Blockade Of Orai1 Store-operated Calcium Entry Protects Against Renal Fibrosis

Renal fibrosis is pathophysiologic process of progressive kidney disease. When sufferring from various pathogenic factors such as trauma, infection, inflammation, blood circulation obstacle, and immune response, the kidney show damaged intrinsic cells and extra cell matrix accumulation?ECM?, accompanied with renal parenchyma clerosis, scarring, and lose-of-function by the end. The damage of kidney, no matter caused by primary glomerulopathy, diabetes, or ureteral obstruction, is closely related to renal fibrosis. Therefore, renal fibrosis is the final result of many chronic kidney diseases?CKD? and is the main cause of end-stage renal failure.Renal fibrosis, including glomerular sclerosis and renal tubular interstitial fibrosis, is essentially characterized by excessive accumulation and deposition of ECM. Epithelial-mesenchymal transition?EMT? is so far considered to be one of the important causes leading to renal fibrosis. When suffering stimulus, renal tubular epithelia cells transform into fibroblasts and produce excessive ECM. The ECM then accumulate in the renal interstitium and lead to a loss of kidney tubules. This process is called epithelial-mesenchymal transition?EMT?. The renal tubular epithelia cells undergoing EMT was associated with morphologic changes, including elongation and hypertrophy, separation from neighboring cells, and decreased protein expression of the epithelial marker E-cadherin, cytokeratin, and zonula occludens 1?ZO-1?. At the same time, the protein abundance of vimentin, a-SMA, S100A4, fibronectin?FN?, fibroblast-specific protein 1?FSP-1?, and collagen I, III, and IV, as mesenchymal markers, is upregulated in renal tubular epithelia cells undergoing EMT. Transforming growth factor-beta 1?TGF-?1? is the strongest inducer of EMT. It mediates EMTthrough smad dependence signal. TGF-?1 stimulates serine/threonine type I and type II receptor by combining with them, which lead to phosphorylation of smad2 and smad3. Phosphorylated smad2/3 and smad4 composed a complex, then transfer to nucleus to regulate the transcription of target genes which are associated with EMT and renal fibrosis. It shows an importance to study the factors which can mediate EMT in renal fibrosis.Calcium is an important second message in cellular signal transduction. The homeostasis of calcium is crucial to the normal physiological functions in cells. Disruption of calcium homeostasis is related to kidney diseases. Early studies suggest an upregulation of the expression of calcium channels or the intracellular calcium concentration([Ca²⁺]i) in fibroblasts, podocytes and renal tubular epithelia cells during renal fibrosis. It shows that changes of [Ca²⁺]i is involved in renal fibrosis. Calcium channels may be the effective target in protecting against renal fibrosis.Calcium ion channel in cytomembrane is the main way to regulate [Ca²⁺]i. There are three kind of calcium channels exist in mammalia cells: voltage-dependent Ca²⁺ channel?VDCC?, receptor-operated calcium channel?ROCC?, and store-operated Ca²⁺channel?SOCC?. VDCC is mainly expressed in excitable cells such as cardiac cells and neurons, participating in physiological functions like neurotransmitter releasing, smooth muscle contraction, and cardiac-pacing. ROCC and SOCC play important roles in excitable and nonexcitable cells. They are the main channels mediated the Ca²⁺ influx. Studies show that ROCC and SOCC participate in some physiological functions such as proliferation, migration, and matrix production in mesangial cells, fibroblasts, and podocytes. The current study suggests Orai1 and stromal interaction molecular1?Stim1? together consititute the SOCC. Orai1 is a kind of membrane protein, containing four transmembrane domains?TM1-TM4? which form a pore for Ca²⁺ influx. Stim1, acts as a Ca²⁺ concentration sensor, is located in the endoplasmic reticulum?ER? with a stable state. When