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.

Cloning of the Human MORG1 Promoter: Differential Regulation by Hypoxia and Prolyl-Hydroxylase Inhibitors.

MAPK-organizer 1 (MORG1) is a molecular scaffold for prolyl-hydroxylase-3 containing a domain (PHD3) protein linking MORG1 to mechanisms of adaptation in hypoxic conditions. In this paper, we report the cloning of the promoter region of the murine and human MORG1 gene. Among other transcriptional factors binding sites, we identified that both (mouse and human) promoter regions contained several putative hypoxia-inducible factor binding motifs. Analyses of the human MORG1 promoter by reporter assays revealed that hypoxia and pharmacological inhibitors of prolyl-hydroxylases under in vitro conditions in HEK 293 cells differentially regulate the MORG1 promoter reporter activity. The exposure of the cells to 10% hypoxia showed inhibition of MORG1 promotor activity at 6 and 12 h, but stimulation after 24 h while treated with prolyl-hydroxylase inhibitors led to a time-independent MORG1 promoter activation. Mutational analyses of the individual HIF binding sites on human MORG1 promoter suggest that the binding sites work in a complex corporation because single mutations were not sufficient to abolish completely the MORG1 reporter activation by PHD inhibitors. Our data provide the first evidence that not only MORG1 regulate HIF stabilization through a PHD complex, but also that, vice versa, HIFs control MORG1 expression directly or indirectly by a complex regulatory mechanism.

Ramipril pretreatment worsened renal injury and survival despite a reduction in renal inflammation in experimentally induced sepsis in mice.

INTRODUCTION: The angiotensin converting enzyme inhibitor ramipril is a standard antihypertensive therapy for many patients. Because angiotensin II may promote inflammation, we were interested in whether basal pretreatment with ramipril may modify renal function and inflammation as well as systemic outcome in experimentally induced sepsis in mice. MATERIAL AND METHODS: Ramipril (10 mg/kg/day) pretreatment or placebo (NaCl) was given intraperitoneally for 5 days to C57BL6/J mice, followed by either sham operation or cecal ligation and puncture sepsis induction. Real-time polymerase chain reaction and immunological stains were used to evaluate renal gene and protein expression, respectively. Plasma creatinine, neutrophil-gelatinase associated lipocalin, and blood urea nitrogen were used as markers for renal function. A clinical severity score was determined. RESULTS: Administration of ramipril before cecal ligation and puncture surgery was associated with reduced renal inflammation but did not improved renal function and structure and even worsened the clinical status of septic mice. CONCLUSIONS: The data suggest that the effects of ramipril pretreatment are complex. Additional studies including monitoring of hemodynamic parameters are necessary to elucidate the exact mechanism(s) of this observation. In addition, the timing of the ramipril administration could be of importance.

Phenotypic and functional differences between senescent and aged murine microglia.

Microglia, the key innate immune cells in the brain, have been reported to drive brain aging and neurodegenerative disorders; however, few studies have analyzed microglial senescence and the impact of aging on the properties of microglia. In the present study, we characterized senescence- and aging-associated phenotypes of murine brain microglia using well-accepted markers, including telomere length, telomerase activity, expression of p16INK4a, p21, p53, senescence-associated ß-galactosidase, and a senescence-associated secretory phenotype. Quantitative real-time polymerase chain reaction analysis and a Telomeric Repeat Amplification Protocol assay indicated shortened telomeres and increased telomerase activity in senescent microglia, whereas telomeres remained unaltered and telomerase activity was reduced in aged microglia. Senescent microglia upregulated p16INK4a, p21, and p53, whereas acutely isolated microglia from the aged brain only exhibited a modest upregulation of p16INK4a. Senescent microglia showed decreased proliferation, while it was unchanged in aged microglia. Furthermore, microglia at late passages strongly upregulated expression of the senescent marker senescence-associated ß-galactosidase. Senescent and aged microglia exhibited differential activation profiles and altered responses to stimulation. We conclude that microglia from the aged mouse brain do not show typical senescent changes because their phenotype and functional response strongly differ from those of senescent microglia in vitro.

Cell cycle-dependent and -independent telomere shortening accompanies murine brain aging.

