AGE-Induced Suppression of EZH2 Mediates Injury of Podocytes by Reducing H3K27me3.

BACKGROUND: Chronic hyperglycemia, a pivotal feature of diabetes mellitus (DM), initiates the formation of advanced glycation end products (AGEs) and the dysregulation of epigenetic mechanisms, which may cause injury to renal podocytes, a central feature of diabetic kidney disease (DKD). Previous data of our group showed that AGEs significantly reduce the expression of NIPP1 (nuclear inhibitor of protein phosphatase 1) in podocytes in vitro as well as in human and murine DKD. NIPP1 was shown by others to interact with enhancer of zeste homolog 2 (EZH2), which catalyzes the repressive methylation of H3K27me3 on histone 3. Therefore, we hypothesized that AGEs can directly induce epigenetic changes in podocytes. METHODS: We analyzed the relevance of AGEs on EZH2 expression and activity in a murine podocyte cell line. Cells were treated with 5 mg/mL glycated BSA for 24 h. To determine the meaning of EZH2 suppression, EZH2 activity was inhibited by incubating the cells with the pharmacological methyltransferase inhibitor 3-deazaneplanocin A; EZH2 expression was repressed with siRNA. mRNA expression was analyzed with real-time PCR, and protein expression with Western blot. EZH2 expression and level of H3K27 trimethylation in podocytes of diabetic db/db mice, a mouse model for type 2 DM, were analyzed using immunofluorescence. RESULTS: Our data demonstrated that AGEs decrease EZH2 expression in podocytes and consequently reduce H3K27me3. This suppression of EZH2 mimicked the AGE effects and caused an upregulated expression of pathological factors that contribute to podocyte injury in DKD. In addition, analyses of db/db mice showed significantly reduced H3K27me3 and EZH2 expression in podocytes. Moreover, the suppression of NIPP1 and EZH2 showed similar effects regarding podocyte injury. CONCLUSIONS: Our studies provide a novel pathway how AGEs contribute to podocyte injury and the formation of the so-called metabolic memory in DKD.

FSP1-specific SMAD2 knockout in renal tubular, endothelial, and interstitial cells reduces fibrosis and epithelial-to-mesenchymal transition in murine STZ-induced diabetic nephropathy.

Extracellular matrix deposition during tubulointerstitial fibrosis (TIF), a central pathological process in patients with diabetic nephropathy (DN), is driven by locally activated, disease-relevant myofibroblasts. Myofibroblasts can arise from various cellular sources, e.g., tubular epithelial cells via a process named epithelial-to-mesenchymal transition (EMT). Transforming growth factor beta 1 (TGF-ß1) and its downstream Smad signaling play a critical role in both TIF and EMT. Whereas Smad3 is one central mediator, the role of the other prominently expressed variant, Smad2, is not completely understood. In this study, we sought to analyze the role of renal Smad2 in the development of TIF and EMT during streptozotocin-induced DN by using a fibroblast-specific protein 1 (FSP1)-promotor-driven SMAD2 knockout mouse model with decreased tubular, endothelial, and interstitial Smad2 expression. In contrast to wild-type diabetic mice, diabetic SMAD2 knockout mice showed the following features: (1) significantly reduced DN and TIF (shown by KIM1 expression; periodic acid Schiff staining; collagen I and III, fibronectin, and connective tissue growth factor deposition); (2) significantly reduced tubular EMT-like changes (e.g., altered Snail1, E-cadherin, matrix metalloproteinase 2, and vimentin deposition); and (3) significantly decreased expression of myofibroblast markers (α-smooth muscle actin, FSP1). As one mechanism for the protection against diabetes-induced TIF and EMT, decreased Smad3 protein levels and, as a possible consequence, reduced TGF-ß1 levels were observed in diabetic SMAD2 knockout mice. Our findings thus support the important role of Smad2 for pro-fibrotic TGF-ß/Smad3 signaling in experimental DN.