calcium store is depleted, Stim1 and Orai1 move together to the EM-PM apposition and combine with each other. Then the pore of Orai1 is opened accompany with Ca²⁺ entry in the cells. This process is considered as the activated mechanism of SOCC.In recent years, more and more researches discuss about Orai1 regulating kidney diseases. Early studies showed that Orai1 is involved in glomerular disease, CKD, and renal tumor. Moreover, Orai1 plays an important role in regulating the proliferation and matrix production of mesangial cells, EMT of fibroblasts, and proliferation and migration of renal clear cell cancer cells. However, there is little known about the regulation of Orai1 in renal tubular epithelia cells and renal fibrosis.Endophilins are a family of enrichment of pyrrole glycine SH3 domains. Endophlin A2 is one of the subfamilies which widely distributes in lots of tissues and organs. Previous studies of Endophilin A2 mainly about the regulation of clathrin mediated endocytosis, synaptic endocytosis, and neurons excitability. Later studies have reported that Endophilin A2 can inhibit the endocytosis of matrix metalloproteinase, promote the degradation of ECM, and regulate the intracellular signal transduction. It's worth noting that recent reports showed Endophilin A2 could also regulate glomerular filtration and nephrotic syndrome by affecting the endocytosis of podocytes. These research findings suggest a new chapter in studying the role of Endophilin A2 in kidney diseases. Nevertheless, the role of Endophilin A2 in renal fibrosis is not yet clear. Recently, we found that Endophilin A2 was downregulated in renal cortex with fibrotic. The same changes of Endophilin A2 protein expression were found in TGF-?1-treated renal tubular epithelia cells, which was opposite to the expression of Orai1 protein. It shows us Orai1 Ca²⁺ channel and Endophilin A2 might be the key mecahnisms of promotion and inhibiton of renal fibrosis.To identify the effect and mechanism of Orai1-mediated calcium influx in renal fibrosis, and discuss if Endophilin A2 regulate renal fibrosis combined with Orai1, we designed the projects as follows: Part I, we detected the changes of fibrotic kidney and fibrotic markers expression after knockdown of Orai1 or inhibition of Orai1 channel-mediated Ca²⁺ influx in mice with renal fibrosis induced by high-fat diet or UUO. Part II, we discussed the mechanisms of Orai1 in renal fibrosis in renal tubular epithelia cells in vitro. Part III, by using renal tubular epithelia cells and transgenic mice, we studied the role of Endophilin A2 in renal fibrosis.Part I Blockade of Orai1 Ca²⁺ channel protects against mice renal fibrosis induced by high-fat diet or UUOTo study the role of Orai1 Ca²⁺ channel in renal fibrosis, we first detected the expression of Orai1 in the fibrotic kidney of mice induced by high-fat diet?HFD? or UUO. Then we investigated the renal structure while blockade of Orai1 Ca²⁺ channel by using adenovirus?Ad? short hair?sh RNA? or Ca²⁺ channel blockers. The results were shown as follows:1. Immunohistochemistry demonstrated that Orai1 was mainly located in the proximal tubules in the cortex of Apo E-/- mice kidney. Little labeling was found in the glomeruli or medullary tubules. Immunofluorescence showed a colocalization of Orai1 with sodium-hydrogen exchanger type 3. The abundance of Orai1 protein expression in cultured HMCs was significantly lower than that in HK2 cells, suggesting a more important role for orai1 in proximal tubule cells, but not mesangial cells in renal diseases.2. We investigated the expression of Orai1 in kidney cortex of 8-week-old Apo E-/-mice with 12 weeks HFD. The results showed the abundance of Orai1 and m RNA in the cortex was significantly increased in Apo E-/- mice fed with HFD when compared with controls.3. 