Replication-based telomere shortening during lifetime is species- and tissue-specific, however, its impact on healthy aging is unclear. In particular, the contribution of telomere truncation to the aging process of the CNS, where replicative senescence alone fails to explain organ aging due to low to absent mitotic activity of intrinsic populations, is undefined. Here, we assessed changes in relative telomere length in non-replicative and replicative neural brain populations and telomerase activity as a function of aging in C57BL/6 mice. Telomeres in neural cells and sub-selected neurons shortened with aging in a cell cycle-dependent and -independent manner, with preponderance in replicative moieties, implying that proliferation accelerates, but is not prerequisite for telomere shortening. Consistent with this telomere erosion, telomerase activity and nuclear TERT protein were not induced with aging. Knockdown of the Rela subunit of NF-κB, which controls both telomerase enzyme and subcellular TERT protein allocation, did also not influence telomerase activity or telomere length, in spite of its naive up-regulation selectively under aging conditions. We conclude that telomere instability is intrinsic to physiological brain aging beyond cell replication, and appears to occur independently of a functional interplay with NF-κB, but rather as a failure to induce or relocate telomerase.

MORG1+/- mice are protected from histological renal damage and inflammation in a murine model of endotoxemia.

BACKGROUND: The MAPK-organizer 1 (MORG1) play a scaffold function in the MAPK and/or the PHD3 signalling paths. Recently, we reported that MORG1+/- mice are protected from renal injury induced by systemic hypoxia and acute renal ischemia-reperfusion injury via increased hypoxia-inducible factors (HIFs). Here, we explore whether MORG1 heterozygosity could attenuate renal injury in a murine model of lipopolysaccharide (LPS) induced endotoxemia. METHODS: Endotoxemia was induced in mice by an intraperitoneal (i.p) application of 5 mg/kg BW LPS. The renal damage was estimated by periodic acid Schiff's staining; renal injury was evaluated by detection of urinary and plasma levels of neutrophil gelatinase-associated lipocalin and albumin/creatinine ratio via ELISAs. Renal mRNA expression was assessed by real-time PCR, whereas the protein expression was determined by immunohistochemistry or Western blotting. RESULTS: LPS administration increased tubular injury, microalbuminuria, IL-6 plasma levels and renal TNF-α expression in MORG1 +/+ mice. This was accompanied with enhanced infiltration of the inflammatory T-cells in renal tissue and activation of the NF-κB transcription factors. In contrast, endotoxemic MORG1 +/- showed significantly less tubular injury, reduced plasma IL-6 levels, significantly decreased renal TNF-α expression and T-cells infiltration. In support, the renal levels of activated caspase-3 were lower in endotoxemic MORG1 +/- mice compared with endotoxemic MORG1 +/+ mice. Interestingly, LPS application induced a significantly higher accumulation of renal HIF-2α in the kidneys of MORG1+/- mice than in wild-type mice, accompanied with a diminished phosphorylation of IκB-α and IKK α,ß and decreased iNOS mRNA in the renal tissues of the LPS-challenged MORG1+/- mice, indicating an inhibition of the NF-κB transcriptional activation. CONCLUSIONS: MORG1 heterozygosity protects against histological renal damage and shows anti-inflammatory effects in a murine endotoxemia model through modulation of HIF-2α stabilisation and/or simultaneous inhibition of the NF-κB signalling. Here, we show for the first time that MORG1 scaffold could represent the missing link between innate immunity and inflammation.

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.

Vitamin D3 Partly Antagonizes Advanced-Glycation Endproducts-Induced NFκB Activation in Mouse Podocytes.

