Rat Kidney Tubular Epithelial Cells Research Articles

Connexin32 gap junction channels deliver miR155-3p to mediate pyroptosis in renal ischemia-reperfusion injury

ObjectivesTo explore whether the gap junction (GJ) composed by connexin32(Cx32) mediated pyroptosis in renal ischemia-reperfusion(I/R) injury via transmitting miR155-3p, with aim to provide new strategies for the prevention and treatment of acute kidney injury (AKI) after renal I/R.Methods8–10 weeks of male C57BL/ 6 wild-type mice and Cx32 knockdown mice were divided into two groups respectively: control group and renal I/R group. MCC950 (50 mg/kg. ip.) was used to inhibit NLRP3 in vivo. Human kidney tubular epithelial cells (HK - 2) and rat kidney tubular epithelial cells (NRK-52E) were divided into high-density group and low-density group, and treated with hypoxia reoxygenation (H/R) to mimic I/R. The siRNA and plasmid of Cx32, mimic and inhibitor of miR155-3p were transfected into HK - 2 cells respectively. Kidney pathological and functional injuries were measured. Western Blot and immunofluorescent staining were used to observe the expression of NLRP3, GSDMD, GSDMD-N, IL - 18, and mature IL-18. The secretion of IL-18 and IL-1β in serum, kidney tissue and cells supernatant were detected by enzyme-linked immuno sorbent assay (ELISA) kit, and the expression of NLPR3 and miR155-3p were detected by RT-qPCR and fluorescence in situ hybridization (FISH).ResultsTubular pyroptosis were found to promote AKI after I/R in vivo and Cx32-GJ regulated pyroptosis by affecting the expression of miR155-3p after renal I/R injury. In vitro, H/R could lead to pyroptosis in HK-2 and NRK-52E cells. When the GJ channels were not formed, and Cx32 was inhibited or knockdown, the expression of miR155-3p was significantly reduced and the pyroptosis was obviously inhibited, leading to the reduction of injury and the increase of survival rate. Moreover, regulating the level of miR155-3p could affect survival rate and pyroptosis in vitro after H/R.ConclusionsThe GJ channels composed of Cx32 regulated tubular pyroptosis in renal I/R injury by transmitting miR155-3p. Inhibition of Cx32 could reduce the level of miR155-3p further to inhibit pyroptosis, leading to alleviation of renal I/R injury which provided a new strategy for preventing the occurrence of AKI.-tWJY53zy_-Wc_anQ2Z7vvVideo

Actin cytoskeletal reorganization is involved in hyperosmotic stress-induced autophagy in tubular epithelial cells

Tubular epithelial cells are routinely exposed to severe changes in osmolarity. Although the autophagic activity of cells is an indispensable process to maintain cellular homeostasis and respond to stressors, the effect of hyperosmotic stress on autophagic activity in tubular epithelial cells remains unknown. The aim of this study was to determine the effect of hyperosmotic stress on autophagy in rat kidney tubular epithelial cells focusing on the role of actin and microtubule cytoskeletons. Normal rat kidney (NRK)-52E cells exposed to mannitol-induced hyperosmotic stress. As a result, NRK-52E cells showed elevated protein levels of the autophagosome marker LC3-II, indicating enhancement of the autophagic flux. Hyperosmotic stress also transiently decreased cell volume and caused the reorganization of actin and microtubule cytoskeletal structures in NRK-52E cells. The inhibition of the actin cytoskeleton reorganization by cytochalasin D impaired the increase in the levels of LC3-II; however, disassembly of the microtubules following treatment with nocodazole did not affect the increase. These results indicate that hyperosmotic stress can induce autophagy mediated by the reorganization of the actin cytoskeleton in tubular epithelial cells.

ZnT8 Exerts Anti-apoptosis of Kidney Tubular Epithelial Cell in Diabetic Kidney Disease Through TNFAIP3-NF-κB Signal Pathways.

Apoptosis of kidney tubular epithelial cells contributes to diabetic kidney disease (DKD) pathophysiology, but the mechanisms are not fully understood. Zinc transporter protein member 8 (ZnT8, SLC30A8) is a susceptive gene in diabetes. Here, we aim to investigate whether ZnT8 has effects on pathophysiology of DKD. The animal groups include control, ZnT8KO mice, STZ-induced, and ZnT8-KO-STZ. STZ-induced DKD was performed in male C57BL/6J mice and in ZnT8-KO mice. High glucose (HG)-induced apoptosis in a normal rat kidney tubular epithelial cell line (NRK-52E cells) was performed in vitro. Transfection of hZnT8-EGFP or TNFAIP3 siRNA was done in NRK-52E cells. Flow cytometry with Annexin V-FITC/PI double staining and TUNEL analysis was performed for the detection of apoptosis. Gene expression at mRNA and protein levels was examined with real-time RT-PCR and Western blot. Urine albumin to creatinine ratio, proinflammatory cytokines, and apoptosis were enhanced in kidneys of STZ and ZnT8-KO-STZ mice compared to control or ZnT8-KO mice. ZnT8 overexpression after hZnT8-EGFP transfection decreased HG-stimulated inflammatory activity and apoptosis in NRK-52E cells. Furthermore, treatment with ZnSO4 blunted HG-induced apoptosis and NF-κB activation. ZnSO4 increased the abundance of zinc-finger protein TNF-α-induced protein 3 (TNFAIP3). Also, ZnT8 over-expression after hZnT8-EGFP transfection significantly ameliorates HG-induced NF-κB-dependent transcriptional activity and apoptotic protein expressions in NRK-52E cells, but the inhibitory effect of ZnT8 was significantly abolished with TNFAIP3 siRNA. Our study provides evidence that ZnT8 has protective effects against apoptosis of renal tubular epithelial cells through induction of TNFAIP3 and subsequent suppression of the NF-κB pathway.