8-week Apo E-/- mice were fed with HFD for 8 weeks, then they were injected with Ad-Orai1-sh RNA?5x109 pfu/mouse? to knockdown Orai1 in vivo, or injected with Ad-GFP-sh RNA?5x109 pfu/mouse? as controls via tail vein. The mice were fed with HFD for another 4 weeks, then they were sacrificed to observe the formation of renal fibrosis. The results showed: compared with the Ad-GFP-sh RNA group, the expression of Orai1 protein and m RNA was decreased significantly in mice treated with Ad-Orai1-sh RNA. We detected the expression of collagens in Apo E-/- mice kidney through Masson Thrichrome and Sirius Red staining. The data showed marked fibrotic lesions in the kidneys of Apo E-/- mice fed with HFD when compared with controls. By contrast, knockdown of Orai1 by Ad-Orai1-sh RNA showed less fibrosis in tubulointerstitial areas than in Ad-GFP-sh RNA controls.injection per day after 8 weeks HFD. In combined with the HFD, 4 weeks later the mice were sacrificed to observe the formation of renal fibrosis. The results as follow: Masson Thrichrome and Sirius Red staining showed inhibition of Orai1 with Ca²⁺ channel blocker SKF96365 decreased fibrosis in tubulointerstitial areas of Apo E-/-mice when compared with PBS controls. 4. 8-week Apo E-/- mice were given SKF96365 10 mg/kg or PBS by intraperitoneal5. Knockdown of Orai1 or inhibition of Orai1 Ca²⁺ channel prevented HFD-induced impaired renal function. Serum creatinine levels and albuminuria were increased in mice on HFD, which was attenuated by inhibition of Orai1 Ca²⁺ channel.6. Fibronectin, a-SMA, TGF-?1, smad3, and collagen I, III, and IV are involved in renal fibrosis. Western blotting and immunohistochemistry demonstrated that protein expression of all these markers was significantly upregulated in the cortexes of kidneys of Apo E-/- mice fed with HFD compared with controls, whereas Ad-Orai1-sh RNA or SKF96365 treatment was associated with reduced protein expression of these markers.7. 12-week C57BL/6 mice were undergone UUO or sham accompany with treatment of Ad-Orai1-sh RNA?5x109 pfu/mouse? or Ad-GFP-sh RNA?5x109 pfu/mouse? on day 1 and day 3 after UUO. Or the mice were given SKF96365?10 mg/kg? or PBS by intraperitoneal injection per day after UUO. The kidneys were harvested at day 7 and day 14 after UUO, respectively. The data showed: Masson Trichrome showed marked fibrotic lesions in the obstructed kidneys of UUO-treated mice when compared with sham-treated mice. By contrast, knockdown of Orai1 by Ad-Orai1-sh RNA or inhibition of Orai1 with Ca²⁺ channel blocker SKF96365 showed less fibrosis in tubulointerstitial areas than in their respective controls.8. Western blotting and immunohistochemistry demonstrated that protein expression of the profibrotic markers fibronectin, a-SMA, TGF-?1, and collagen I, III, and IV was significantly upregulated in the renal cortex of obstructed mice compared with sham mice, whereas Ad-Orai1-sh RNA or SKF96365 treatment was associated with reduced protein expression of the above markers. Moreover, UUO was associated with significantly increased abundance of Orai1 and STIM1 protein expression.?chronic tubule-interstitial nephritis, proliferative sclerotic lg A nephropathy, focal proliferative sclerotic lg A nephropathy? and 1 patient with glomerular minimal change diseases, which was considered as control. Masson Trichrome staining showed fibrotic lesions in kidneys from patients with focal proliferative sclerosis and tubule-interstitial nephritis, but little in minimal change disease. Immunocytochemistry demonstrated that Orai1 protein was expressed in tubular epithelia, glomerular cells, endothelia of blood vessels, and fibroblasts. The intensity of Orai1 immunostaining in the tubular epithelium was much higher in specimens from patients with fibrotic kidney diseases, compared with that from patients with glomerular minimal change diseases, indicating the involvement of Orai1 in the development of human renal fibrosis. 9. We collected the kidney biopsy tissue from 3 different patients with renal fibrosisSummary: 1. Orai1 was located mainly in tubular epithelium of renal cortex but not glomerulus and renal medulla. 