Matrix Metalloproteinase-9 Expression Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced by Albumin in In Vitro Primary Parietal Cell Culture

As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy. Albumin overload may induce MMP-9 expression and secretion by PECs via the activation of p44/42 MAPK pathway.     

Formyl peptide receptor 1 promotes podocyte injury through regulation of mitogen-activated protein kinase pathways

Podocyte injury contributes to glomerular injury and is implicated in the pathogenesis of diabetic nephropathy. Formyl peptide receptor (FPR) 1 is abundantly expressed in neutrophils and mediates intracellular transport of Ca ²⁺. Intracellular Ca ²⁺ regulates pathological process in renal podocyte and plays a role in diabetic nephropathy. However, the role of formyl peptide receptor 1 in podocyte injury of diabetic nephropathy has not been reported yet. Firstly, a rat model with diabetic nephropathy was established by streptozotocin injection, and a cell model was established via high glucose treatment of mouse podocytes (MPC5). Formyl peptide receptor 1 was enhanced in streptozotocin-induced rats and high glucose-treated MPC5. Secondly, streptozotocin injection promoted the glomerular injury with decreased nephrin and podocin. However, tail injection with adenovirus containing shRNA for silencing of formyl peptide receptor 1 attenuated streptozotocin-induced glomerular injury and the decrease in nephrin and podocin. Moreover, silencing of formyl peptide receptor 1 repressed cell apoptosis of podocytes in diabetic rats and high glucose-treated MPC5. Lastly, protein expression levels of p-p38, p-ERK, and p-JNK protein were up-regulated in streptozotocin-induced rats and high glucose-treated MPC5. Silencing of formyl peptide receptor 1 attenuated high glucose-induced increase in p-p38, p-ERK, and p-JNK in MPC5, and over-expression of formyl peptide receptor 1 aggravated high glucose-induced increase in p-p38, p-ERK, and p-JNK. In conclusion, inhibition of formyl peptide receptor 1 preserved glomerular function and protected against podocyte dysfunction in diabetic nephropathy.     

Requirement for class II phosphoinositide 3-kinase C2alpha in maintenance of glomerular structure and function

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.     

Effect of the knockdown of Cabin1 on p53 in glomerular podocyte

Calcineurin binding protein 1 (Cabin1) is a natural inhibitor of calcineurin (CN). Moreover, Cabin1 retards tumor cell apoptosis by regulating p53. This study was designed to observe the expression of Cabin1 during podocyte injury, as well as its relationship with p53. Sprague-Dawley rats were used for the establishment of 5/6 nephrectomized rat model. Sham-operated rats underwent ventral laparotomy without nephrectomy. Then, rats were sacrificed at 8 and 12 weeks after nephrectomy. WT-1, a podocyte nuclear protein, was used for indicating the localization of Cabin1 in glomeruli. As tacrolimus protects podocyte via inhibiting AngiotensinII (AngII) induced CN activation. Cultured podocytes were injured by AngII or restored by tacrolimus. The protein expression and localization was detected by western blot or immunofluorescence staining. Cabin1 was knocked down by siRNA in cultured podocytes. In 5/6 nephrectomized rats, the colocalization of Cabin1 and WT-1 became more obviously in podocyte nuclei. Cabin1 protein was markedly increased in rats at 8 and 12 weeks after nephrectomy, as well as in AngII injured podocytes at 48?h (0.99?±?0.12 in AngII group versus 0.80?±?0.16 in control group). Cabin1 and p53 colocalized in cultured podocyte nuclei, p53 expression was significantly decreased (0.21?±?0.05 in siRNA group versus 0.31?±?0.05 in negative control group) after Cabin1 was being knocked down. In conclusion, Cabin1 expression significantly increases during podocyte injury. Knockdown of Cabin1 induces p53 expression decrease in cultured podocyte. Cabin1 may provide a new target to investigate podocyte injury.     