BACKGROUND/AIMS: We have previously shown that advanced glycation-endproducts (AGEs) induced NFκB activation in differentiated mouse podocytes. This NFκB activation may contribute to the progression of renal disease and mediation of fibrosis by various mechanisms. This study was undertaken to test whether this detrimental response may be reversed by vitamin D3 or its analogue paricalcitol. METHODS: Differentiated mouse podocytes were challenged with glycated bovine serum albumin (AGE-BSA), or non-glycated control BSA (in the presence or absence of various concentrations of vitamin D3 (decostriol, 1α,25-dihydroxyvitamin D3)) or its active analog paricalcitol. Quantitative mRNA expressions were measured by real-time PCR, whereas protein expressions were determined by Western blotting followed by densitometry. Cytoplasmic and nuclear protein expression of the NFκB subunit p65 (Rel A) were determined by Western blotting. Furthermore, the ratio of phosphorylated to non-phosphorylated IκB-α was measured using specific antibodies. Electrophoretic mobility shift assays and a capture ELISA assay were used to assess NFκB transactivation in vitro. In addition, NFκB transactivation was also monitored in HEK-NFκBIA reporter cells using live cell luminometry. RESULTS: Podocytes expressed the receptor for vitamin D. The vitamins did not suppress receptor for AGEs (RAGE) expression; instead, they rather upregulated RAGE. Although vitamin D3 and paricalcitol partly and differentially modified some of the studied parameters, both hormones inhibited AGE-BSA-induced NFκB transactivation, presumably by various mechanisms including the upregulation of IκB-α protein, keeping NFκB sequestered in an inactive state in the cytoplasm. CONCLUSION: Vitamin D3 or its analog paricalcitol partly prevented AGE-mediated NFκB activation, an important feature of diabetic nephropathy (DN). Whether this in vitro finding is of clinical relevance to prevent/treat DN requires further studies.

Preconditioned suppression of prolyl-hydroxylases attenuates renal injury but increases mortality in septic murine models.

BACKGROUND: Septic conditions contribute to tissue hypoxia, potentially leading to multiple organ failure, including acute kidney injury. The regulation of cellular adaptation to low oxygen levels is regulated by hypoxia-inducible transcription factors (HIFs). While the role of HIFs in ischaemia/reperfusion is more studied, their function in sepsis-induced renal injury is not well characterized. In this study, we investigated whether pharmacological activation of HIFs by suppression of prolyl-hydroxylases (PHDs) protects against septic acute kidney injury. METHODS: Two models of sepsis-caecal ligation and punction and peritoneal contamination and infection-were induced on 12-week-old C57BL6/J mice. Pharmacological inhibition of PHDs, leading to HIF activation, was achieved by intraperitoneal application of 3,4-dihydroxybenzoate (3,4-DHB) before sepsis. A quantitative real-time reverse transcription polymerase chain reaction, immunohistology and enzyme-linked immunosorbent assays were utilized to detect gene expression, renal protein levels and renal functional parameters, respectively. Tissue morphology was analysed by periodic acid-Schiff reaction. Early kidney injury was estimated by kidney injury molecule-1 analyses. Apoptosis was detected in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling stain. The systemic effect of 3,4-DHB pretreatment in sepsis was analysed by 72-h survival studies. RESULTS: Pharmacological activation of HIFs before sepsis induction attenuated sepsis-related vacuolization and dilation of the proximal tubules, reduced tubular apoptosis and correlated to lower T-cell infiltration in renal tissue compared with the non-treated septic animals. PHD suppression elevated the basal renal HIF-1α expression and basal plasma concentrations of HIF targets erythropoietin and vascular endothelial growth factor. Whereas it preserved renal structure in both models, it improved renal function in a model-dependent manner. Moreover, inhibition of PHDs led to increased mortality in both models. Analysis of liver function showed increased organ destruction with massive glycogen loss and hepatocyte's apoptosis due to 3,4-DHB administration before sepsis induction. CONCLUSIONS: In summary, the pharmacological activation of HIFs by 3,4-DHB administration, although it showed renoprotective effects in sepsis-related kidney injury, induced more severe problems in other organs such as the liver during sepsis, leading to increased mortality.

Role of Neuropilin-1 in Diabetic Nephropathy.

Diabetic nephropathy (DN) often develops in patients suffering from type 1 or type 2 diabetes mellitus. DN is characterized by renal injury resulting in proteinuria. Neuropilin-1 (NRP-1) is a single-pass transmembrane receptor protein devoid of enzymatic activity. Its large extracellular tail is structured in several domains, thereby allowing the molecule to interact with multiple ligands linking NRP-1 to different pathways through its signaling co-receptors. NRP-1's role in nervous system development, immunity, and more recently in cancer, has been extensively investigated. Although its relation to regulation of apoptosis and cytoskeleton organization of glomerular vascular endothelial cells was reported, its function in diabetes mellitus and the development of DN is less clear. Several lines of evidence demonstrate a reduced NRP-1 expression in glycated-BSA cultured differentiated podocytes as well as in glomeruli from db/db mice (a model of type 2 Diabetes) and in diabetic patients diagnosed with DN. In vitro studies of podocytes implicated NRP-1 in the regulation of podocytes' adhesion to extracellular matrix proteins, cytoskeleton reorganization, and apoptosis via not completely understood mechanisms. However, the exact role of NRP-1 during the onset of DN is not yet understood. This review intends to shed more light on NRP-1 and to present a link between NRP-1 and its signaling complexes in the development of DN.