2. HFD and UUO could induce renal fibrosis in mice. Orai1 was upregulated in the fibrotic kidneys compared to their controls. 3. Orai1 was higher expressed in the kidneys from patients with fibrotic kidney diseases when compared to controls. 4. Knockdown of Orai1 or inhibition of Orai1 Ca²⁺ channel protected against renal fibrosis of mice induced by HFD and UUO, and improved the kidney function.Part II Mechanisms of Orai1 Ca²⁺ channel regulating renal fibrosisIn order to further illustrate the mechanisms of Orai1 Ca²⁺ channel contributing to renal fibrosis, we first discussed the role of TGF-?1 in the change of [Ca²⁺]i in tubular epithelial cells and the expression of profibrotic markers. And then we studied the mechanisms of Orai1 Ca²⁺ channel promoting renal fibrosis through TGF-?/smad signaling pathway, Ca²⁺-Ca MKII signaling pathway, and Ca²⁺-calcineurin signaling pathway. We found that:significantly decreased when the cells were transfected with Ad-Orai1-sh RNA?200 MOI?. TGF-?1?5 ng/ml? significantly increased [Ca²⁺]i in HK2 cells, knockdown of Orai1 or STIM1 markedly attenuated TGF-?1-evoked [Ca²⁺]i compared with controls. Similarly, angiotensin II?Ang II??100 nmol/l? induced [Ca²⁺]i was also significantly prevented by Orai1 inhibition. 1. The protein expression of Orai1 in human tubular epithelial cells?HK2? was2. The protein expression of fibronectin and collagen IV was markedly increased in HK2 cells when treated with TGF-?1?5 ng/ml? or Ang II?100 nmol/l? for 48 hours. Whereas, inhibition of Orai1 with Ad-Orai1-sh RNA attenuated the increased expression of fibronectin and collagen IV induced by TGF-?1 and Ang II.3. Treatment with TGF-?1?5 ng/ml? for 30 minutes significantly increased the protein abundance of phosphorylated smad2/3, whereas smad2/3 protein abundance was maintained in HK2 cells. Knockdown of Orai1 with Ad-Orai1-sh RNA remarkably suppressed the TGF-?1-induced increased expression level of phosphorylated smad2/3. Similarly, SKF96365?20 ?mol/l? or 2-APB?100 ?mol/l? inhibited TGF-?1-induced increased phosphorylated smad2/3 expression but didn't increase the expression of smad2/3. These results indicate that Orai1 Ca²⁺ channel is involved in TGF-?/smad signaling pathway in HK2 cells.4. Pre-incubation with the calcineurin inhibitor FK-506?10 ?mol/l? suppressed TGF-?1–stimulated smad2/3 phosphorylation,fibronectin, and collagen IV expression in HK2 cells. Whereas, Ca MK II inhibitor KN-93?10 ?mol/l? didn't affect the expression level of smad2/3 phosphorylation, fibronectin and collagen IV after TGF-?1 treatment. Neither FK-506 nor KN-93 affect the expression of smad2/3 in HK2 cells. These findings indicate that it is Ca²⁺ calcineurin signaling but not Ca²⁺-Ca MK II signaling contributes to TGF-?1-induced renal fibrosis.5. In HK2 cells, treatment with TGF-?1?5 ng/ml? for 48 hours was associated with morphologic changes, including elongation and hypertrophy, separation from neighboring cells, and decreased protein expression of the epithelial marker E-cadherin. At the same time, the protein abundance of a-SMA, vimentin, and S100A4, as mesenchymal markers, was significantly upregulated in HK2 cells treated with TGF-?1. Knockdown of the Orai1 Ca²⁺ channel markedly preventedmorphologic changes induced by TGF-?1 in HK2 cells. Inhibition of the Orai1 Ca²⁺ channel with Ad-Orai1-sh RNA, SKF96365 or 2-APB markedly reversed the effect of TGF-?1 on EMT by restoring the expression of E-cadherin and by attenuating the expression of a-SMA, vimentin, and S100A4 in HK2 cells. These data suggest a potential role of the Orai1 Ca²⁺ channel in tubular epithelial cells EMT induced by TGF-?1.Summary 1. Both TGF-?1 and Ang II promoted the level of [Ca²⁺]i and increased the expression of fibrotic markers in HK2 cells. TGF-?1showed more significant effects than Ang II. 2. Inhibition of the Orai1 Ca²⁺ channel significantly suppressed the increased level of [Ca²⁺]i and fibrotic markers expression induced by TGF-?1and Ang II in HK2 cells. 