Overexpression of BMP­7 reverses TGF­ß1­induced epithelial­mesenchymal transition by attenuating the Wnt3/ß­catenin and TGF-ß1/Smad2/3 signaling pathways in HK­2 cells.

Tubular epithelial cells undergoing epithelial­mesenchymal transition (EMT) is a crucial event in the progression of renal interstitial fibrosis (RIF). Bone morphogenetic protein­7 (BMP­7) has been reported to exhibit anti­fibrotic functions in various renal diseases. However, the function of BMP­7 in regulating EMT and the progression of RIF remains largely unknown. The aim of the present study was to examine the potential effect of BMP­7 on transforming growth factor ß1 (TGF­ß1)­induced EMT and the underlying mechanisms by which BMP­7 exerted its effects. Human renal proximal tubular epithelial cells (HK­2) were treated with TGF­ß1 for various time periods and at various concentrations and lentiviral vectors were used to overexpress BMP­7. Cell Counting Kit­8 and Transwell assays were used to evaluate the viability and migration of HK­2 cells in vitro. EMT was estimated by assessing the changes in cell morphology and the expression of EMT markers. In addition, the activation of the Wnt3/ß­catenin and TGF­ß1/Smad2/3 signaling pathways were analyzed using western blotting. TGF­ß1 induced EMT in a time­ and dose­dependent manner in HK­2 cells. Treatment with TGF­ß1 induced morphological changes, decreased cell viability and the expression of E­cadherin, increased cell migration and the expression of α­smooth muscle actin, fibroblast­specific protein 1, collagen I and vimentin, and activated the Wnt3/ß­catenin and TGF­ß1/Smad2/3 signaling pathways in HK­2 cells. However, BMP­7 overexpression notably reversed all these effects. These results suggest that BMP­7 effectively suppresses TGF­ß1­induced EMT through the inhibition of the Wnt3/ß­catenin and TGF­ß1/Smad2/3 signaling pathways, highlighting a potential novel anti­RIF strategy.

Erythropoietin Protects Erythrocytes Against Oxidative Stress-Induced Eryptosis In Vitro.

BACKGROUND: Excessive eryptosis has been found in maintained hemodialysis or peritoneal dialysis patients. Signaling of triggering eryptosis includes oxidative stress, increased cytosolic Ca2+-activity, and ceramide. Erythropoietin (EPO) possesses the property of an antioxidant. The aim of this study was to investigate the ability of hydrogen peroxide (H2O2) on erythrocytes in vitro, and to assess the possible effects of recombinant human erythropoietin (rhEPO) on eryptosis. METHODS: One percent erythrocyte suspension was cultured in vitro in three kinds of media: Control group (Group C), H2O2 group (Group H), and EPO group (Group E). Erythrocytes were sampled at 24 hours and 60 hours. Phosphatidylserine (PS) was estimated with annexin-V, reactive oxygen species (ROS) with 2',7'-dichlorodihydrofuorescein diacetate (DCFDA) and cytosolic Ca2+ activity ([Ca2+]i) with Fluo3. RESULTS: Eryptosis in Group C increased as the incubating time extended (2.05 ± 0.06 at 24 hours, and 10.00 ± 0.08 at 60 hours). Eryptosis increased in Group H compared with Group C (10.86 ± 0.06 at 24 hours, p < 0.01; 12.46 ± 0.14 at 60 hours, p < 0.01, respectively), while it decreased in Group E compared with Group H (8.80 ± 0.08 at 24 hours, p < 0.01; 11.29 ± 0.04 at 60 hours, p < 0.01, respectively). Meanwhile, ROS increased in Group H compared with Group C (9.37 ± 0.04 versus 5.49 ± 0.09 at 24 hours, p < 0.01;19.82 ± 0.05 versus 13.51 ± 0.10 at 60 hours, p < 0.01). [Ca2+]i increased in Group H compared with Group C (10.91 ± 0.12 versus 2.53 ± 0.06 at 24 hours, p < 0.01;14.55 ± 0.05 versus 4.63 ± 0.08 at 60 hours, p < 0.01). ROS decreased in Group E compared with Group H (6.80 ± 0.05 at 24 hours, p < 0.01; 16.82 ± 0.06 at 60 hours, p < 0.01). [Ca2+]i decreased in Group E compared with Group H (7.63 ± 0.14 at 24 hours, p < 0.01; 10.72 ± 0.07 at 60 hours, p < 0.01). CONCLUSIONS: Our research showed eryptosis was triggered by H2O2 and paralleled by increased ROS and [Ca2+]i which was partially reversed by EPO. It indicated that EPO could protect erythrocytes against oxidative stress-induced eryptosis.

