| Objectives: Tubulointerstitial fibrosis is the principal hallmark of the most types of progressive renal disease. It has been largely demonstrated that the severity of tubulointerstitial changes correlated better with renal function compared with glomerulosclerosis. The appearance and activation of myofibroblasts are thought to play a key role in the progression of chronic renal fibrosis. These cells express the mesenchymal markerα-smooth muscle actin(α-SMA). Despite some debates regarding the origin of renal myofibroblasts, emerging evidence suggests that, under pathologic conditions, these cells may be derived mainly from epithelial cell-myofibroblast transdifferentiation (EMT) of tubular epithelial cells. The precise mechanism and signaling pathway initiating EMT remain unclear. Janus kinase/signal transducer and activators of transcription(JAK/STAT)is an important signaling pathway, which has been known to mediate the signaling of numerous cytokinase and growth factors and to be implicated in the regulation of a wide range of cellular processes such as proliferation, differentiation and apoptosis. It was reported that high glucose and angiotensin II can induce activation of JAK/STAT in the glomerular mesangial cells of rat cultrued in vitro. There are few reports about the JAK/STAT signaling pathway in tubulointerstitial fibrosis. Experimental and clinical investigations indicated that antagonists of renin-angiotensin system, such as angiotensin-converting enzyme inhibitor (ACEI) and angiotensin recepter blockade (ARB), have renal protective effects and have become one of the treatment strategies against fibrosis in progressive renal disease. In this study, SD rats were used to induce experimental renal fibrosis (5/6 subtotal nephrectomy, STNX) and human renal proximal tubular epithelial cells (HKCs) were cultured in high glucose medium to investigate the activation of JAK/STAT signaling pathway and effects of AT1Ra and Bushenhuoxue Fang .Methods:1. Detection of EMT and JAK/STAT signaling pathway of HKCs cultured in high glucose mediumHKCs were divided into four groups: low glucose group(5.5mmol/L glucose, LG), high glucose group(30mmol/L glucose , HG), high mannitol group(5.5mmol/L glucose+24.5mmol/L mannitol, LG+M), and HG+AG490 group(30mmol/L glucose+10μmol/L AG490, a specific inhibitor of JAK2, AG). The cells were cultured in 24-pore plate and 25cm2 plastic culture flask. After treatment for 6, 12, 24, 48 and 72h, the supernatants was collected and cells were harvested for immuno- flurescence, protein extraction and RNA isolation. Immunoprecipitation and Western blot analysis were used to determine the expression of tryosine phosphorylated Janus kinase 2 (p-JAK2). The protein expressions of STAT1, STAT3, p-STAT1, p-STAT3,α-SMA and E-Cadherin were observed by immunoflurescence and Western blot . The contents of TGF-β1 and type I collagen in the supernatants of the cultured HKC were detected by enzyme-linked immunoadsorbent assay(ELISA). TGF-β1 mRNA was measured by reverse transcription and polymerase chain reaction ( RT-PCR) .2. Detection of EMT and JAK/STAT signaling pathway of HKCs cultured in high glucose medium after treatment with valsartanHKCs were divided into three groups: LG group(5.5mmol/L glucose, LG); HG group(30mmol/L glucose, HG)and HG+Val group(30mmol/L glucose+10μmol/L valsartan, HG+Val). The supernatants was collected and cells were harvested for immunoflurescence, protein extraction and RNA isolation after treatment for 6, 12, 24, 48 and 72h, respectively. The tyrosine phosphorylation of JAK2 was detected by immunoprecipitation and Western blot analysis. The protein expression of STAT1, STAT3, p-STAT1, p-STAT3,α-SMA and E-Cadherin were observed by immunoflurescence and Western blot . The contents of TGF-β1 and type I collagen in the supernatants of the HKCs were detected by enzyme-linked immunoadsorbent assay ( ELISA). TGF-β1 mRNA was measured by reverse transcription and polymerase chain reaction ( RT-PCR) .3. Detection of EMT and JAK/STAT signaling pathway in 5/6 subtotal nephrectomy (STNX) rats after treatment with losartan5/6 subtotal nephrectomy was performed in Male Sprague-Dawley rats to induce experimental renal fibrosis. 54 rats were randomly divided into three group: sham group, STNX group and STNX +losartan group. losartan was ig administrated to rats in the losartan-treated group in a dose of 20mg/kg for 12weeks. Six rats from each group were respectively sacrificed at weeks 4,8,12 after the induction of 5/6 subtotal nephrectomy. Blood, urine and kidney samples were collected. The concentration of 24h urine protein, serum urea nitrogen (BUN) and creatinine(Cr) were determined, respectively. Partial renal tissure was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to isolate proximal tubules and extract total RNA and protein. The levels of expression of JAK2,STAT1,p-STAT1,STAT3 , p-STAT3 andα-SMA were evaluated by immunohistochemistry and Western blot. The tyrosine phosphorylation of JAK2 was detected by immunoprecipitation and Western blot analysis. The expression of STAT1 mRNA, STAT3 mRNA and TGF-β1mRNA were measured by reverse transcription and polymerase chain reaction (RT-PCR).4. Detection of EMT and JAK/STAT signaling pathway in 5/6 subtotal nephrectomy (STNX) rats after treatment with Bushenhuoxue Fang5/6 subtotal nephrectomy was performed in Male Sprague-Dawley rats to induce experimental renal fibrosis. 