Reactive oxygen species in diabetic nephropathy: friend or foe?

Based on the numerous cellular and animal studies over the last decades, it has been postulated that reactive oxygen species (ROS) are important secondary messengers for signalling pathways associated with apoptosis, proliferation, damage and inflammation. Their adverse effects were considered to play a leading role in the onset and progression of type 1 and type 2 diabetes mellitus as well as in the complication of diabetic disease leading to vascular-, cardiac-, neuro-degeneration, diabetic retinopathy and diabetic nephropathy. All these complications were mostly linked to the generation of the superoxide anion, due to a prolonged hyperglycaemia in diabetes, and this anion was almost 'blamed for everything', despite the fact that its measurement and detection in life systems is extremely complicated due to the short lifespan of the superoxide anion. Therefore, a tremendous amount of research has been focused on finding ways to suppress ROS production. However, a recent report from Dugan et al. shed new insights into the life detection of superoxide generation in diabetes and raised the question of whether we treat the diabetes-related complications correctly or the target is somewhat different as thought. This review will focus on some aspects of this novel concept for the role of ROS in diabetic nephropathy.

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.

Advanced glycated end-products affect HIF-transcriptional activity in renal cells.

Advanced glycated end-products (AGEs) are ligands of the receptor for AGEs and increase in diabetic disease. MAPK organizer 1 (Morg1) via its binding partner prolyl-hydroxylase domain (PHD)-3 presumably plays a role in the regulation of hypoxia-inducible factor (HIF)-1α and HIF-2α transcriptional activation. The purpose of this study was to analyze the influence of AGEs on Morg1 expression and its correlation to PHD3 activity and HIF-transcriptional activity in various renal cell types. The addition of glycated BSA (AGE-BSA) significantly up-regulated Morg1 mRNA levels in murine mesangial cells and down-regulated it in murine proximal tubular cells and differentiated podocytes. These effects were reversible when the cells were preincubated with a receptor for α-AGE antibody. AGE-BSA treatment induced a relocalization of the Morg1 cellular distribution compared with nonglycated control-BSA. Analysis of PHD3 activity demonstrated an elevated PHD3 enzymatic activity in murine mesangial cells but an inhibition in murine proximal tubular cells and podocytes after the addition of AGE-BSA. HIF-transcriptional activity was also affected by AGE-BSA treatment. Reporter gene assays and EMSAs showed that AGEs regulate HIF- transcriptional activity under nonhypoxic conditions in a cell type-specific manner. In proximal tubular cells, AGE-BSA stimulation elevated mainly HIF-1α transcriptional activity and to a lesser extent HIF-2α. We also detected an increased expression of the HIF-1α and the HIF-2α proteins in kidneys from Morg1 heterozygous (HZ) placebo mice compared with the Morg1 wild-type (WT) placebo-treated mice, and the HIF-1α protein expression in the Morg1 HZ streptozotocin-treated mice was significantly higher than the WT streptozotocin-treated mice. Analysis of isolated mesangial cells from Morg1 HZ (±) and WT mice showed an inhibited PHD3 activity and an increased HIF-transcriptional activity in cells with only one Morg1 allele. These findings are important for a better understanding of the molecular mechanisms of diabetic nephropathy.

Angiotensin II differentially regulates Morg1 expression in kidney cells.