The Calcineurin Inhibitor Tacrolimus Reduces Proteinuria in Membranous Nephropathy Accompanied by a Decrease in Angiopoietin-Like-4

Tacrolimus is an anticalcineurinic agent with potent immunosuppressive activity that has recently been shown to have the added benefit of reducing proteinuria in membranous nephropathy (MN) patients. However, its potential mechanisms remain unknown. To reveal the mechanism, rat cohorts were administered tacrolimus or vehicle from days 7 to 28 after the induction of passive Heymann nephritis (PHN). PHN induction resulted in heavy proteinuria and increased expression of desmin, a marker of injured podocytes. We also showed that the glomerular expression of angiopoietin-like-4 (Angptl4) was markedly upregulated in PHN rats and human MN followed by an increase in urine Angptl4 excretion. In addition, increased Angptl4 expression may be related to podocyte injury and proteinuria. Furthermore, upregulated Angptl4 expression primarily colocalized with podocytes rather than endothelial or mesangial cells, indicating that podocytes may be the source of Angptl4, which then gradually migrated to the glomerular basement membrane over time. However, tacrolimus treatment markedly reduced glomerular and urinary Angptl4, accompanied by a reduction in the established proteinuria and the promotion of podocyte repair. Additionally, glomerular immune deposits and circulating IgG levels induced by PHN clearly decreased following tacrolimus treatment. In conclusion, this is the first demonstration that the calcineurin inhibitor tacrolimus can reduce Angptl4 in podocytes accompanied by a decrease in established proteinuria and promotion of podocyte repair in MN.     

Emerging role of podocyte autophagy in the progression of diabetic nephropathy

Glomerular podocytes are pivotal in maintaining glomerular filtration barrier function. As severe podocyte injury results in proteinuria in patients with diabetic nephropathy, determining the pathogenesis of podocyte injury may contribute to the development of new treatments. We recently showed that autophagy is involved in the pathogenesis of diabetes-related podocyte injury. Insufficient podocyte autophagy and podocyte loss are observed in diabetic patients with massive proteinuria. Podocyte loss and massive proteinuria occur in high-fat diet-induced diabetic mice with podocyte-specific autophagy deficiency, with podocytes of these mice and of diabetic rats having huge damaged lysosomes. Sera from diabetic patients and from rodents with massive proteinuria cause autophagy insufficiency, resulting in lysosome dysfunction and apoptosis of cultured podocytes. These findings suggest the importance of autophagy in maintaining lysosome homeostasis in podocytes under diabetic conditions. Impaired autophagy may be involved in the pathogenesis of podocyte loss, leading to massive proteinuria in diabetic nephropathy.     

Autoradiographic studies on the proliferation of glomerular and tubular cells of the rat kidney in early diabetes

Although Osterby and coworkers have shown that the onset of diabetes is associated with glomerular hypertrophy and enlargement of the filtration surface, the pathogenesis of this lesion and the behaviour of the glomerular tuft cells during the increase in glomerular filtration surface area remain unclear. Autoradiography and electron microscopic morphometry in kidneys of male Sprague-Dawley rats with streptozotocin-induced diabetes indicate that glomerular enlargement is combined with glomerular cell hyperplasia. 6 days after continuous 3H-thymidine infusion there is a significant increase in DNA-synthesis in tuft cells indicating cell proliferation. Since podocyte volume only increases in acute diabetes (as shown by Osterby and Gundersen in 1980), and the mean podocyte nuclear area remains unchanged, we conclude that the increased DNA-synthesis results in podocyte hypercellularity so that an increased glomerular filtration surface area can be covered by a greater number of cells.     

Glomerular Endothelial Cell Injury and Damage Precedes That of Podocytes in Adriamycin-Induced Nephropathy

