Protective Effect Of Curcumin On Podocyte In Diabetic Nephropathy Via Regulating Caveolin-1

Background and ObjectiveDiabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD), with a significant increase in morbidity and mortality in patients with diabetes. Many patients continue to show progressive renal damage despite strict blood glucose and/or blood pressure control. Up to now, the pathophysiological mechanism responsible for DN has not been fully understood and DN hasn’t been treated well yet. Therefore, it is extremely important to identify the mechanism of DN and find effective drugs to halt the progression of DN.Although DN is traditionally considered as a nonimmune disease, an increasing number of clinical and animal model studies have implicated that the activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of DN. In recent years, the role of TLRs in the pathogenesis of DN has been gradually concerned. TLRs are pattern-recognition receptors which primarily function as initiators of the innate immune response leading to the expression of pro-inflammatory cytokines and chemokines. In the case of diabetes, it has been reported that TLRs sense high glucose (HG) as danger signals and subsequently mediate the inflammatory cascade to promote disease progression. It is reported that TLR4 also plays an important role in various kidney disorders, such as glomerulonephritis, renal ischemia and diabetic tubular inflammation.The podocyte is a terminally differentiated and highly specialized glomerular visceral epithelial cell that synthesizes components of the glomerular basement membrane. Emerging evidence has established that podocyte injury is a common trigger leading to the disruption of the filtration barrier and protein leakage, and ultimately resulting in glomerulosclerosis. Reduced podocyte density and podocyte injury have been documented in both experimental and clinical diabetic nephropathy and are among the strongest predictors of its progression. In response to several stimuli, podocytes undergo a range of adaptive changes. If the injury is progressive and chronic, podocytes are able to undergo epithelial-to-mesenchymal transition (EMT) to escape from podocyte apoptosis, which results in phenotypic changes associated with the loss of highly specialized podocyte features and expression of new mesenchymal markers. EMT has been proposed by recent studies as a possible engagement for podocyte depletion in diabetic nephropathy.Caveolin-1 (cav-1) is the main constituent molecule of caveolae, which are omega-shaped plasma membrane invaginations enriched in cholesterol and sphingolipids. Caveolae are thought to be signalling platforms regulating the activation of several signalling pathways. Cav-1 Tyr14 phosphorylation may be function to facilitate cav-1 interaction with other proteins in a stimulus specific fashion. Recent studies implicated the key role of cav-1 in regulating innate immunity and inflammation. It has been reported that cav-1 confers its anti-inflammatory effects through direct binding of TLR4. Recent studies also implicated the key role of cav-1 phosphorylation at Tyr14 in activating the TLR4 signaling pathway and, hence, mediated the inflammatory response. However, little is known about cav-1 or whether cav-1 phosphorylation influences TLR4 activation in podocytes. Many experiments also found that cav-1 could be precipitated with E-cadherin and interacts with P-catenin. Thus, cav-1 might have a more general role in regulating cell junctions. The importance of linking the alteration of cav-1 and β-catenin complex expression may be a new way to understand the EMT of DN.Curcumin is the main active component of turmeric extracted from the roots of the curcuma longa plant, and has been used for centuries in treating inflammatory ailments and conditions. Curcumin has been shown to have anti-inflammatory, anti-oxidant, anti-apoptotic and anti-fibrotic effects. But the molecular mechanisms by which curcumin supplementation may inhibit or delay diabetes-associated kidney inflammation and podocytes EMT are not known.In current study, we hypothesize that cav-1 or cav-1 phosphorylation may be changed in the kidney of diabetic rats and involve in activation of TLR4 and inflamation. And curcumin treatment ameliorates DN via inhibition of inflammatory gene expression by reversing cav-1 or cav-1 phosphorylation influenced TLR4 activation; curcumin may inhibit renal fibrosis as assessed by EMT through modulating the expression of cav-1 and the crosstalk between cav-1 and P-catenin signalling pathway. We determined the expression of inflammatory gene, EMT, cav-1, cav-1 phosphorylation, TLR4 and β-catenin in the kidney of diabetic rats, which could find the associated changes in vivo. And then we tried to reveal the relationship between cav-1, TLR4 and P-catenin in high glucose (HG)-induced podocyte EMT.Methods1. To make clear the effect and mechanism of curcumin on kidney inflammation in diabetic rats, we used STZ-induced diabetic rats as a diabetes mellitus (DM) model, and the rats were divided into three groups:the control group, DM group and DM+curcumin (100 mg·kg-1·d-1 body weight). All rats were sacrificed at 12 weeks after the induction of diabetes for analysis. Podocytes were pretreated with curcumin (1,5,10 μM) or vehicle for 1 h and then incubated with either LG (5.5mM) or HG(30mM) for 72 h in vitro. Blood glucose,24 h urine protein quantitative (UP 24 h), creatinine clearance (Ccr) and