BACKGROUND: The mitogen-activated protein kinase organizer 1 (Morg1) belongs to the WD-40 repeat protein family and is a scaffold molecule for the extracellular regulated kinase signaling pathway. Morg1 also binds to prolyl-hydroxylase 3 (PHD3) and regulates the hypoxia-inducible factor-1α (HIF-1α) expression via PHD3 stabilization. Morg1 has been detected in the kidney as well as in other cell tissues but its expression in renal cells has not been well investigated. It has been widely shown that angiotensin II (ANG II) mediates renal damage. We have previously shown that ANG II downregulates the expression of PHD3 in PC12 cells. The aim of this study was to analyze whether ANG II regulates Morg1 expression in mouse mesangial cells (MMC), mouse proximal tubular cells (MTC) and in differentiated podocytes. The correlation between the expression of Morg1 and PHD3 activity was also addressed. METHODS: Effect of ANG II on the Morg1 mRNA expression level was assessed by real-time PCR. Morg1 and HIF-1α cellular localization was analyzed by immunohistochemistry. HIF-1α promoter activity was investigated using a reporter gene system. PHD3 hydroxylase activity test was measured with a hydroxylation-coupled decarboxylation assay. RESULTS: ANG II differentially regulates Morg1 expression in MMC, MTC and differentiated podocytes. We detected a biphasic effect of ANG II on Morg1 mRNA expression which was time dependent. While 9-hour ANG II treatment downregulated Morg1 expression in MMC, it induced Morg1 expression in MTC. Conversely, 24-hour ANG II stimulation upregulated the expression of Morg1 mRNA in MMC, but showed an opposite effect in MTC and differentiated podocytes. In addition, we found that ANG II signals mostly through the AT(1) receptor subtype in MMC and via the AT(2) subtype in MTC. PHD3 activity correlated to Morg1 expression patterns. Our data also demonstrate that HIF-1α transcriptional activity in MTC contrasted to PHD3 activity at 9 and 24 h, whereas in the MMC and in podocytes we did not find any correlation between PHD3 HIF-1α hydroxylation ability and HIF-1α transcriptional activation, suggesting a different mechanism of regulation in these cell types. Interestingly, the reduced expression of Morg1 in mesangial cells isolated from Morg1 (+/-) heterozygous mice correlated with a reduced PHD3 enzymatic activity and an increased HIF-1α transcriptional activity compared with mesangial cells originated from wild-type (Morg1 +/+) mice. CONCLUSIONS: We show for the first time in various renal cells that ANG II modulates Morg1 expression and HIF-1α transcriptional activity via cell type-specific mechanisms, demonstrating a novel mechanism by which ANG II may contribute to renal disease.

Advanced glycation end products inhibit adhesion ability of differentiated podocytes in a neuropilin-1-dependent manner.

Podocyte injury can occur by a number of stimuli. Maintaining of an intact podocyte structure is essential for glomerular filtration; therefore, podocyte damage severely impairs renal function. Recently, we have reported that addition of glycated BSA [advanced glycation end products (AGE)-BSA] to differentiated murine podocytes inhibited neuropilin-1 (NRP1) expression and dramatically influenced podocyte migration ability (Bondeva T, Ruster C, Franke S, Hammerschmid E, Klagsbrun M, Cohen CD, Wolf G. Kidney Int 75: 605-616, 2009; Bondeva T, Wolf G. Am J Nephrol 30: 336-345, 2009). The present study analyzes the influence of AGEs and NRP1 on podocyte adhesion and cytoskeleton reorganization. We show that treatment with AGE-BSA significantly reduced podocyte adhesion to collagen IV, laminin, and fibronectin compared with Co-BSA (nonglycated BSA)-incubated cells, which was further augmented by transient inhibition of NRP1 expression using NRP1 short interference (si) RNA. On the other hand, forced overexpression of NRP1 markedly increased the adhesion ability of podocytes to the ECMs despite the AGE-BSA treatment. No changes were observed when podocyte adhesion to collagen I was assayed. These findings were also manifested with disorganization of podocyte actin stress fibers and decreased lamellipodia formation processes due to AGE-BSA treatment or NRP1 suppression. In addition, AGE-BSA or suppression of NRP1 both reduced the phosphorylation of focal adhesion kinase (FAK) and Erk1/2 in PMA-stimulated differentiated podocytes. Analysis of RhoA family GTPase activity demonstrated that treatment with AGE-BSA or NRP1 depletion inhibited as well the activation of the Rac-1 and Cdc42 but did not affect RhoA activity. All these effects were reversed by forced overexpression of full-length NRP1 cloned into the pcDNA3 vector in differentiated podocytes. Our study demonstrates that AGEs, in part via suppression of NRP1 expression, decreased podocyte adhesion and contribute to reduction of Rac-1 and Cdc42 GTPase activity. These effects may be further responsible for the podocytes damage and loss in diabetic nephropathy. Our findings suggest a role for NRP1 in regulating the podocyte actin cytoskeleton, and therefore reduction of NRP1 expression could be critical for podocyte function.