The role of podocytes in the development and progression of glomerular disease has been extensively investigated in the past decade. However, the importance of glomerular endothelial cells in the pathogenesis of proteinuria and glomerulosclerosis has been largely ignored. Recent studies have demonstrated that endothelial nitric oxide synthatase (eNOS) deficiency exacerbates renal injury in anti-GBM and remnant kidney models and accelerates diabetic kidney damage. Increasing evidence also demonstrates the importance of the glomerular endothelium in preventing proteinuria. We hypothesize that endothelial dysfunction can initiate and promote the development and progression of glomerulopathy. Administration of adriamycin (ADR) to C57BL/6 mice, normally an ADR resistant strain, with an eNOS deficiency induced overt proteinuria, severe glomerulosclerosis, interstitial fibrosis and inflammation. We also examined glomerular endothelial cell and podocyte injury in ADR-induced nephropathy in Balb/c mice, an ADR susceptible strain, by immunostaining, TUNEL and Western blotting. Interestingly, down-regulation of eNOS and the appearance of apoptotic glomerular endothelial cells occurred as early as 24 hours after ADR injection, whilst synaptopodin, a functional podocyte marker, was reduced 7 days after ADR injection and coincided with a significant increase in the number of apoptotic podocytes. Furthermore, conditioned media from mouse microvascular endothelial cells over-expressing GFP-eNOS protected podocytes from TNF-α-induced loss of synaptopodin. In conclusion, our study demonstrated that endothelial dysfunction and damage precedes podocyte injury in ADR-induced nephropathy. Glomerular endothelial cells may protect podocytes from inflammatory insult. Understanding the role of glomerular endothelial dysfunction in the development of kidney disease will facilitate in the design of novel strategies to treat kidney disease.     

Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes

The adhesion molecule integrin alpha3beta1 is the major receptor of podocyte to the glomerular capillary basement membrane (GBM). Since progressive alteration of the glomerular extracellular matrix (ECM) compartment leading to GBM thickening is common in diabetic nephropathy, we investigated the cellular distribution of alpha3beta1 integrin in podocytes of patients with diabetic nephropathy and streptozotocin-induced diabetic rats, and we evaluated the effects of high glucose on the cultured rat podocytes. Both human and rat kidneys were stained using the immunoelectron microscopy and immunoperoxidase technique with mouse monoclonal antibodies to human integrin alpha3 subunit. The results showed that both the number of immunogold particles and the staining of integrin alpha3 subunit on podocytes were weaker in patients with diabetic nephropathy than those of control kidneys. The staining of alpha3 on podocytes in the poorly-controlled diabetic rats was also weaker after one and three months of hyperglycemia. However, the staining was identical to controls in rats with only one week of hyperglycemia. High glucose (25 mM) but not streptozotocin in vitro suppressed the alpha3 expression of cultured rat podocytes. Our results demonstrated that the expression of integrin alpha3beta1 on podocytes was suppressed in both human and rats with diabetes, possibly due to the effects of hyperglycemia, and the suppression became more severe with the duration of diabetes.     

AMPK signalling: Implications for podocyte biology in diabetic nephropathy

Diabetic nephropathy is a major long‐term complication of diabetes mellitus and one of the most common causes of end‐stage renal disease. Thickening of the glomerular basement membrane, glomerular cell hypertrophy and podocyte loss are among the main pathological changes that occur during diabetic nephropathy, resulting in proteinuria. Injury to podocytes, which are a crucial component of the glomerular filtration barrier, seems to play a key role in the development of diabetic nephropathy. Recent studies have suggested that dysregulation of AMP‐activated kinase protein, which is an essential cellular energy sensor, may play a fundamental role in this process. The purpose of this review is to highlight the molecular mechanisms associated with AMP‐activated protein kinase (AMPK) in podocytes that are involved in the pathogenesis of diabetic nephropathy.     

Podocyte injury and overexpression of vascular endothelial growth factor and transforming growth factor-beta 1 in adriamycin-induced nephropathy in rats

The aim of this study is to investigate the expression of nephrin, vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-β1), and podocyte number in adriamycin (ADR)-induced nephropathy. A total of 60 male Sprague-Dawley rats were randomly divided into the control group and the ADR nephropathy group. The nephropathy was induced by tail-vein injection of ADR (4 mg/kg) twice at a 14-day interval. The expression levels of nephrin, VEGF, and TGF-β1 in glomeruli were assessed by immunohistochemistry and western blotting. The podocyte number was also evaluated after anti-Wilms' tumor-1 (WT1) immunohistochemical staining. In addition, the urinary protein content, biochemical parameters in serum samples and glomerular sclerosis index (SI) were compared between groups. In the ADR nephropathy group, the expression levels of nephrin was significantly decreased with the fusion of podocyte foot processes at 6 weeks after the first ADR injection, which was associated with a marked proteinuria. A decrease in podocyte number and an increase in SI with the overexpression of both VEGF and TGF-β1 were also observed in the glomeruli at 10 weeks after the first ADR injection. This was associated with focal segmental glomerulosclerosis (FSGS). The study data suggest that podocyte injury and decreased nephrin, as well as increased VEGF and TGF-β1, may contribute to the development of proteinuria and FSGS in ADR-induced nephropathy in rats.     

Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis

Subepithelial immune complex deposition in glomerular disease causes local inflammation and proteinuria by podocyte disruption. A rat model of membranous nephropathy, the passive Heymann nephritis, suggests that Abs against specific podocyte Ags cause subepithelial deposit formation and podocyte foot process disruption. In this study, we present a mouse model in which a polyclonal sheep anti-mouse podocyte Ab caused subepithelial immune complex formation. Mice developed a nephrotic syndrome with severe edema, proteinuria, hypoalbuminemia, and elevated cholesterol and triglycerides. Development of proteinuria was biphasic: an initial protein loss was followed by a second massive increase of protein loss beginning at approximately day 10. By histology, podocytes were swollen. Electron microscopy revealed 60-80% podocyte foot process effacement and subepithelial deposits, but no disruption of the glomerular basement membrane. Nephrin and synaptopodin staining was severely disrupted, and podocyte number was reduced in anti-podocyte serum-treated mice, indicating severe podocyte damage. Immunohistochemistry detected the injected anti-podocyte Ab exclusively along the glomerular filtration barrier. Immunoelectron microscopy localized the Ab to podocyte foot processes and the glomerular basement membrane. Similarly, immunohistochemistry localized mouse IgG to the subepithelial space. The third complement component (C3) was detected in a linear staining pattern along the glomerular basement membrane and in the mesangial hinge region. However, C3-deficient mice were not protected from podocyte damage, indicating a complement-independent mechanism. Twenty proteins were identified as possible Ags to the sheep anti-podocyte serum by mass spectrometry. Together, these data establish a reproducible model of immune-mediated podocyte injury in mice with subepithelial immune complex formation.     

Expression and regulation of adrenomedullin in renal glomerular podocytes

Adrenomedullin (AM) is postulated to exert organ-protective effects. It is expressed in the renal glomeruli, but its roles in the glomerular podocytes have been poorly elucidated. In the present study, we investigated the expression and regulation of AM in recently established conditionally immortalized mouse podocyte cell line in vitro and podocyte injury model in vivo. The cultured differentiated podocytes expressed AM mRNA and secreted measurable amount of AM. AM secretion from the podocytes was increased by H(2)O(2), hypoxia, puromycin aminonucleoside (PAN), albumin overload, and TNF-alpha. Real-time RT-PCR analysis revealed that AM mRNA expression in the podocytes was enhanced by PAN and TNF-alpha, both of which were suppressed by mitochondrial antioxidants. Furthermore, AM expression was upregulated in the glomerular podocytes of PAN nephrosis rats. These results indicated that AM expression in the podocytes was upregulated by stimuli or condition relevant to podocyte injury, suggesting its potential role in podocyte pathophysiology.     

Thioredoxin-interacting protein mediates NALP3 inflammasome activation in podocytes during diabetic nephropathy

Numerous studies have shown that the NALP3 inflammasome plays an important role in various immune and inflammatory diseases. However, whether the NALP3 inflammasome is involved in the pathogenesis of diabetic nephropathy (DN) is unclear. In our study, we confirmed that high glucose (HG) concentrations induced NALP3 inflammasome activation both in vivo and in vitro. Blocking NALP3 inflammasome activation by NALP3/ASC shRNA and caspase-1 inhibition prevented IL-1β production and eventually attenuated podocyte and glomerular injury under HG conditions. We also found that thioredoxin (TRX)-interacting protein (TXNIP), which is a pro-oxidative stress and pro-inflammatory factor, activated NALP3 inflammasome by interacting with NALP3 in HG-exposed podocytes. Knocking down TXNIP impeded NALP3 inflammasome activation and alleviated podocyte injury caused by HG. In summary, the NALP3 inflammasome mediates podocyte and glomerular injury in DN, moreover, TXNIP participates in the formation and activation of the NALP3 inflammasome in podocytes during DN, which represents a novel mechanism of podocyte and glomerular injury under diabetic conditions.     

Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice

To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4–5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease.