| BackgroundTubulointerstitial fibrosis is an integral part of the structural changes of kidney in progression of chronic kidney disease. Tubulointerstitial fibrosis has evolved as the most consistent predictor of an irreversible loss of renal function and progression to ESRD.Recent studies have demonstrated that a critical step in the pathogenesis of tubulointerstitial fibrosis is epithelial-to-mesenchymal transition, whereby renal tubular epithelial cells change phenotypically and functionally into myofibroblasts. Activation of tubulointerstitial myofibroblast leads to the production of excessive extracellular matrix with a predominance of interstitial collagens, and plays a critical role in the progression of chronic kidney diseases.Therefore, EMT in the kidney should be of significant interest as a t herapeutic target, and in this regard, it is quite important to prevent t ubular epithelial-to-mesenchymal transition in the therapy of tubulointer stitial fibrosis.Traditional Chinese medicine has a rich history of treating chronic kidney diseases over thousands of years and some herbal medicine and extracts have been proven containing anti-renal fibrosis properties both in vivo and in vitro. While specific therapies of preventing the progression of chronic kidney diseases are still lacking, seeking drug candidates or herbal compounds which can effectively prevent or inhibit EMT would provide an new strategie in the treatment of chronic kidney disease.Uremic clearance granule was produced by Guangzhou Consun pharmaceuti cals company and including Radix et Rhizoma Rhei, Radix Astragali, Radix Salviae Miltiorrhizae, Radix Sophorae Flavescentis, Rhizoma Atractylodis Macrocephalae and radix-polygoni multiflori, et al. Uremic clearance gran ule has been widely used for the treatment of chronic kidney diseases in traditional Chinese medicine. Although the clinical efficacy of Uremic cl earance granule had been established, its exact mechanism was still uncer tain. Whether Uremic clearance granule has protective effect in tubular e pithelial-to-mesenchymal transition is still lack of systematic study.ObjectiveTo investigate the effect of Uremic clearance granule on epithelial to-mesenchymal transition in vivo with unilateral ureteral obstruction mo del and in vitro with TGF-betal induced epithelial-to-mesenchymal transit ion of HK-2 Cells. To detect the inhibitory effect of Uremic clearance gr anule on tubulointerstitial fibrosis.Methods1. We used control study methods, included chronic kidney disease patients (stage 3 to stage 5) who were hospitalized in our hospital from 2008 to 2009, analysised the Distribution of TCM symptoms and the using of Uremic clearance granule.2.50 SD rats were random divided into three groups:sham operation group(n=10), UUO group(n=20), and UUO with Uremic clearance granule treated group(UUO+UCG, n=20). Unilateral ureteral obstruction model(UUO) was performed by double ligating the left ureter using 4-0 silk after a left lateral abdominal incision. Uremic clearance granule was given at a dose of 4.5g/kg body weight per day by gavage after the surgery. The sham operation group and UUO group rats were received the same volume of saline solution. Rats were sacrificed 7 and 14 days (n=10 per group) after surgery. Kidney sections were prepared and stained with Masson trichrome staining to detect the renal morphologic changes and the degree of tubulointerstitial fibrosis. Tissue immunofluorescence staining and western-blot analysis were used to detect the localization and expression of E-cadheirn, Vimentin andα-SMA in frozen sections and kidney tissues.3. Acquired rat serum containing Uremic clearance granule, to detect whether the healthy rat serum or the rat serum containing Uremic clearance granule had cytotoxic responses to HK-2 cells. Using 10ng/ml of TGF-β1 cultured for 48 or 72h in HK-2 cells to induce epithelial-to-mesenchymal transition, and detected whether simultaneous incubation of rat serum containing Uremic clearance granule had inhibitory effect. Microscope was used to observed the morphologic changes of HK-2 cells. Immunofluorescence staining and western-blot analysis were used to detect the localization and the expression of E-cadherin and Vimentin in cultured cells.Results1. A total of 193 patients of chronic kidney disease patients(stage3 to stage 5, non dialysis) were fit in our study. We