Resveratrol ameliorates hyperglycemia-induced renal tubular oxidative stress damage via modulating the SIRT1/FOXO3a pathway.

AIMS: Oxidative stress plays an important role in the development and progression of diabetic nephropathy (DN). We aimed to investigate if resveratrol (RSV) could ameliorate hyperglycemia-induced oxidative stress in renal tubules via modulating the SIRT1/FOXO3a pathway. METHODS: The effects of RSV on diabetes rats were assessed by periodic acid-Schiff, Masson staining, immunohistochemistry, and western blot analyses. Additionally, oxidative indicators (such as catalase, superoxide dismutase, reactive oxygen species, and malondialdehyde), the deacetylase activity of SIRT1 and protein expressions of SIRT1, FOXO3a, and acetylated-FOXO3a were measured. These indicators were similarly evaluated in an in vitro study. Furthermore, the silencing of SIRT1 was used to confirm its role in the resistance to oxidative stress and the relationship between SIRT1 and FOXO3a in vitro. RESULTS: After 16weeks of RSV treatment, the renal function and glomerulosclerosis of rats with DN was dramatically ameliorated. RSV treatment increased SIRT1 deacetylase activity, subsequently decreasing the expression of acetylated-FOXO3a and inhibiting the oxidative stress caused by hyperglycemia both in vivo and in vitro. The silencing of SIRT1 in HK-2 cells aggravated the high glucose-induced oxidative stress and overexpression of acetylated-FOXO3a; RSV treatment failed to protect against these effects. CONCLUSIONS: RSV modulates the SIRT1/FOXO3a pathway by increasing SIRT1 deacetylase activity, subsequently ameliorating hyperglycemia-induced renal tubular oxidative stress damage. This mechanism provides the basis for a new approach to developing an effective DN treatment, which is of great clinical significance for reducing the morbidity and mortality associated with DN.

Adipose-derived stem cells ameliorate renal interstitial fibrosis through inhibition of EMT and inflammatory response via TGF-ß1 signaling pathway.

Adipose-derived stem cells (ADSCs) have been successfully used to treat acute kidney injury or acute renal failure. However, the effect of ADSCs on treating renal interstitial fibrosis remains unknown. Here, we assessed the therapeutic efficacy of ADSCs on renal interstitial fibrosis induced by unilateral ureter obstruction (UUO) and explored the potential mechanisms. After 7days of UUO, rats were injected with ADSCs (5×106) or vehicle via tail vein. We found that ADSCs administration significantly ameliorated renal interstitial fibrosis, the occurrence of epithelial-mesenchymal transition (EMT) and inflammatory response. Furthermore, ADSCs administration could inhibit the activation of transforming growth factor-ß1 (TGF-ß1) signaling pathway, which might play a crucial role in renal interstitial fibrosis of the UUO model rats. These results suggested that ADSCs treatment attenuates renal interstitial fibrosis possibly through inhibition of EMT and inflammatory response via TGF-ß1 signaling pathway. Therefore, ADSCs may be an effective therapeutic strategy for the treatment of renal interstitial fibrosis.

Identification and isolation of kidney-derived stem cells from transgenic rats with diphtheria toxin-induced kidney damage.