Erythropoietin protects podocytes from damage by advanced glycation end-products.

BACKGROUND: Podocyte damage and accumulation of advanced glycation end-products (AGEs) are implicated in the development and progression of diabetic nephropathy. We have previously shown that changes in podocyte pathophysiology, such as hypertrophy and reduced migration, are closely linked with the induction of the cell cycle inhibitor p27(Kip1) and a decrease in neuropilin-1 (NRP1) expression. We investigated whether the erythropoietin receptor activators CERA and epoetin-ß may prevent AGE-mediated changes in podocytes. METHODS: Differentiated mouse podocytes in culture were challenged by AGE-modified bovine serum albumin (BSA) or control BSA in the presence or absence of CERA as well as epoetin-ß. Cell cycle analysis and determination of apoptosis markers were performed. p27(Kip1) and NRP1 expression was measured by RT-PCR and Western blots. RESULTS: Differentiated mouse podocytes in culture expressed erythropoietin receptors which were phosphorylated after incubation with CERA or epoetin-ß. CERA or epoetin-ß prevented the p27(Kip1)-dependent cell cycle arrest and cellular hypertrophy induced by AGE-BSA incubation. Furthermore, the p27(Kip1)-dependent AGE-BSA-induced decrease in cell viability and decrease in cell proliferation was ameliorated in the presence of CERA or epoetin-ß. Following erythropoietin treatment, AGE-BSA failed to further reduce NRP1 expression, resulting in improved podocyte migration. CONCLUSION: Treatment with the erythropoietin receptor activators epoetin-ß or CERA protected podocytes from AGE-BSA-mediated damage via an effect on p27(Kip1) and NRP1 expression. Consequently, early treatment with erythropoietin may help to prevent diabetic nephropathy.

Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells.

INTRODUCTION: Advanced glycation end products (AGEs) have been introduced to be involved in the pathogenesis of osteoarthritis (OA). The influence of AGEs on osteoarthritic fibroblast-like synovial cells (FLS) has been incompletely understood as yet. The present study investigates a potential influence of AGE-modified bovine serum albumin (AGE-BSA) on cell growth, and on the expression of proinflammatory and osteoclastogenic markers in cultured FLS. METHODS: FLS were established from OA joints and stimulated with AGE-BSA. The mRNA expression of p27Kip1, RAGE (receptor for AGEs), nuclear factor kappa B subunit p65 (NFkappaB p65), tumor necrosis factor alpha (TNF-alpha, interleukin-6 (IL-6), receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin was measured by real-time PCR. The respective protein expression was evaluated by western blot analysis or ELISA. NFkappaB activation was investigated by luciferase assay and electrophoretic mobility shift assay (EMSA). Cell cycle analysis, cell proliferation and markers of necrosis and early apoptosis were assessed. The specificity of the response was tested in the presence of an anti-RAGE antibody. RESULTS: AGE-BSA was actively taken up into the cells as determined by immunohistochemistry and western blots. AGE-induced p27Kip1 mRNA and protein expression was associated with cell cycle arrest and an increase in necrotic, but not apoptotic cells. NFkappaB activation was confirmed by EMSAs including supershift experiments. Anti-RAGE antibodies attenuated all AGE-BSA induced responses. The increased expression of RAGE, IL-6 and TNF-alpha together with NFkappaB activation indicates AGE-mediated inflammation. The decreased expression of RANKL and osteoprotegerin may reflect a diminished osteoclastogenic potential. CONCLUSIONS: The present study demonstrates that AGEs modulate growth and expression of genes involved in the pathophysiological process of OA. This may lead to functional and structural impairment of the joints.

Advanced glycation end products suppress neuropilin-1 expression in podocytes by a reduction in Sp1-dependent transcriptional activity.