3. Orai1 Ca²⁺ channel-mediated Ca²⁺ influx regulated TGF-?/smad signaling pathway throug Ca²⁺-calcineurin signaling pathway. 4. Inhibition of the Orai1 Ca²⁺ channel protected against renal fibrosis by preventing HK2 cells EMT induced by TGF-?1.Part III Endophilin A2 protected against renal fibrosis as downstream signal molecules of Orai1To further investigate the mechanisms of Orai1 Ca²⁺ channel regulating renal fibrosis, we detected the effect of Endophilin A2 on matrix expression in HK2 cells after finding that Endophilin A2 protein expression was inhibited by Orai1 Ca²⁺ channel. Next,. We further investigated the process of renal fibrosis in Endophilin A2 transgenic mice.1. Endophilin A2 was found mainly in renal tubule epithelial cells, but not glomerulus or medulla in the kidneys, which was the same as Orai1 protein distribution in the kidney.2. The expression of Endophilin A2 was decreased in UUO-induced fibrotic kidneys. Nevertheless, we found the abundance of Endophilin A2 in Ad-Orai1-sh RNA treatedmice was increased when compared with controls. Endophilin A2 protein expression was time dependence downregulated with TGF-?1 treatment in HK2 cells, accompanied with upregulation of Orai1 protein expression. It suggested Endophilin A2, associated with Orai1, was involved in renal fibrosis formation and morphology changes of renal tubule epithelial cells.3. We found that Orai1 Ca²⁺ channel regulated HK2 cells EMT by affecting TGF-?/smad signal pathway. To futher discus the role of Endophilin A2 in renal fibrosis, we detected the effect of Endophilin A2 on TGF-?/smad signal pathway and matrix expression in HK2 cells. The results showed: overexpression of Endophilin A2 suppressed the TGF-?1-induced increased expression level of phosphorylated smad3, FN, and collagen IV, with no change of smad3 protein expression in HK2 cells. On the contrary, the upregulation of phosphorylated smad3, FN, and collagen IV induced by TGF-?1 was increased by knockdown of Endophilin A2 in HK2 cells, with on change of smad3 protein expression neither. The results showed that Endophilin A2 prevented renal tubule epithelial cells from transiting to mesenchyme.4. PCR products showed that there was a 270 bp target band in Endophilin A2 transgenic mice. Western blotting results demonstrated that the expression of Endophilin A2 was increased in kidneys from transgenic mice.5. There was no obvious difference of the physiological morphology between the Endophilin A2 Tg mice kidneys and the wildtype?WT? mice kidneys. However, Endophilin A2 Tg mice were associated with lower expression of FN, a-SMA, TGF-?1, and collagen I, III, and IV when compared to WT mice in the kidneys. UUO was carried out to further examine the role of Endophilin A2 in renal fibrosis. Endophilin A2 Tg mice showed less fibrotic lesions in the tubulointerstitial areas and less expression of FN, a-SMA, TGF-?1, and collagen I, III, and IV than in their respective controls.Summary 1. Endophilin A2, which was negatively correlated with Orai1 expression, expressed less in fibrotic kidneys and renal tubule epithelial cells undergoing EMT.2. Inhibition of Orai1 Ca²⁺ channel increased Endophilin A2 protein expression in the kidney. 3. Endophilin A2 inhibited EMT induced by TGF-?1 in renal tubule epithelial cells, and it played an important role in protecting against renal fibrosis.Conclusion 1. Orai1 was mainly expressed in tubular epithelial cells. And the expression of Orai1 was significantly increased in fibrotic kidneys. 2. Inhibition of Orai1 Ca²⁺ channel alleviated renal fibrosis and improved the impaired kidney function induced by HFD. 3. Orai1 Ca²⁺ channel regulated renal fibrosis by mediating TGF-?/smad signaling pathway and EMT in tubular epithelial cells. 4. Endophilin A2 expression was regulated by Orai1 Ca²⁺ channel. Overexpression of Endophilin A2 blocked TGF-?/smad signal pathway and protected against renal fibrosis.