54 rats were randomly divided into three group: sham group, STNX group and STNX + Bushenhuoxue Fang group. Bushenhuoxue Fang was ig administrated to rats in the Bushenhuoxue Fang-treated group in a dose of 20ml/kg for 12weeks. Six rats from each group were respectively sacrificed at weeks 4,8,12 after 5/6 subtotal nephrectomy. Blood, urine and renal samples were collected. The concentration of 24h urine protein, blood BUN and blood Scr were determined, respectively. Partial renal tissure was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to isolate proximal tubular and extract total RNA and protein. Immunohistochemistry, Western blot and immunoprecipitation analysis were used to investigate the expression of JAK2, p-JAK2, STAT1, p-STAT1, STAT3 , p-STAT3 andα-SMA. RT-PCR was used to detect the expression of STAT1mRNA, STAT3mRNA and TGF-β1mRNA in renal.Results:1. Effects of activation of JAK/STAT on high glucose-induced EMT in HKCs①Immunofluorescence cytochemical staining showed that four STATs proteins were observed in nuclei and cytoplasm of HKCs. Compared with low glucose group, the expression of p-STAT1 and p-STAT3 was increased in high glucose group,and the increased expression of STAT1 and STAT3 was observed mainly in nuclei suggesting nuclear translocation of STAT1 and STAT3. The levels of expression of p-STAT1 and p-STAT3 in AG490 (a specific JAK2 inhibitor) group were obviously lower than those of the high glucose group. The nuclear translocation of STAT1 and STAT3 was inhibited in AG490 group.②Compared with low glucose group, phosphorylation level of JAK2 was increased in high glucose group(P<0.01)and phosphorylation of JAK2 was inhibited by AG490 (P<0.01)。③Compared with low glucose group, the expression of p-STAT1 and p-STAT3 was increased(P <0.01) in high glucose group in time dependent manner. The expression of p-STAT1 and p-STAT3 in AG490 group was decreased compared with high glucose group (P<0.01). There was no significantly difference in the expressions of STAT1 and STAT3 between high glucose group and AG490 group(P﹥0.05).④Immunofluorescence and westernblot showed that the expression ofα-SMA was significantly increased in the high glucose group in time-depentdent manner, which was markedly attenuated by the treatment with AG490(P<0.01, respectively). The expression of E-cadherin was significantly decreased in high glucose group(compared with low glucose group, P<0.01) and significantly increased in AG490 group(compared with high glucose group, P<0.01).⑤RT-PCR showed that the relative expression level of TGF-β1 mRNA was increased in high glucose group (P<0.01). After treatment with AG490, the relative expression level of TGF-β1 mRNA was decreased(compared with high glucose group, P<0.01).⑥The concentration of TGF-β1, and type I collagen in supernatants of the HKCs exposed to high glucose was higher than those in low glucose group at different times from 6h to 72h. After treament with AG490, the concentration of TGF-β1, and type I collagen was decreased(compared with high glucose group, P<0.05).2. Effects of valsartan on high glucose-induced EMT in HKCs and role of JAK/STAT signaling pathway①Immunoprecipitation and Western blot analysis indicated that phosphorylation level of JAK2 was increased in high glucose group(compared with low glucose group, P <0.01), and was decreased in valsartan group (compared with high glucose group, P<0.01).②Immunofluorescence cytochemical staining and Western blot analysis showed that the level of phosphorylation of STAT1 and STAT3 was increased in high glucose group in time-depentdent manner and decreased in valsartan treated group(compared with high glucose group, P<0.05), but the expression of STAT1 and STAT3 showed no significant difference among different groups.③Compared with the high glucose group, the expression ofα-SMA in valsartan-treated group was significantly decreased as shown by immunofluorescence cytochemistry and westernblot (P<0.01).④RT-PCR analysis indicated that the expression TGF-β1 mRNA was higher in high glucose group than that in low glucose group, while valsartan could down-regulate the expression TGF-β1 mRNA in HKCs cultured in high glucose medium. The secretion of TGF-β1 and type I collagen in HKCs cultured in high glucose medium could be inhibited by valsartan(compared with high glucose group, P <0.01).3. Renal morphological changes, expression ofα-SMA,TGF-β1 mRNA and JAK/STAT signaling protein in 5/6 subtotal nephrectomy (STNX) rats After 5/6 subtotal nephrectomy, rat developed chronic renal injures including glomerular sclerosis, tubulo-interstitial fibrosis and a progressive decline in renal function.