BACKGROUND: Neuropilin-1 (NRP1) is a transmembrane glycoprotein, initially defined as a receptor for members of the semaphorin family. We observed that NRP1 expression was downregulated by the addition of advanced glycation end products-modified bovine serum albumin (AGE-BSA). The present study was undertaken to unravel the molecular mechanisms underlying AGE-BSA-mediated NRP1 suppression. METHODS: Expression of NRP1 was analyzed in podocytes. The transcriptional activity of the NRP1 promoter was investigated using wild-type and mutant NRP1 promoter reporter constructs. Electrophoretic mobility assays were performed. RESULTS: NRP1 expression was downregulated in podocytes by the addition of AGE-BSA. In contrast, phorbolester induced NRP1 mRNA and protein expression. The wild-type promoter transcriptional activity was significantly reduced when podocytes were treated with AGE-BSA compared with control, unmodified BSA. Point mutations of proximal and distal Sp1-like sites inhibited basal NRP1 promoter activity. AGE-BSA failed to further suppress transcriptional activity of these constructs. Double mutation of the Sp1A and Sp1B binding sites completely abolished NRP1 transcriptional activity. Gel shift analysis showed a specific binding of the Sp1 transcription factor. Treatment of podocytes with AGE-BSA revealed a decrease in Sp1 binding to consensus sequences, but no effect on AP1 binding. CONCLUSIONS: AGE-BSA inhibits NRP1 promoter transcriptional activity in podocytes by reducing the binding ability of the Sp1 transcription factor to attach to the NRP1 promoter.

Angiotensin II upregulates RAGE expression on podocytes: role of AT2 receptors.

BACKGROUND: Advanced glycation end products (AGEs) play an important role in diabetic nephropathy. The receptor for AGEs, called RAGE, is present on podocytes. We investigated whether angiotensin II (ANG II) modulates RAGE expression on cultured differentiated podocytes. RESULTS: Cultured podocytes expressed AT1 and AT2 receptors. Surprisingly, ANG II induced RAGE mRNA and protein expression through AT2 receptors. ANG II had no influence on proliferation or protein content of podocytes. The increase in RAGE expression depended on stimulated transcriptional activity. Using various mutant reporter constructs of the RAGE promoter region, it was shown that a NF-kappaB binding site at -1519 was essential for ANG II-induced transcriptional activity. Preincubation with ANG II increased the expression of tumor necrosis factor-alpha mRNA and protein expression induced by AGE, indicating that the ANG II-mediated upregulation of RAGE has functional consequences. AGE-BSA was incorporated into cells as measured by Western blots for N epsilon-carboxymethyllysine, but ANG II did not influence this process. ANG II in the absence or presence of AGE-BSA did not induce apoptosis of podocytes. CONCLUSION: Our study revealed aninteraction between the renin-angiotensin system and the AGE/RAGE axis in podocytes. Since intraglomerular ANG II levels are increased in diabetic nephropathy, this interaction may have pathophysiological consequences for podocyte injury and inflammation associated with the development of diabetic nephropathy.

Advanced glycation end-products suppress neuropilin-1 expression in podocytes.

Advanced glycation end products (AGEs) have been linked to the pathogenesis of diabetic nephropathy. Here we tested the effect of AGE-modified bovine serum albumin (AGE-BSA) on differentiated mouse podocytes in culture. Differential display and real-time PCR analyses showed that in addition to neuropilin-1, the entire signaling receptor complex of neuropilin-2, semaphorin-3A, and plexin-A1, was significantly reduced by AGE-BSA as was neuropilin-1 protein. The effect was specific for podocytes compared to isolated mesangial and tubular epithelial cells. Further, AGE-BSA was not toxic to podocytes. Neuropilin-1 expression was decreased in glomeruli of diabetic db/db mice compared to their non-diabetic littermates. Transcripts of both neuropilins were found to be decreased in renal biopsies from patients with diabetic nephropathy compared to transplant donors. Podocyte migration was inhibited by AGE-BSA with similar results found in the absence of AGE-BSA when neuropilin-1 expression was down-regulated by siRNA. In contrast, podocyte migration was stimulated by overexpression of neuropilin-1 even in the presence of AGE-BSA. Our study shows that AGE-BSA inhibited podocyte migration by down-regulating neuropilin-1. The decreased migration could lead to adherence of uncovered areas of the glomerular basement membrane to Bowman's capsule contributing to focal glomerulosclerosis.