kidney weight-to-body weight ratio (KW/B W) were detected by laboratory methods; PAS staining, masson staining and immunohistochemistry were performed to assess glomerular sclerotic injury and the expression of FN, Collagen Ⅰ; the expression of ED-1, MCP-1, TLR4 in kidney tissues of diabetic rats were measured by immunohistochemistry; expressions of pro-inflammatory cytokines (IL-6 and TNFa) at protein level were determined by ELISA; expressions of pro-inflammatory cytokines (IL-6 and TNFa) and TLR4 at gene level were determined by real time RT-PCR; western blot was performed to detect the protein expression of cav-1 phosphorylation at Tyr14, cav-1 and TLR4 in the kidney of diabetic rats. Podocytes transfected with TLR4 siRNA, or NC siRNA, were incubated with LG (5.5mM) or HG (30mM) for 24 h to test the association between TLR4 and inflammation. Gene and protein expression of IL-6 and TNF-a were determined by real-time PCR and ELISA. To further evaluate the effect of cav-1 phosphorylation at Tyr14 on HG-induced podocyte inflammation response and TLR4 activation, a recombinant plasmid GFP-Cav-1 Y14F whose phosphorylation site of cav-1 is mutated, was constructed by GeneChem and transfected into cultured podocytes. Western blot was performed to detect the protein expression of cav-1 phosphorylation at Tyr14, cav-1 and TLR4 in podocytes, and ELISA was performed to detect the protein expression of IL-6 and TNFa.2. To make clear the effect and mechanism of curcumin on podocyte EMT in diabetic rats, we used STZ-induced diabetic rats as a DM model, and the rats were divided into three groups:the control group, DM group and DM+curcumin (100 mg-kg-1·d-1 body weight). All rats were sacrificed at 12 weeks after the induction of diabetes for analysis. Podocytes were pretreated with curcumin (1,5,10 μM) or vehicle for 1 h and then incubated with either LG (5.5mM) or HG (30mM) for 72 h in vitro. Blood glucose, UP 24 h, creatinine clearance (Ccr) and kidney weight-to-body weight ratio (KW/BW) were detected by laboratory mathods; PAS and masson staining were performed to assess glomerular sclerotic injury. The level of podocyte process effacement was examined by electron microscopy. Protein and gene expression of FSP-1, a-SMA, P-cadherin, synaptopodin in kidneys of diabetic rats and podocytes were determined by immunohistochemistry, western blot and real time RT-PCR. Western blot and immunohistochemistry were performed to detect the protein expression of cav-1 phosphorylation at Tyr14, cav-1, active β-catenin and β-catenin. Immunoprecipitation was performed to detect the association of cav-1 and β-catenin. Immunofluorescence analysis showed subcellular localization of cav-1 and β-catenin after treated with HG and/or curcumin.Results1. Curcumin prevents diabetic nephropathy against inflammatory response via reversing cav-1 Tyr14 phosphorylation influenced TLR4 activation.1.1 Curcumin ameliorated physical and biochemical parameters of diabetic rats There were no significant differences in fasting blood-glucose (FBG) levels between the DM group and DM+Cur group. After curcumin treatment, UP 24 h and KW/BW were significantly reduced compared with the DM group, with higher ccr in DM+Cur group.1.2 Curcumin treatment improved histological abnormalities and fibrosis of diabetic kidneyPAS staining demonstrated that curcumin reduced diabetes-induced ECM deposition in the mesangium in rat kidneys. Masson staining showed that in DM rats, renal fibrosis was present, and after 12 weeks of treatment with curcumin, renal fibrosis was significantly improved compared with that in untreated diabetic rats. Immunohistochemical staining also showed that the levels of FN and Collagen I protein in the kidney tissues of the DM+Cur group were significantly decreased compared with the DM group.1.3 Curcumin treatment improved renal macrophages infiltration and MCP-1 expressionCompared with the DM group, curcumin treatment significantly reduced renal ED-1+ macrophage infiltration in the diabetic rats. The immunostaining for MCP-1 shows significantly increase in the glomeruli of DM group, but remarkably reduced by curcumin treatment.1.4 Curcumin inhibited renal inflammatory gene expression in diabetic ratsCompared with the control group, the expression of renal IL-6, TNF-α at protein level and renal IL-6, TNF-α and IL1β at gene level as revealed by ELISA and RT-qPCR respectively in the DM group was all significantly increased. However, curcumin treatment significantly decreased the overexpression of these cytokines.1.5 Curcumin inhibited TLR4 activation via regulating cav-1 phosphorylation at Tyr14 in the diabetic kidneyImmunohistochemical staining, real time RT-PCR and western blot showed that the expression of TLR4 was significantly upregulated in the DM group compared with the control group, which was significantly attenuated in the curcumin treated group compared with the DM group. In the present study we also showed that compared with the control group, cav-1 phosphorylation at Tyr14 in the DM group was significantly increased. However, curcumin treatment reduced the phosphorylation of cav-1.1.6 Curcumin reduced the inflammatory gene expression in HG-stimulated podocytesCompared with the normal group, the expression of IL-6, TNF-α and IL-1β at gene and the expression of IL-6, TNF-a levels at protein level in podocytes as revealed by RT-qPCR and ELISA respectively in the DM group were all significantly increased. However, curcumin treatment significantly decreased the overexpression of these factors.1.7 TLR4 mediated the pro-inflammatory effect induced by HG in podocytesTransfection with TLR4 siRNA decreased endogenous expression of TLR4 protein by 60% and mRNA level by 72% in podocytes. Transfection with TLR4 siRNA had no effect on endogenous expression of IL-6 and TNF-a but significantly decreased HG induced upregulation of IL-6 and TNF-a gene expression and protein secretion. 