic abnormality, in ILK-deficient podocytes. ILK deficiency caused an aberrant distribution of nephrin and alpha-actinin-4 in podocytes, whereas the localization of podocin and synaptopodin remained relatively intact. Co-immunoprecipitation demonstrated that ILK physically interacted with nephrin to form a ternary complex, and alpha-actinin-4 participated in ILK/nephrin complex formation. Therefore, ILK plays an essential role in specifying nephrin and alpha-actinin-4 distribution and in maintaining the slit diaphragm integrity and podocyte architecture. These results also illustrate that the integrin and slit diaphragm signals in podocytes are intrinsically coupled through an ILK-dependent mechanism.     

The podocyte's response to injury: role in proteinuria and glomerulosclerosis

The terminally differentiated podocyte, also called glomerular visceral epithelial cell, are highly specialized cells. They function as a critical size and charge barrier to prevent proteinuria. Podocytes are injured in diabetic and non-diabetic renal diseases. The clinical signature of podocyte injury is proteinuria, with or without loss of renal function owing to glomerulosclerosis. There is an exciting and expanding literature showing that hereditary, congenital, or acquired abnormalities in the molecular anatomy of podocytes leads to proteinuria, and at times, glomerulosclerosis. The change in podocyte shape, called effacement, is not simply a passive process following injury, but is owing to a complex interplay of proteins that comprise the molecular anatomy of the different protein domains of podocytes. These will be discussed in this review. Recent studies have also highlighted that a reduction in podocyte number directly causes proteinuria and glomerulosclerosis. This is owing to several factors, including the relative inability for these cells to proliferate, detachment, and apoptosis. The mechanisms of these events are being elucidated, and are discussed in this review. It is the hope that by delineating the events following injury to podocytes, therapies might be developed to reduce the burden of proteinuric renal diseases.     

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.     

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.     

Permanent genetic tagging of podocytes: fate of injured podocytes in a mouse model of glomerular sclerosis

Injured podocytes lose differentiation markers. Therefore, the true identity of severely injured podocytes remains unverified. A transgenic mouse model equipped with a podocyte-selective injury induction system was established. After induction of podocyte injury, mice rapidly developed glomerulosclerosis, with downregulation of podocyte marker proteins. Proliferating epithelial cells accumulated within Bowman's space, as seen in collapsing glomerulosclerosis. In this study, the fate of injured podocytes was pursued. Utilizing Cre-loxP recombination, the podocyte lineage was genetically labeled with lacZ in an irreversible manner. After podocyte injury, the number of lacZ-labeled cells, which were often negative for synaptopodin, progressively declined, correlating with glomerular damage. Parietal epithelial cells, but not lacZ-labeled podocytes, avidly proliferated. The cells proliferating within Bowman's capsule and, occasionally, on the outer surface of the glomerular basement membrane were lacZ-negative. Thus, when podocytes are severely injured, proliferating parietal epithelial cells migrate onto the visceral site, thereby mimicking proliferating podocytes.     

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.     

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.     

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.     

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.