Adult stem cells have been well characterized in numerous organs, with the exception of the kidneys. Therefore, the present study aimed to identify and isolate kidney-derived stem cells. A total of 12 Fischer 344 transgenic rats expressing the human diphtheria toxin receptor in podocyte cells of the kidney, were used in the present study. The rats were administered 5-bromo-2'-deoxyuridine (BrdU) in order to detect cellular proliferation. After 60 days, the rats were treated with the diphtheria toxin (DT), in order to induce kidney injury. Immunohistochemical analysis indicated that the number of BrdU-positive cells were increased following DT treatment. In addition, the expression of octamer-binding transcription factor 4 (Oct-4), a stem cell marker, was detected and suggested that kidney-specific stem cells were present in the DT-treated tissue samples. Furthermore, tissue samples exhibited repair of the DT-induced injury. Further cellular culturing was conducted in order to isolate the kidney-specific stem cells. After 5 weeks of culture, the majority of the cells were non-viable, with the exception of certain specialized, unique cell types, which were monomorphic and spindle-shaped in appearance. The unique cells were isolated and subjected to immunostaining and reverse transcription-polymerase chain reaction analyses in order to reconfirm the expression of Oct-4 and to detect the expression of Paired box 2 (Pax-2), which is necessary for the formation of kidney structures. The unique cells were positive for Oct-4 and Pax-2; thus suggesting that the identified cells were kidney-derived stem cells. The results of the present study suggested that the unique cell type identified in the kidneys of the DT-treated rats were kidney-specific stem cells that may have been involved in the repair of DT-induced tissue injury. In addition, these cells may provide a useful cell line for studying the fundamental characteristics of kidney stem cells, as well as identifying kidney-specific stem cell markers.

Serum response factor induces endothelial-mesenchymal transition in glomerular endothelial cells to aggravate proteinuria in diabetic nephropathy.

We investigated the expression and function of serum response factor (SRF) in endothelial-mesenchymal transition (EndMT) in glomerular endothelial cells (GEnCs) of diabetic nephropathy (DN). The expression of SRF, endothelial markers (VE-cadherin, CD31), and mesenchymal markers (α-SMA, FSP-1, fibronectin) was examined in GEnCs following high glucose or in renal cortex tissues of DN rats. SRF was upregulated by SRF plasmids and downregulated by CCG-1423 (a small molecule inhibitor of SRF) to investigate how SRF influenced EndMT in GEnCs of DN. Streptozocin (STZ) was used to generate diabetes mellitus DM in rats. In GEnCs after high glucose treatment and in renal cortex tissues of diabetic rats, SRF, α-SMA, FSP-1, and fibronectin increased, while VE-cadherin and CD31 declined. SRF overexpression in GEnCs induced expression of Snail, an important transcription factor mediating EndMT. Blockade of SRF reduced Snail induction, protected GEnCs from EndMT, and ameliorated proteinuria. Together, increased SRF activity provokes EndMT and barrier dysfunction of GEnCs in DN. Targeting SRF by small molecule inhibitor may be an attractive therapeutic strategy for DN.

Nitro-oleic acid ameliorates oxygen and glucose deprivation/re-oxygenation triggered oxidative stress in renal tubular cells via activation of Nrf2 and suppression of NADPH oxidase.

Nitroalkene derivative of oleic acid (OA-NO2), due to its ability to mediate revisable Michael addition, has been demonstrated to have various biological properties and become a therapeutic agent in various diseases. Though its antioxidant properties have been reported in different models of acute kidney injury (AKI), the mechanism by which OA-NO2 attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO2 in an in vitro model of renal ischemia/reperfusion (I/R) injury. Human tubular epithelial cells were subjected to oxygen and glucose deprivation/re-oxygenation (OGD/R) injury. Pretreatment with OA-NO2 (1.25 µM, 45 min) attenuated OGD/R triggered reactive oxygen species (ROS) generation and subsequent mitochondrial membrane potential disruption. This action was mediated via up-regulating endogenous antioxidant defense components including superoxide dismutase (SOD1), heme oxygenase 1 (HO-1), and γ-glutamyl cysteine ligase modulatory subunits (GCLM). Moreover, subcellular fractionation analyses demonstrated that OA-NO2 promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO2 inhibited NADPH oxidase activation and NADPH oxidase 4 (NOX4), NADPH oxidase 2 (NOX2) and p22phox up-regulation after OGD/R injury, which was not relevant to Nrf2. These results contribute to clarify that the mechanism of OA-NO2 reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.

SIRT1 activator ameliorates the renal tubular injury induced by hyperglycemia in vivo and in vitro via inhibiting apoptosis.