1.8 Curcumin suppressed the HG-induced activation of TLR4 via regulating cav-1 phosphorylation at Tyr14 in podocytesUnder HG conditions, TLR4 was significantly increased in mRNA and protein expression, compared with that in podocytes treated with LG. Furthermore, HG significantly induced cav-1 phosphorylation at Tyr14. Pre-treatment with curcumin (1, 5,10μM) decreased the HG-induced increase of TLR4 and cav-1 phosphorylation at Tyr14. GFP-Cav-1 Y14F significantly decreased HG induced upregulation of cav-1 phosphorylation at Tyr14, TLR4, and IL-6, TNF-a protein expression than that of cells transfected with the empty vector GFP-C1.2. Curcumin ameliorates EMT of podocytes in vivo and in vitro via regulating cav-12.1 Effects of curcumin on metabolic parameters and renal histopathological changes in diabetic ratsThere were no significant differences in fasting blood-glucose (FBG) levels between the DM group and DM+Cur group. After curcumin treatment, UP 24 h and KW/BW were significantly reduced compared with the DM group, with higher ccr in DM+Cur group. PAS staining demonstrated that curcumin reduced diabetes-induced ECM deposition in the mesangium in rat kidneys. Masson staining showed that renal fibrosis was significantly improved after treated with curcumin compared with that in untreated diabetic rats. Compared with normal controls, electron microscopy exhibited thickening of GBM and foot process effacement in DM group. These morphologic injuries were attenuated in diabetic rats received curcumin than those untreated.2.2 Effects of curcumin on the renal EMT in diabetic ratsImmunohistochemical staining, western blot and RT-PCR showed the expression of P-cadherin, synaptopodin was noticeably dropped and FSP-1, a-SMA, snail1 expression was detected significantly up-regulated in the DM group compared with the normal group, but was significantly attenuated in the curcumin-treated group compared with the DM group.2.3 Effects of curcumin on phosphorylation of cav-1 in diabetic ratsWestern blot showed that compared with those in the control group, phosphorylation of cav-1 was significantly increased and β-catenin, active β-catenin were increased in DM rats. Immunohistochemical staining showed that cav-1 expression in three groups had no significant difference, and β-catenin was increased in DM group compared with those in the control group. After curcumin treatment for 12 weeks, the diabetic rats showed a significant down-regulation in cav-1 phosphorylation, β-catenin and/or active P-catenin compared with the DM group.2.4 HG-induced EMT of podocytes and effects of curcumin on the podocytes EMTCompared with the control group, HG induced substantial decrease in protein and mRNA expression of β-cadherin and synaptopodin in a time-dependent manner. Furthermore, there was significant increase in protein and mRNA expression of FSP-1 and α-SMA in podocytes treated with HG compared with the control group. Pretreatment with curcumin (1,5,10 μM) significantly blocked HG induced changes in the protein and mRNA expression of P-cadherin, synaptopodin and FSP-1,α-SMA in podocytes compared with the control group.2.5 Curcumin treatment reversed HG-induced EMT of pococytes via modulating the phosphorylation of cav-1 and increasing stabilization of cav-1 and P-cateninUnder HG conditions, phosphorylation of cav-1 was significantly increased, compared with that in podocytes treated with LG. Furthermore, there was significant increase in protein expression of active β-catenin and P-catenin in podocytes treated with HG compared with podocytes under LG incubation. Pretreatment with curcumin could down-regulate the HG-induced increase of phosphorylation of cav-1, and decrease active β-catenin and β-catenin levels. Immunoprecipitation analysis showed that the association between cav-1 and β-catenin was markedly decreased in podocytes treated with HG for 72 h. But the presence of curcumin caused a greater extent association. Fluorescence microscopy showed that treatment with HG led to a decrease of co-localization between cav-1 and β-catenin at the cell borders compared with that of control. However, curcumin treatment blocked this effect.Conclusion1. Curcumin can attenuate the kidney injury in rats with diabetes, as revealed by the reduction of proteinuria and amelioration of renal function.2. Curcumin ameliorates glomerular fibrosis, downregulates the thickening of GBM and the foot process effacement in STZ-induced diabetic rats.3. The mechanism of curcumin mediated beneficial effects is related to inhibition of the activation of TLR4. Curcumin treatment ameliorates DN via reversing cav-1 Tyr14 phosphorylation influenced TLR4 activation, and down-regulating the pro-inflammatory cytokines expression.4. The mechanism of curcumin-mediated beneficial effects is related to inhibition podocyte EMT. The protective role of curcumin in counteracting HG-induced EMT in podocytes in vivo and in vitro is via decreasing the phosphorylation of cav-1 associated with dissociation of cav-1 and β-catenin.