①Light microscopically the glomeruli were enlarged and proximal tubular epithelial cells were swelling in the residual kidneys in 5/6 subtotal nephrectomy group at weeks 4. Glomerular sclerotic lesion, dilation and atrophy of the tubules, interstitial fibrosis and inflammatory changes were found at week 8. The extent of glomerular damage and interstitial fibrosis was increased at week 12.②The renal function indexes such as serum urea nitrogen, creatinine and 24h urine protein were significantly increased in 5/6 subtotal nephrectomy group ( compared with sham-operated group, P<0.05).③RT-PCR and western blot analysis indicated that the levels of exprssion of TGF-β1 mRNA andα-SMA protein in 5/6 subtotal nephrectomy group were markedly increased than those in sham-operated group.④Compared with sham-operated group, the level of tyrosine phosphorylation of JAK2 was increased at week 4 in 5/6 subtotal nephrectomy group(P <0.01), and progressively increased with time duration. Meanwhile, immunohistochemical staining and western blot analysis indicated that the expression of STAT1, STAT3, p-STAT1 and p-STAT3 were also increased in time dependent manner in 5/6 subtotal nephrectomy group (P <0.01).4. Effects of losartan on EMT and activation of JAK/STAT signaling pathway in 5/6 subtotal nephrectomy (STNX) rats①The pathological changes of the residual kidneys including interstitial cellular infiltration, tubular dilation and atrophy, or microcysts, mesangial proliferation and glomerular sclerosis, were markedly less evident in the 5/6 subtotal nephrectomy group treated with losartan. Meanwhile, serum creatinine, BUN, and urinary protein excretion were lower in losartan-treated than in untreated nephrectomized rats. The expression ofα-SMA was also decreased in losartan-treated group(P<0.05).②The phosphorylation level of JAK2 was decreased after losartan treatment (P <0.05). The expressions of p-STAT1 and p-STAT3 were down-regulated in losartan-treated group( p-STAT1: STNX group 3.28±0.19, losartan-treated group 1.79±0.24; p-STAT3: STNX group 3.47±0.21, losartan treatment group 1.57±0.31 P all <0.05 ). Losartan could not affect the expression of JAK2, STAT1, and STAT3.③The expression of TGF-β1 mRNA in losartan -treated group was down-regulated than in STNX group(P <0.05).5. Effects of Bushenhuoxue Fang on EMT and activation of JAK/STAT signaling pathway in 5/6 subtotal nephrectomy (STNX) rats①Microscopy showed much less tubulo-interstitial damage in the rats of Bushenhuoxue Fang group than the rats in STNX group. Similarly, administration of Bushenhuoxue Fang soup could improve the renal function. Serum creatinine, BUN and urinary protein excretion were lower in Bushenhuoxue Fang-treated group than in untreated nephrectomized group(P <0.05). The expression ofα-SMA also decreased in Bushenhuoxuefang-treated group(P <0.05).②The phosphorylation level of JAK2 was decreased (P <0.05). The expression of p-STAT1 and p-STAT3 was down-regulated ( p-STAT1: STNX group 2.56±0.19, Bushenhuoxue Fang-treated group 1.76±0.14; p-STAT3: STNX group2.34±0.27, Bushenhuoxue Fang-treated group 1.86±0.42 P <0.05 ).③The expression of TGF-β1 mRNA in Bushenhuoxue Fang-treated group was down-regulated than in STNX group(P <0.05).Conclusions①The expressions of TGF-β1mRNA,α-SMA were increased and the content of TGF-β1 and type I collagen in the supernatants was also increased in the HKCs cultured in high glucose medium, suggesting that high glucose could induce EMT of HKCs. The levels of phosphorylation of JAK2, STAT1 and STAT3 were enhanced in the HKCs cultured in high glucose medium, suggesting that high glucose could activate JAK/STAT signaling pathway of HKCs. AG490, could decrease the level of phosphorylation of STAT1, STAT3 and decrease the secretion of TGF-β1, type I collagen in the HKCs cultured in high glucose medium. From these results it can be deduced that JAK/STAT signaling pathway might be involved in high glucose-induced EMT in HKCs.②Valsartan could inhibit activation of JAK/STAT signaling pathway and down-regulate the expression of TGF-β1mRNA, TGF-β1 and -SMA. It could be concluded that valsartan could attenuate high glucose-induced EMT in HKCs. The underlying mechanism might be attributed to inhibiting the activation of JAK/STAT signaling pathway.③5/6 subtotal nephrectomy can result in chronic renal interstitial fibrosis and a progressive decline in renal function in rats. Treatment with Losartan could inhibit phosphorylation of JAK2, STAT1 and STAT3, down-regulate the expression of TGF-β1mRNA , attenuate EMT in residual kidney, slow the progression of chronic renal insufficiency in STNX rats. These suggest that the protective effects of AT1Ra on renal function might be partly through inhibiting JAK/STAT signaling pathway.④Long-term treatment with Bushenhuoxue Fang can inhibit the expressions of TGF-β1 andα-SMA, and the progression of chronic renal failure caused by 5/6 subtotal nephrectomy, thus preserve the renal function. However, the exact protective mechanism of Bushenhuoxue Fang for renal function remains further investigation.
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