We aimed to explore the role of SIRT1 in apoptosis in human kidney proximal tubule epithelial (HK-2) cells, and to determine whether resveratrol (RSV, a SIRT1 activator) could ameliorate apoptosis in rats with streptozotocin-induced diabetes mellitus (DM) and/or in high glucose (HG, 30mM) - stimulated HK-2 cells. Rats were distributed randomly into three groups: 1) control group, 2) DM group, and 3) DM with RSV group (DM+RSV; rats treated with 30mg/kg/d of RSV for 16 weeks). The physical, biochemical, and morphological parameters were then examined. Additionally, the deacetylase activity of SIRT1, and the expression levels of SIRT1 and of representative apoptosis markers, such as p53, acetylated p53, cleaved caspase-3, caspase-9, and cleaved PARP, were measured. HK-2 cells were stimulated by HG for different lengths of time to study the effect of HG on apoptosis. HK-2 cells were treated with or without RSV (25µM) to investigate if RSV has a protective effect on HG-induced apoptosis. A gene-specific small interfering RNA against SIRT1 was used to study the role of SIRT1 in apoptosis. More apoptosis was found in the DM rats than in the control rats. Similarly, the expression levels of cleaved caspase-3, cleaved PARP, and acetylated p53 were significantly higher, and the level of SIRT1 was significantly lower, in the HK-2 cells that were cultured under HG conditions than those in the HK-2 cells that were cultured under low glucose (5.5mM) conditions. Notably, treatment with RSV lessened the HG-induced changes in the levels of apoptosis indicators, and this inhibition of HG-induced apoptosis in HK-2 cells by RSV treatment was abolished by SIRT1 silencing. Our study showed that hyperglycemia contributes to apoptosis in rat kidney and HK-2 cells. SIRT1 activation by RSV can reduce urinary albumin excretion and proximal tubule epithelial apoptosis both in vitro and in vivo. Based on our study, SIRT1/p53 axis played an important role in the hyperglycemia induced apoptosis. These findings indicated that the increased expression of SIRT1, mediated by RSV, is a possible mechanism by which RSV prevents renal tubular injury in diabetic nephropathy (DN). So RSV has great clinical significance and could provide the basis for the new way to effective treatment to contain the morbidity and mortality associated with DN.

Serum response factor provokes epithelial-mesenchymal transition in renal tubular epithelial cells of diabetic nephropathy.

We investigated the role of serum response factor (SRF) in epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs) in diabetic nephropathy (DN). The expression of SRF, epithelial markers (E-cadherin and ZO-1), and mesenchymal markers (fibronectin, collagen-1, α-SMA, FSP-1) was examined in human proximal renal tubular epithelial cells (HK-2 cells) or renal medulla tissues following high glucose. SRF was upregulated by SRF plasmids and downregulated by CCG-1423 (a small molecule inhibitor of SRF) to investigate how SRF influenced EMT in TECs of DN. Streptozotocin was used to generate DM in rats. In HK-2 cells after high-glucose treatment and renal medulla tissues of diabetic rats, SRF, fibronectin, collagen-1, α-SMA, and FSP-1 increased, while E-cadherin and ZO-1 declined. SRF overexpression in HK-2 cells induced expression of Snail, an important transcription factor mediating EMT. Blockade of SRF by CCG-1423 reduced Snail induction and protected TECs from EMT both in vitro and in vivo. Together, increased SRF activity promotes EMT in TECs and dysfunction in DN. Targeting SRF by small molecule inhibitor may be an attractive therapeutic strategy for DN.

The role of long-term label-retaining cells in the regeneration of adult mouse kidney after ischemia/reperfusion injury.

BACKGROUND: Label-retaining cells (LRCs) have been recognized as rare stem and progenitor-like cells, but their complex biological features in renal repair at the cellular level have never been reported. This study was conducted to evaluate whether LRCs in kidney are indeed renal stem/progenitor cells and to delineate their potential role in kidney regeneration. METHODS: We utilized a long-term pulse chase of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in C57BL/6J mice to identify renal LRCs. We tracked the precise morphological characteristics and locations of BrdU(+)LRCs by both immunohistochemistry and immunofluorescence. To examine whether these BrdU(+)LRCs contribute to the repair of acute kidney injury, we analyzed biological characteristics of BrdU(+)LRCs in mice after ischemia/reperfusion (I/R) injury. RESULTS: The findings revealed that the nuclei of BrdU(+) LRCs exhibited different morphological characteristics in normal adult kidneys, including nuclei in pairs or scattered, fragmented or intact, strongly or weakly positive. Only 24.3 ± 1.5 % of BrdU(+) LRCs co-expressed with Ki67 and 9.1 ± 1.4 % of BrdU(+) LRCs were positive for TUNEL following renal I/R injury. Interestingly, we found that newly regenerated cells formed a niche-like structure and LRCs in pairs tended to locate in this structure, but the number of those LRCs was very low. We found a few scattered LRCs co-expressed Lotus tetragonolobus agglutinin (LTA) in the early phase of injury, suggesting differentiation of those LRCs in mouse kidney. CONCLUSIONS: Our findings suggest that LRCs are not a simple type of slow-cycling cells in adult kidneys, indicating a limited role of these cells in the regeneration of I/R injured kidney. Thus, LRCs cannot reliably be considered stem/progenitor cells in the regeneration of adult mouse kidney. When researchers use this technique to study the cellular basis of renal repair, these complex features of renal LRCs and the purity of real stem cells among renal LRCs should be considered.

