The Role Of ELMO1in Renal Tubuloinsterstitial Fibrosis And Its Mechanism

Progressive tubulointerstitial fibrosis is the final common pathway for all kidney diseases leading to chronic renal failure. The histopathology of tubulointerstitial fibrosis features deposition of interstitial matrix in association with inflammatory cells, tubular cell loss, fibroblast accumulation, and rarefaction of the peritubular microvasculature. Excessive deposition of extracellular matrix, particularly the presence of collagenous fibers, is the most striking and name-lending feature of tubulointerstitial fibrosis.Many studies have shown that progressive renal fibrosis is mediated by multiple mediators including growth factors, cytokines, metabolic toxins, and stress molecules via multiple mechanisms and pathways. Among them, transforming growth factor-β1(TGF-β1) has been recognized as a key mediator in the pathogenesis of renal fibrosis Active TGF-β binds its receptors and functions as autocrine and paracrine manners to exert its biological and pathological activities via Smad-dependent and independent signaling pathways. It is now well established that the binding of TGF-β1to its receptor Ⅱ (TβRⅡ) can activate the TGF-β receptor type Ⅰ (TβRI)-kinase, resulting in phosphorylation of Smad2and Smad3, two receptor-associated Smads (R-Smads). Subsequently, phosphorylated Smad2and Smad3bind to the common Smad4and form the Smad complex, which translocates into the nucleus to regulate the target gene transcription. Many fibrogenic genes, such as (collagen Ⅰ and collagen Ⅲ) and tissue inhibitor of MMP-1(TIMP-1), are the downstream targets of TGF-β/Smad3signaling, suggesting that Smad3may be a critical mediator of TGF-β/Smad signaling in fibrosis. The non-canonical TGF-β signaling pathways may interact with Smad signaling in a tissue-or cell type-specific manner. TGF-βinduced the production of the collagen Ⅰ through a PI3K-Rac1-ERK signaling pathway.TGF-β induction appears to be a convergent pathway that integrates, directly or indirectly, the effects of many other fibrogenic factors. Some of these, such as angiotensin Ⅱ and high glucose, act as an upstream TGF-β inducer, whereas others such connective tissue growth factor work as its downstream effector.Rac1is a member of Rho family of small guanosine triphosphatases(GTPases). Rac1is ubiquitously expressed and exist in two conformational states, an inactive GDP-bound form and an active GTP-bound form. In response to extracellar signals, the interconversion of these states via guanine nucleotide exchange factors(GEFs) which convert Rac1to its active form, and GTPase-activating proteins(GAPs), which inactive Rac1.A study suggested that TGF-β1could induce the production of collagen Ⅰ via activate Rac1and ERK. Besides, active Racl could increase the production and secretion of Matrix Metalloproteinases (MMPs), activate NF-kappaB and facilitate the neuclear translocation of β-catenin. Thus, Rac1signaling and TGF-β1signaling seem to be a convergent pathway. And Racl may be critical in the non-canonical TGF-β signaling pathways. DOCK180is a member of DOCK family of Rho GEFs. DOCK180interacted with ELMO to robustly and highly specifically activated Rac. ELMO could facilitate the accumulation of DOCK180protein by preventing its degradation by the ubiquitin-proteasome systerm. Besides, ELMO facilitate the co-localization of DOCK180and Rac at the membrane and therefore indirectly endorses Rac GEF activity of the complex.ELMO1expression was weakly detectable mainly in tubular and glomerular epithelial cells in normal mouse kidney, and was clearly elevated in the kidney of diabetic mice. ELMO1expression was elevated in COS cells cultured under high glucose condition compared with cells under normal glucose conditions. Furthermore, the expression of collagen Ⅰ and fibronectin were increased in cells that overexpress ELMO1.Now that high glucose act as a upstream TGF-β inducer, ELMO1interacted with DOCK180to synergistically activate Racl, TGF-βcould induce the production of extracellar matrix through Rac1. Thus, whether ELMO1is vital in TGF-β-induced extracellar matrix? If so, which pathway did TGF-β induce them by? And because of the pivotal role TGF-β of in renal tubuloinsterstitial fibrosis, ELMO1may play a role in tubuloinsterstitial fibrosis.In order to answer these questions, our research use a rat unilateral ureteral occlusion (UUO) model that represents renal tubuloinsterstitial fibrosis to observe the expression and the location of ELMO1. And the influence on expression of ELMO1in a tubular epithelial cell lines, NRK52E induced by TGF-β were investigated. Futhermore, ELMO1was also overexpressed and silenced to explore its impact on the expression of collagen I and Racl activity. The primary aim of this research is to study the role of ELMO1in renal tubuloinsterstitial fibrosis and its possible molecular mechanism to provide novel target for intervention.Methods:a) The performance of rat unilateral ureteral occlusion (UUO)24male SD rats, weighted range from250to280g, were performed a sham surgery (6rats)or UUO surgery (18rats). The rats were feeded routinely with normal food and water,6rats in the UUO group were sacrificed3,7and14days after the surgery respectively, the sham group were sacrificed14days after the surgery. Half of the left kidney were used for immunohistochemistry, the other half were frozen in liquid nitrogen and transfered to-80℃freezer for Western blot.2. The expression level of ELMO1, collagen Ⅰ and α-SMA in the kidney of rats Western blotting were performed to test the protein level of ELMO1, collagen Ⅰ and α-SMA in the kidney tissue from sham, UUO day3, day7,day14.3. The expression and location of ELMO1protein in the rat kidney tissue.ELMO1protein in the rat kidney tissue from sham and UUO day7were stained by immunohistochemistry.4. Cell cultureThe NRK52E cells were cultured in F12/DMEM medium with10%FCS at37℃,5%CO2.5. The influence on ELMO1and collagen Ⅰ protein expression in NRK52E cells induced by TGF-β1.The NRK52E were plated in35mm dishes with appropriate proportion, when60%confluence was reached, the culture medium were replaced by serum-free F12/DMEM medium. After starvation for24hours, the cells were treated with10ng/ml TGF-β1. And harvested after0,24,48,72hours. Then Western blotting were performed to detect the protein levels of ELMO1and collagen Ⅰ.6. The influence on the collagen Ⅰ protein level by overexpressing ELMO1The NRK52E were plated in35mm dishes with appropriate proportion, when60%confluence was reached, the pcDNA-HA-ELMO1dilueted by OPTI-MEM was mixed with lipofectamine2000dilueted by OPTI-MEM, then was transfected to NRK52E. After5hours later, the medium was replaced by F12/DMEM medium supplemented with10%FCS. And the cells were cultured for another18hours before its starvation for12hours. Then, the cells were treated with or without10ng/ml TGF-β1for48hours.Western blot was applied to detect the protein expression of ELMO1, collagen Ⅰ.7. The influence on the Rac1activity by overexpressing ELMO1The NRK52E were plated in35mm dishes with appropriate proportion, when60%confluence was reached, the pcDNA-HA-ELMO1dilueted by OPTI-MEM was mixed with lipofectamine2000dilueted by OPTI-MEM, then was transfected to NRK52E. After5hours later, the medium was replaced by F12/DMEM medium supplemented with10%FCS. And the cells were cultured for48hours before the Rac1activity was testedCell lysate were treated with Pull-down experiment and Western blot to detect the active Rac1. An equal amount of whole cell lysate was also blotted to evaluate total Rac1.8. The influence on the Rac1activity by ELMO siRNAThe NRK52E were plated in35mm dishes with appropriate proportion, when60%confluence was reached, the NC siRNA or ELMO1siRNA dilueted with OPTI-MEM was mixed with lipofectamine2000dilueted with OPTI-MEM, then was transfected to NRK52E. After5hours later, the medium was replaced by F12/DMEM medium supplemented with10%FCS. And the cells were cultured for48hours before the Rac1activity was testedCell lysate were treated with Pull-down experiment and Western blot to detect the active Racl. An equal amount of whole cell lysate was also blotted to evaluate total Rac1.9. Statistical Analysis All experiments were performed in triplicate. Continuous variables, expressed as mean±standard deviation, were compared using one-way ANOVA or Welch method when the assumption of equal variances did not hold. Pairwise comparisons were evaluated by LSD or Dunnett’s T3procedure when the assumption of equal variances did not hold. The difference of variables bwteen two samples were compared using independent samples t test. Two-tailed P values,0.05were considered statistically significant. Statistical analysis were performed with SPSS13.0.Results1. The protein levels of ELMO1, collagen Ⅰ and α-SMA were elevated in rat renal tissue from the UUO groups(day3, day7, day14after UUO), compared with that from the sham group.The results suggest that it is statistically significantly between groups mentioned above for the protein levels of ELMO1, collagen Ⅰ and α-SMA. The assumption of equal variances hold(ELMO1P=0.174; collagen Ⅰ P=0.167; a-SMA P=0.102), Oneway ANOVA was used, ELMO1F=139.315, P<0.001; collagen Ⅰ F=21.052, P<0.001; α-SMA F=74.619, P<0.001. The expression of ELMO1, collagen Ⅰ and α-SMA in UUO day3group were3.1±0.33times (P<0.001);1.7±0.54times (P<0.05);19.8±1.88(P<0.001) that of sham group respectively. As time went by after the occlusion, the expression of those proteins was increasingly elevated. At day7after UUO, ELMO1, collagen Ⅰ and α-SMA expression was4.4±0.48times (P<0.001),2.0±0.33times (P<0.05),19.9±2.40(P<0.001), compared with sham group. At day14after UUO, ELMO1, collagen Ⅰ and α-SMA expression8.3±0.67times (P<0.001);3.2±0.30times (P<0.001);23.7±2.75(P<0.001),compared with sham group.The results of immunohistochemistry alao showed the ELMO1expression at day7after UUO was higher than that of sham, and ELMO1was mostly located in the proximal tubular epithelial cells.2. The time effect of TGF-β1on the ELMO1, collagen Ⅰ protein expression in NRK52E cells.The results showed it is statistically significantly between different time points. The assumption of equal variances hold(ELMO1P=0.455; collagen Ⅰ P=0.495), One-Way ANOVA was used, ELMO1F=40.027, P<0.001; collagen Ⅰ F=176.200, P<0.001. Treating the cells with TGF-β1for24hours, ELMO1and collagen Ⅰ expression was2.4±0.15times (P<0.001),3.3±0.14times (P<0.001) that of0hours; After48hours, ELMO1and collagen Ⅰ expression was2.4±0.26times (P<0.001);3.6±0.26times (P<0.001). When72hours after treatment, the ELMO1and collagen I expression was2.5±0.43times(P<0.001),4.2±0.20times(P<0.001)that of0hours.3. The effect of TGF-β1on the Rac1activity in NRK52E cellsThe results showed the constitutive Rac1activity of the control group was rather low. With10ng/ml TGF-β1treatment for48hours, it is s statistically significantly between the control and TGF-β1group. The Rac1activity of TGF-β1group was4.4±0.23times that of control, two independent samples t-test was used, the assumption of equal variances was hold (F=2.525, P=0.187), P<0.001.4. The influence of ELMO1overexpression on the Collagen Ⅰ expresssionNRK52E cells were transfected with pcDNA-HA-ELMO1to overexpress ELMO1. Normal (N) and the ELMO1-overexpressed (ELMO1-wt) cells were treated with or without10ng/ml TGF-β for48h. It is statistically significant between each group for the differences of ELMO1, collagen Ⅰ expression. The assumption of equal variances hold (ELMO1P=0.108; collagen Ⅰ P=0.052;) Oneway ANOVA was used. ELMO1F=43.255, P<0.001; Collagen Ⅰ F=100.333, P<0.001. ELMOl-wt group compared with N group, which ELMO1expression is9.5±1.35times that of N group (P<0.001), which means ELMO1overexpression is successful, collagen Ⅰ expression level was not statistically significant compared with N group (P=0.092), show that only overexpressing ELMO1can’t cause collagen Ⅰ expression to rise. However, N+TGF-β group of collagen Ⅰ expression is4.6±0.83times that of N group (P<0.001); And ELMO1-wt+TGF-β group is9.5±0.93times that of N group (P<0.001). This suggests that ELMO1overexpression can further enhance collagen I production induced by TGF-β.5. The influence of ELMO1overexpression on the Racl activityNRK52E cells were transfected with pcDNA-HA-ELMO1to overexpress ELMO1. The Rac1activity were tested for Normal (N) and the ELMO1-overexpressed (ELMO1-wt) cells. Pull-down and western blot methods were applied to detect racl activity in NRK52E cells. ELMO1expression between the two groups have statistical differences, the two independent sample t-test was used, The assumption of equal variances hold (F=0.581, P=0.074), t=-5.324,P<0.05, ELMO1-wt group is4.4±1.11times that of the N group which suggested overexpressing ELMO1is successful. Rac1activity between the two groups have statistical differences, the two independent sample t-test was used. The assumption of equal variances hold (F=7.268, P=0.054), t=-5.790, P<0.05, ELMO1-wt group is3.5±0.75times that of N group. This suggests that overexprssing ELMO1in NRK52E cells can further promote Rac1activation5. The influence of ELMO1siRNA on the Rac1activityNRK52E cells were transfected with NCsiRNA or ELMOl to silence ELMOl. The Racl activity were tested for NCsiRNA and the ELMO1siRNA cells. Pull-down and western blot methods were applied to detect racl activity in NRK52E cells. ELMOl expression between the two groups have statistical differences, the two independent sample t-test was used, The assumption of equal variances hold (F=1.467, P=0.292), t=-10.290, P<0.05, ELMO1-wt group is46%that of the N group which suggested ELMO1siRNA is effective. Racl activity between the two groups have statistical differences, the two independent sample t-test was used. The assumption of equal variances hold (F=0.738, P=0.439), t=10.735, P<0.05, ELMO1-wt group is34%that of N group. This suggests that overexprssing ELMO1in NRK52E cells can further promote Rac1activationConclusion1. This study has successfully established a model of renal interstitial fibrosis model of UUO rats.2. ELMO1expression was significantly increased in the kidney of UUO rats. And ELMO1was mainly located in tubular epithelial cells3. TGF-β1-induced tubular epithelial cells to express ELMO14. TGF-β1induced Rac1activation in tubular epithelial cells5. ELMO1promote the activation of Racl6. ELMO1promote collagen Ⅰ synthesis in tubular epithelial cells induced by TGF-β1...