found that the TCM synptoms of chronic kidney disease patients were mainly spleen and kidney Qi, wet muddy stasis, spleen deficiency and damp heat stasis. The utilization rate of Uremic clearance granule also reached to 80%.2. In vivo study:(1). Uremic clearance granule inhibited renal tubulointerstitial fibrosis in UUO rats. Rats of sham operation group showed normal glomerular and tubulointerstitial morphology. Rats of UUO group showed the enlargement of tubular lumen, tubular atrophy, the expanded tubular interstitial volume and tubulointerstitial fibrosis at day 7 and more severe at d 14 when almost all of the tubules were destroyed. Meanwhile, Uremic clearance granule improved histopathologic changes in UUO rats at day 7 and day 14. The tubulointerstitial fibrosis score was reduced by 17.5%±1.1% at day 7 and 20.0%±1.2% at day 14 in Uremic clearance granule treated group, compared with UUO group (P<0.05, respectively)(2). Uremic clearance granule inhibited EMT in UUO rats. We demonstrated de novo Vimentin andα-SMA expression in the expanded tubular interstitial volume at day7, whileα-SMA was detected only exclusively in vascular smooth muscle cells and Vimentin was almost not expressed in the sham operation group rats. As the disease progressed, the number ofα-SMA-positive cells and Vimentin-positive cells were increased, which were mainly expressed in the fibrotic Area. E-cadherin was expressed in most of the epithelial cells in sham operation group rats. The expression of E-cadherin was significantly decreased at day7, and was continuously downregulated with lesion progression at day 14. According to the immunofluorescence staining and western-blot analysis results, we provided that Uremic clearance granule could partly inhibit the expression of Vimentin andα-SMA and improved the expression of E-cadherin at day7 and day 14, compared with UUO group (P<0.05, respectively)3. In vitro study:(1). Both the healthy rat serum or rat serum containing Uremic clearance granule used in this study would not mediate by cytotoxic response. The rat serum containing Uremic clearance granule was safe to use in the further culture.(2). Our results showed morphologic changes after 10ng/ml of TGF-β1 cultured for 48 or 72h in HK-2 cells, with cells becoming elongated in shape, disassociating from neighboring cells and losing their cobblestone monolayer pattern.After HK-2 cells incubated in medium containing lOng/ml of TGF-β1 for 48 or 72h, the protein level of Vimentin was increased, while reduction expression of E-cadherin was detected, using immunofluorescence staining and western-blot analysis. Simultaneous incubation of rat serum containing Uremic clearance granule with TGF-β1 could partly prevent the HK-2 cells from changing to fibroblast phenotype and maintained the epithelial morphology of the HK-2 cells. Both 5% and 10% rat serum containing Uremic clearance granule could significantly prevent the change in Vimentin and E-cadherin, with increasing expression of E-cadherin and reducing expression of Vimentin, compared with TGF-β1 controls. These results indicated that rat serum containing Uremic clearance granule not only prevented the de novo expression of the fibroblast marker Vimentin in HK-2 cells but also prohibits the loss of the epithelial marker E-cadherin.Conclusions1. The TCM synptoms of 193 chronic kidney disease patients were mainly spleen and kidney Qi, wet muddy stasis, spleen deficiency and damp heat stasis. The utilization rate of Uremic clearance granule also reached to 80%. Uremic clearance granule meeted the TCM group distribution, however, its molecular and cellular mechanisms were not yet clea. We carryed the proposed follow-up animal and cell experiments.2. UUO rats of 7 days and 14 days turned to the typical tubular epith elial-to- mesenchymal transdifferentiation (EMT), with decreasing express ion of E-cadherin and increasing expression of Vimentin,α-SMA. Uremic c learance granule could maintain the expression of E-cadherin and suppress ed Vimentin, alpha-SMA expression at day 7 and day 14, compared with UUO group.3. In vitro experiment our data revealed that rat serum containing Ur emic clearance granule could partly inhibit TGF-betal induced myofibrobla st phenotype and maintained the epithelial morphology. It was partially t hrough modulating Vimentin increase and E-cadherin reduction.
|
PDF Full Text Request