Curcumin attenuates high glucose-induced podocyte apoptosis by regulating functional connections between caveolin-1 phosphorylation and ROS.

AIM: Caveolin-1 (cav-1) is a major multifunctional scaffolding protein of caveolae. Cav-1 is primarily expressed in mesangial cells, renal proximal tubule cells and podocytes in kidneys. Recent evidence shows that the functional connections between cav-1 and ROS play a key role in many diseases. In this study we investigated whether regulating the functional connections between cav-1 and ROS in kidneys contributed to the beneficial effects of curcumin in treating diabetic nephropathy in vitro and in vivo. METHODS: Cultured mouse podocytes (mpc5) were incubated in a high glucose (HG, 30 mmol/L) medium for 24, 48 or 72 h. Male rats were injected with STZ (60 mg/kg, ip) to induce diabetes. ROS generation, SOD activity, MDA content and caspase-3 activity in the cultured cells and kidney cortex homogenate were determined. Apoptotic proteins and cav-1 phosphorylation were analyzed using Western blot analyses. RESULTS: Incubation in HG-containing medium time-dependently increased ROS production, oxidative stress, apoptosis, and cav-1 phosphorylation in podocytes. Pretreatment with curcumin (1, 5, and 10 µmol/L) dose-dependently attenuated these abnormalities in HG-treated podocytes. Furthermore, in HG-containing medium, the podocytes transfected with a recombinant plasmid GFP-cav-1 Y14F (mutation at a cav-1 phosphorylation site) exhibited significantly decreased ROS production and apoptosis compared with the cells transfected with empty vector. In diabetic rats, administration of curcumin (100 or 200 mg/kg body weight per day, ig, for 8 weeks) not only significantly improved the renal function, but also suppressed ROS levels, oxidative stress, apoptosis and cav-1 phosphorylation in the kidneys. CONCLUSION: Curcumin attenuates high glucose-induced podocyte apoptosis in vitro and diabetic nephropathy in vivo partly through regulating the functional connections between cav-1 phosphorylation and ROS.

Critical role of serum response factor in podocyte epithelial-mesenchymal transition of diabetic nephropathy.

PURPOSE: To investigate the expression and function of serum response factor in podocyte epithelial-mesenchymal transition of diabetic nephropathy. METHODS: The expression of serum response factor, epithelial markers and mesenchymal markers was examined in podocytes or renal cortex tissues following high glucose. Serum response factor was upregulated by its plasmids and downregulated by CCG-1423 to investigate how it influenced podocyte epithelial-mesenchymal transition in diabetic nephropathy. Streptozotocin was used to generate diabetes mellitus in rats. RESULTS: In podocytes after high glucose treatment, serum response factor and mesenchymal markers increased, while epithelial markers declined. Similar changes were observed in vivo. Serum response factor overexpression in podocytes induced expression of Snail, an important transcription factor mediating epithelial-mesenchymal transition. Blockade of serum response factor reduced Snail induction, protected podocytes from epithelial-mesenchymal transition and ameliorated proteinuria. CONCLUSION: Together, increased serum response factor activity provokes podocytes' epithelial-mesenchymal transition and dysfunction in diabetic nephropathy. Targeting serum response factor by small-molecule inhibitor may be an attractive therapeutic strategy for diabetic nephropathy.

Nitro-oleic acid attenuates OGD/R-triggered apoptosis in renal tubular cells via inhibition of Bax mitochondrial translocation in a PPAR-γ-dependent manner.