Heterozygosity of mitogen-activated protein kinase organizer 1 ameliorates diabetic nephropathy and suppresses epithelial-to-mesenchymal transition-like changes in db/db mice.

BACKGROUND: Progressive diabetic nephropathy (DN) is characterized by tubulointerstitial fibrosis that is caused by accumulation of extracellular matrix. Induced by several factors, matrix-producing myofibroblasts may to some extent originate from tubular cells by epithelial-to-mesenchymal transition (EMT). Although previous data document that activation of hypoxia-inducible factor (HIF) signalling can be renoprotective in acute kidney disease, this issue remains controversial in chronic kidney injury. Here, we studied whether DN and EMT-like changes are ameliorated in a mouse model of type 2 diabetes mellitus with increased stability and activity of the HIF. METHODS: We used db/db mice that were crossed with transgenic mice expressing reduced levels of mitogen-activated protein kinase organizer 1 (MORG1), a scaffold protein interacting with prolyl hydroxylase domain 3 (PHD3), because of deletion of one MORG1 allele. RESULTS: We found significantly reduced nephropathy in diabetic MORG1+/- heterozygous mice compared with the diabetic wild-types (db/dbXMORG1+/+). Furthermore, we demonstrated that EMT-like changes in the tubulointerstitium of diabetic wild-type MORG1+/+ mice are present, whereas diabetic mice with reduced expression of MORG1 showed significantly fewer EMT-like changes. CONCLUSIONS: These findings reveal that a deletion of one MORG1 allele inhibits the development of DN in db/db mice. The data suggest that the diminished interstitial fibrosis in these mice is a likely consequence of suppressed EMT-like changes.

Growth arrest specific 2-like protein 1 expression is upregulated in podocytes through advanced glycation end-products.

Background: Growth arrest specific 2-like protein 1 (GAS2L1) protein is a member of the GAS2 family of proteins, known to regulate apoptosis and cellular cytoskeleton reorganization in different cells. Recently we identified that Gas2l1 gene expression in podocytes is influenced by advanced glycation end product-bovine serum albumin(AGE-BSA). Methods: The study was performed employing cultured podocytes and diabetic ( db/db ) mice, a model of type 2 diabetes. Akbuminuria as wellas urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion as measured with specific ELISAs. Gene expression was analysed via semiquantitative and real-time polymerase chain reaction. The protein levels were determined by western blotting and immunostaining. Results: We found that the Gas2l1 α isoform is expressed in podocytes. Treatment with AGE-BSA induced Gas2l1 α and Gas2 mRNA levels compared with controls incubated with non-glycated control BSA (Co-BSA). Moreover, application of the recombinant soluble receptor of AGEs (sRAGE), a competitor of cellular RAGE, reversed the AGE-BSA effect. Interestingly, AGE-BSA also increased the protein levels of GAS2L1α in a RAGE-dependent manner, but did not affect the GAS2 expression. Periodic acid-Schiff staining and albuminuria as well as urinary NGAL excretion revealed that db/db mice progressively developed diabetic nephropathy with renal accumulation of N ε -carboxy-methyl-lysine (immunohistochemistry, western blots). Analyses of GAS2L1α and GAS2 proteins in diabetic mice revealed that both were significantly elevated relative to their non-diabetic littermates. In addition, GAS2L1α and GAS2 proteins positively correlated with the accumulation of AGEs in the blood plasma of diabetic mice and the administration of sRAGE in diabetic mice reduced the glomerular expression of both proteins. Conclusions: We show for the first time that the protein expression of GAS2L1α in vitro and in vivo is regulated by the AGE-RAGE axis. The suppression of AGE ligation with their RAGE in diabetic mice with progressive nephropathy reversed the GAS2L1α expression, thus suggesting a role of GAS2L1α in the development of diabetic disease, which needs to be further elucidated.

Activation of the receptor for advanced glycation end products induces nuclear inhibitor of protein phosphatase-1 suppression.