BACKGROUND: Nitroalkene derivatives of oleic acid (OA-NO2) serve as high-affinity ligand for PPAR-γ, which regulates apoptosis, oxidation and inflammation and plays a central role in ischemia-reperfusion injury. In the present study, we elucidated the protective mechanisms of OA-NO2 against renal ischemia-reperfusion injury. METHODS: HK-2 cells were subjected to oxygen and glucose deprivation followed by re-oxygenation (OGD/R) to mimic renal ischemia-reperfusion injury. Cell apoptosis was analyzed by flow cytometry. Bax mitochondrial translocation, cytochrome c and apoptosis-inducing factor (AIF) cytosolic leakage and Akt/Gsk 3ß phosphorylation were evaluated by Western blotting. Bax activation was visualized by immunocytochemistry. GW9662 and siRNA transfection were employed to examine the involvement of PPAR-γ. RESULTS: OGD/R injury promoted mitochondrial translocation and activation of Bax, leakage of cytochrome c and AIF, subsequent caspase-3 activation, and eventually cell apoptosis. Pre-incubation with OA-NO2 (1.25 µM, 45min) inhibited Bax activation and blocked apoptotic cascade, while the protective effects were negated by GW9662 or PPAR-γ siRNA. Moreover, OA-NO2 restored Akt and Gsk 3ß phosphorylation in a PPAR-γ-dependent way. CONCLUSION: These findings suggest that OA-NO2 attenuates OGD/R-induced apoptosis by inhibiting Bax translocation and activation and the subsequent mitochondria-dependent apoptotic cascade in a PPAR-γ dependent manner.

Connective tissue growth factor induces tubular epithelial to mesenchymal transition through the activation of canonical Wnt signaling in vitro.

BACKGROUND: Overwhelming evidences suggest epithelial to mesenchymal transition (EMT) of tubular epithelial cells contributes to renal fibrosis of chronic kidney disease (CKD). Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of EMT. However, the molecular mechanisms that regulate cell behaviors are not clear. OBJECTIVE: The purpose of this study was to investigate whether CTGF induces EMT via activation of canonical Wnt signaling in renal tubular epithelial cells. METHODS: Human renal proximal tubular epithelial cells (HK-2) were divided into control group, CTGF group and dickkopf (Dkk)-1 plus CTGF group. We assessed the biological changes of canonical Wnt signaling, including phosphorylation of low-density lipoprotein receptor-related protein (LRP6) and glycogen synthase kinase-3ß (GSK-3ß) and accumulation and nuclear localization of ß-catenin. Meanwhile, morphological changes of the three groups were observed and tubular EMT was further confirmed by detecting the expression of α-SMA and E-cadherin. RESULTS: The phosphorylation levels of LRP6 and GSK-3ß and the expression of ß-catenin in CTGF group were higher than control group (p < 0.05). The accumulation and nuclear localization of ß-catenin was induced in CTGF group. Meanwhile, CTGF group cells showed a mesenchymal morphological phenotype and exhibited increased expressions of E-cadherin and decreased expressions of α-SMA compared to control group (p < 0.05), suggesting tubular EMT. Furthermore, we also found that Dkk-1 blocked the above CTGF's effects by binding with LRP6. CONCLUSION: CTGF induces EMT via activation of canonical Wnt signaling in HK-2 cells in vitro, which may play an important role in the renal fibrosis of CKD.

TRB3 mediates renal tubular cell apoptosis associated with proteinuria.

Proteinuria may contribute to progressive renal damage by inducing tubulointerstitial inflammation, fibrosis and tubular cell apoptosis, but the underlying mechanisms remain largely unknown. TRB3 is a kinase-like molecule that can modify cellular survival and interfere with signal transduction pathways. We seek to determine the role of TRB3 in renal tubular cell apoptosis associated with proteinuria. Herein, we reported that in a rat tubular cell line, high concentration of albumin augmented TRB3 expression and induced apoptosis, while TRB3 silencing with special small interference RNA significantly attenuated apoptosis. In addition, we found that albumin-induced apoptosis was related to inhibition of Akt phosphorylation, which was, however, partially reversed by TRB3 silencing, indicating that TRB3 worked through Akt pathway in this apoptotic signaling cascade. In vivo, we observed increased TRB3 expression in kidneys of streptozotocin-induced diabetic nephropathy model and albumin-overload nephropathy model, both of which showed overt proteinuria. Notably, proteinuria induced apoptosis in renal tubules, which was less severe after genetically inhibition of TRB3. Taken together, these results suggest that TRB3 mediates renal tubular cell apoptosis induced by protein overload, broadening our understanding of the pathogenesis of progressive proteinuric kidney diseases.

Curcumin ameliorates epithelial-to-mesenchymal transition of podocytes in vivo and in vitro via regulating caveolin-1.

AIMS: Epithelial-mesenchymal transition (EMT) is recognized to play a key role in diabetic nephropathy (DN). Curcumin, the main active component of turmeric extracted from the roots of the Curcuma longa plant, has been reported for its anti-fibrotic effects in kidney fibrosis. The purpose of our study was to investigate the effects of curcumin in reversing epithelial-to-mesenchymal transition (EMT) of podocytes in vivo and in vitro. MATERIALS/METHODS: In vivo streptozotocin (STZ)-induced diabetic rats received vehicle or curcumin, and podocytes were treated with high glucose (HG) in the presence or absence of curcumin in vitro. And we investigated the effect of curcumin on HG-induced phosphorylation of cav-1 on the stability cav-1 and ß-catenin using immunoprecipitation and fluorescence microscopy analysis. RESULTS: Curcumin treatment dramatically ameliorated metabolic parameters, renal function, morphological parameters in diabetic rats. We found that HG treatment led to significant down-regulation of p-cadherin and synaptopodin, as well as remarkable up-regulation of α-SMA and FSP-1 in vivo and in vitro. Furthermore, curcumin inhibited HG-induced caveolin-1 (cav-1) Tyr(14) phosphorylation associating with the suppression of stabilization of cav-1 and ß-catenin. CONCLUSIONS: In summary, these findings suggest that curcumin prevents EMT of podocytes, proteinuria, and kidney injury in DN by suppressing the phosphorylation of cav-1, and increasing stabilization of cav-1 and ß-catenin.

Berberine improves kidney function in diabetic mice via AMPK activation.

Diabetic nephropathy is a major cause of morbidity and mortality in diabetic patients. Effective therapies to prevent the development of this disease are required. Berberine (BBR) has several preventive effects on diabetes and its complications. However, the molecular mechanism of BBR on kidney function in diabetes is not well defined. Here, we reported that activation of AMP-activated protein kinase (AMPK) is required for BBR-induced improvement of kidney function in vivo. AMPK phosphorylation and activity, productions of reactive oxygen species (ROS), kidney function including serum blood urea nitrogen (BUN), creatinine clearance (Ccr), and urinary protein excretion, morphology of glomerulus were determined in vitro or in vivo. Exposure of cultured human glomerulus mesangial cells (HGMCs) to BBR time- or dose-dependently activates AMPK by increasing the thr172 phosphorylation and its activities. Inhibition of LKB1 by siRNA or mutant abolished BBR-induced AMPK activation. Incubation of cells with high glucose (HG, 30 mM) markedly induced the oxidative stress of HGMCs, which were abolished by 5-aminoimidazole-4-carboxamide ribonucleoside, AMPK gene overexpression or BBR. Importantly, the effects induced by BBR were bypassed by AMPK siRNA transfection in HG-treated HGMCs. In animal studies, streptozotocin-induced hyperglycemia dramatically promoted glomerulosclerosis and impaired kidney function by increasing serum BUN, urinary protein excretion, and decreasing Ccr, as well as increased oxidative stress. Administration of BBR remarkably improved kidney function in wildtype mice but not in AMPKα2-deficient mice. We conclude that AMPK activation is required for BBR to improve kidney function in diabetic mice.

Curcumin prevents diabetic nephropathy against inflammatory response via reversing caveolin-1 Tyr14 phosphorylation influenced TLR4 activation.

Inflammation is involved in the development and/or progression of diabetic nephropathy (DN). Curcumin has been reported for its anti-inflammation activity in DN. However, the mechanisms involved in the renoprotective effects of curcumin have not been clearly demonstrated. In this study, we hypothesized that curcumin affected high glucose (HG)-induced inflammation profiles in vivo and in vitro and then prevented renal injury in diabetic rats via reversing cav-1 Tyr(14) phosphorylation that influenced TLR4 activation. Streptozotocin (STZ)-induced diabetic rats received vehicle or curcumin for twelve weeks and podocytes were treated with HG in the presence or absence of curcumin in vitro. To further evaluate the effect of cav-1 phosphorylation at Tyr(14) on HG-induced podocyte inflammation response and TLR4 activation, a recombinant plasmid GFP-Cav-1 Y14F with a mutated phosphorylation site of cav-1, was transfected into cultured podocytes. In vivo, curcumin improved histological abnormalities and fibrosis of a diabetic kidney, inhibited renal inflammatory gene expression and reduced cav-1 phosphorylation at Tyr(14) and the expression of TLR4. Pretreatment of podocytes with curcumin reduced HG-stimulated production of proinflammatory cytokines, TLR4 and the phosphorylation of cav-1. But immunohistochemistry in rat kidney showed that the elevation of TLR4 expression is more evident in the renal interstitum than in the glomerulus where podocytes are located, and the possibility that the anti-inflammatory effects of curcumin on other cells in the kidney may be mediated through the same molecular pathways as in podocytes. Our study suggests that curcumin treatment ameliorates DN via inhibition of inflammatory gene expression by reversing caveolin-1 Tyr(14) phosphorylation that influenced TLR4 activation.

TRB3, up-regulated in kidneys of rats with type1 diabetes, mediates extracellular matrix accumulation in vivo and in vitro.

AIMS: Fibrosis is the final disorder of most chronic kidney disease including diabetic nephropathy (DN), but the mechanisms are not fully understood. The present study aims to determine whether TRB3 participates in fibrogenesis in DN. METHODS: Type1 diabetes was induced in male Wistar rats via intraperitoneal injection of streptozotocin (STZ). The expression of TRB3 and extracellular matrix (ECM) protein collagen I and fibronectin was investigated in kidneys of rats with diabetes and NRK-52E cells (a rat proximal tubular cell line) stimulated with albumin-overload. Rats without diabetes and NRK-52E cells without albumin stimulation served as control. Then gene silencing was used to study whether TRB3 participated in accumulation of collagen I and fibronectin in vivo and in vitro. RESULTS: TRB3 is up-regulated in renal tubules of kidneys of rats with diabetes, especially proximal tubules. Albumin-overload can augments TRB3 expression and increase collagen I and fibronectin secretion in NRK-52E cells. Importantly, silencing of TRB3 alleviates collagen I and fibronectin accumulation in kidneys of rats with diabetes and NRK-52E cells induced by albumin-overload. CONCLUSIONS: TRB3 mediates ECM accumulation in kidneys of rats with STZ-induced type1 diabetes and proximal tubular cells induced by albumin-overload, suggesting a potential target for treatment of DN.

Cyclosporine A combined with medium/low dose prednisone in progressive IgA nephropathy.

The aim of the present study was to evaluate the efficacy of cyclosporine A (CsA) combined with medium/low dose prednisone in the treatment of progressive immunoglobulin A nephropathy (IgAN). Ninety-six patients who satisfied the inclusion criteria were enrolled in a prospective controlled clinical study. They were assigned into two groups and initially given either 0.6-0.8 mg/kg/day prednisone (maximum 40 mg/day) plus 3 mg/kg/day CsA (CsA group), or 1 mg/kg/day prednisone (maximum 60 mg/day) alone (steroid group). During therapy, the dose of prednisone was reduced in both groups and the dose of CsA was gradually tailed off over the first 3 months and maintained at 2 mg/kg/day in the CsA group. Urinary protein excretion, serum biochemical indexes, clinical efficacy and side effects of CsA were assayed. A significant decline in mean 24-hour urinary protein excretion (p < 0.05) was observed 1 month after treatment in patients in the CsA group, which was observed 2 months after treatment in the steroid group. The decline in mean 24-hour urinary protein excretion in the CsA group was more significant than in the steroid group. Serum albumin level increased significantly in the CsA group 2 months after therapy (p < 0.05). Moreover, at the end of the course, a higher remission rate was observed in patients with Lee's Grade III IgAN after combined treatment with prednisone and CsA (p < 0.05). No significant difference in clinical efficacy was observed in patients with Lee's Grade IV and Grade V IgAN between the two groups (p > 0.05). CsA at a dose of 2-3 mg/kg/day in combination with medium/low dose prednisone was effective in inducing remission of IgAN, especially for patients with Lee's Grade III IgAN, and is a safe and effective choice for short-term treatment of patients with progressive IgAN.