The activation of the receptor for advanced glycation end products (RAGE) is involved in the development of diabetic nephropathy. Analysis of protein phosphatase-1 indicated that advanced glycation end products did not affect its expression, but increased its phosphatase activity. Using differential display analysis we previously demonstrated that stimulation of RAGE in podocytes modulates the expression of numerous genes, among others nuclear inhibitor of protein phosphatase-1 (NIPP1). Here we found that silencing of NIPP1 induced podocyte hypertrophy, cell cycle arrest, and significantly increased protein phosphatase-1 activity. NIPP1 downregulation was associated with increased p27(Kip1) protein expression. Reporter assays revealed a transcriptional activation of nuclear factor-κB in podocytes after suppression of NIPP1. The protein level of NIPP1 was also significantly reduced in podocytes of diabetic mice. Blocking the RAGE in vivo by a soluble analog elevated the NIPP1 protein in podocytes of diabetic mice. Thus, activation of the RAGE by advanced glycation end products or other ligands suppresses NIPP1 expression in diabetic nephropathy, contributes to podocyte hypertrophy, and glomerular inflammation.

Erythropoietin ameliorates podocyte injury in advanced diabetic nephropathy in the db/db mouse.

Podocyte damage and accumulation of advanced glycation end products (AGEs) are characteristics of diabetic nephropathy (DN). The pathophysiology of AGE-challenged podocytes, such as hypertrophy, apoptosis, and reduced cell migration, is closely related to the induction of the cell cycle inhibitor p27(Kip1) and to the inhibition of neuropilin 1 (NRP1). We have previously demonstrated that treatment with erythropoietin is associated with protective effects for podocytes in vitro. db/db mice with overt DN aged 15-16 wk were treated with either placebo, epoetin-ß, or continuous erythropoietin receptor activator (CERA) for 2 wk. db/db mice compared with nondiabetic db/m control mice revealed the expected increases in body weight, blood glucose, albumin-to-creatinine ratio, and AGE accumulation. Whereas there were no differences in body weight, hyperglycemia and AGEs were observed among diabetic mice that received epoetin-ß compared with CERA and placebo treatment, indicating that epoetin-ß/CERA treatment does not interfere with the development of diabetes in this model. However, the albumin-to-creatinine ratio was significantly lower in db/db mice treated with epoetin-ß or CERA. Furthermore, kidney weights in db/db mice were increased compared with db/m control mice, indicating renal hypertrophy, whereas the increase in renal weight in epoetin-ß- or CERA-treated db/db mice was significantly lower than in placebo-treated control mice. Induction of p27(Kip1) and suppression of NRP1 were significantly reduced in the epoetin-ß treatment group versus the CERA treatment group. Furthermore, erythropoietin treatment diminished the diabetes-induced podocyte loss. Together, independently from hematopoetic effects, epoetin-ß or CERA treatment was associated with protective changes in DN, especially that NRP1 and p27(Kip1) expressions as well as numbers of podocytes returned to normal levels. Our data show, for the first time, that medication of overt DN with erythropoietin for a short time can ameliorate albuminuria and podocyte loss.

The Role of Hypoxia on the Trimethylation of H3K27 in Podocytes.

Epigenetic alterations contribute to the pathogenesis of chronic diseases such as diabetes mellitus. Previous studies of our group showed that diabetic conditions reduce the trimethylation of H3K27 in podocytes in a NIPP1- (nuclear inhibitor of protein phosphatase 1) and EZH2- (enhancer of zeste homolog 2) dependent manner. It has been previously reported that in differentiated podocytes, hypoxia decreases the expression of slit diaphragm proteins and promotes foot process effacement, thereby contributing to the progression of renal disease. The exact mechanisms are, however, not completely understood. The aim of this study was to analyze the role of hypoxia and HIFs (hypoxia-inducible factor) on epigenetic changes in podocytes affecting NIPP1, EZH2 and H3K27me3, in vitro and in vivo. In vivo studies were performed with mice exposed to 10% systemic hypoxia for 3 days or injected with 3,4-DHB (dihydroxybenzoate), a PHD (prolyl hydroxylase) inhibitor, 24 h prior analyses. Immunodetection of H3K27me3, NIPP1 and EZH2 in glomerular podocytes revealed, to the best of our knowledge for the first time, that hypoxic conditions and pharmacological HIFs activation significantly reduce the expression of NIPP1 and EZH2 and diminish H3K27 trimethylation. These findings are also supported by in vitro studies using murine-differentiated podocytes.

Collagen VIII influences epithelial phenotypic changes in experimental diabetic nephropathy.

Epithelial-to-mesenchymal transition (EMT) is an important mechanism of renal tubulo-interstitial fibrosis in diabetic nephropathy (DN). Inducers of EMT, among others, are transforming growth factor-ß(1) (TGF-ß(1)) as well as extracellular collagens. In renal cells of diabetic mice and in kidneys of patients with DN, the expression of collagen VIII (gene: Col8α1/α2) is enhanced and characteristic features of DN in streptozotocin (STZ)-induced diabetic Col8α1/α2 knockout-(KO) mice are attenuated compared with diabetic wild-type mice. This study aimed to investigate whether collagen type VIII may influence the induction of EMT. DN was induced in wild-type and Col8α1/α2-KO mice using the established and widely accepted low-dose STZ model [treatment for 5 consecutive days (50 mg/kg)]. Healthy and diabetic mice were analyzed for changes in renal function and the expression of EMT-related genes and proteins. Renal morphology, fibrosis, and various EMT markers were studied in kidneys using immunohistological and molecular biological methods. Knockout of Col8α1/α2 attenuated albuminuria, extracellular matrix production, as well as fibrosis. Furthermore, the kidneys of diabetic Col8α1/α2-KO mice showed a marked reduction in interstitial myofibroblasts, and in tubular cells the inhibition of the expression of epithelial markers as well as the expression of typical mesenchymal markers was reduced. The present study demonstrates that in contrast to diabetic wild-type mice EMT-like changes were attenuated in diabetic Col8α1/α2-KO mice, which indicates that either collagen VIII may be one of the major inducers of EMT-like changes in kidneys of diabetic wild-type mice or/possibly the lack of Col8α1/α2 disrupts TGF-ß(1)-induced EMT-like changes.

Sex Differences in Diabetes- and TGF-ß1-Induced Renal Damage.

While females are less affected by non-diabetic kidney diseases compared to males, available data on sex differences in diabetic nephropathy (DN) are controversial. Although there is evidence for an imbalance of sex hormones in diabetes and hormone-dependent mechanisms in transforming growth factor ß1 (TGF-ß1) signaling, causes and consequences are still incompletely understood. Here we investigated the influence of sex hormones and sex-specific gene signatures in diabetes- and TGF-ß1-induced renal damage using various complementary approaches (a db/db diabetes mouse model, ex vivo experiments on murine renal tissue, and experiments with a proximal tubular cell line TKPTS). Our results show that: (i) diabetes affects sex hormone concentrations and renal expression of their receptors in a sex-specific manner; (ii) sex, sex hormones and diabetic conditions influence differences in expression of TGF-ß1, its receptor and bone morphogenetic protein 7 (BMP7); (iii) the sex and sex hormones, in combination with variable TGF-ß1 doses, determine the net outcome in TGF-ß1-induced expression of connective tissue growth factor (CTGF), a profibrotic cytokine. Altogether, these results suggest complex crosstalk between sex hormones, sex-dependent expression pattern and profibrotic signals for the precise course of DN development. Our data may help to better understand previous contradictory findings regarding sex differences in DN.

Flow cytometric quantification of chlamydial infection in cell culture.

A flow cytometric method was developed, which allows fast and efficient analysis of cell cultures infected with chlamydiae. The proportion of positive cells increased with the infectious dose and correlated with chlamydia copy numbers calculated from real-time PCR. While retaining the advantages of single-cell analysis, flow cytometry allows handling of large sample numbers and counterstaining for additional marker proteins.