Cardamomin protects from diabetes-induced kidney damage through modulating PI3K/AKT and JAK/STAT signaling pathways in rats

BackgroundDiabetic nephropathy is one of the common complications of diabetes mellitus, which seriously affects the life quality and health of patients. In this study, we aimed to investigate the function of cardamonin (CAD) in diabetes-induced kidney damage in rats. MethodsThe normal rat kidney tubular epithelial cells (NRK-52E) were pre-treated with different doses of CAD and then stimulated with methylglyoxal (MGO). Streptozotocin (STZ) induced diabetes rat model were received different doses of CAD treatment. MTT, EdU, Transwell, and flow cytometry was used to detect cell viability, proliferation, migration, and apoptosis. Western blot analysis was used to detect the expression of apoptosis related proteins, advanced glycation end-products (AGEs), receptor for AGEs (RAGE), epithelial mesenchymal transition (EMT) related proteins, phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway related proteins, and janus kinas/signal transducer and activator of transcription 3 (JAK/STAT3) related proteins. ELISA assay was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were detected using commercial kit. Hematoxylin and eosin staining was used to assess pathological changes in rat kidney. ResultsCompared with control group, MGO reduced cell viability and proliferation, enhanced migration and apoptosis of NRK-52E cells, while CAD inhibited these effects induced by MGO in NRK-52E cells. Moreover, CAD increased Bcl-2 expression and decreased the expression of Bax and cleaved caspase-3 in MGO-treated NRK-52E cells. Compared with control group, MGO increased the AGEs formation, the expression of RAGE and p-p65, the levels of TNF-α, IL-6, IL-1β, MDA in NRK-52E cells and reduced the levels of GSH and SOD, while treatment of CAD dose-dependently prevented these results. In addition, CAD attenuated MGO-induced EMT of MGO-treated NRK-52E cells. Mechanically, we identified that CAD repressed PI3K/AKT and JAK/STAT3 signaling in NRK-52E cells. Importantly, the kidney injury of diabetes rats was attenuated by CAD. Besides, STZ-induced inflammatory response, oxidative stress, and phosphorylation levels of PI3K, AKT, JAK2, and STAT3 were reduced by CAD in the rats. ConclusionCAD protects from diabetes-induced kidney damage through modulating PI3K/AKT and JAK/STAT signaling pathways in rats.

Exosomes derived from mesenchymal stem cells ameliorate renal fibrosis via delivery of miR-186-5p.

Evidence has shown that mesenchymal stem cells' (MSCs) therapy has potential application in treating chronic kidney disease (CKD). In addition, MSCs-derived exosomes can improve the renal function and prevent the progression of CKD. However, the mechanisms by which MSCs-derived exosomes (MSCs-Exo) ameliorate renal fibrosis in CKD remain largely unclear. To mimic an in vitro model of renal fibrosis, rat kidney tubular epithelial cells (NRK52E) were stimulated with transforming growth factor (TGF)-β1. In addition, we established an in vivo model of unilateral ureteric obstruction (UUO)-induced renal fibrosis. Meanwhile, we exploited exosomes derived from MSCs for delivering miR-186-5p agomir into NRK52E cells or kidneys in vitro and in vivo. In this study, we found that level of miR-186-5p was significantly downregulated in TGF-β1-stimulated NRK52E cells and the obstructed kidneys of UUO mice. In addition, miR-186-5p can be transferred from MSCs to NRK52E cells via exosomes. MSCs-delivered miR-186-5p markedly reduced the accumulation of extracellular matrix (ECM) protein, and inhibited epithelial-to-mesenchymal transition (EMT) and apoptosis in TGF-β1-stimulated NRK52E cells. Moreover, exosomal miR-186-5p from MSCs attenuated kidney injury and fibrosis in a UUO mouse model via inhibition of the ECM protein accumulation and EMT process. Meanwhile, dual-luciferase assay showed that miR-186-5p downregulated Smad5 expression via direct binding with the 3'-UTR of Smad5. Collectively then, these findings indicated that exosomal miR-186-5p derived from MSCs could attenuate renal fibrosis in vitro and in vivo by downregulation of Smad5. These findings may help to understand the role of MSCs' exosomes in alleviating renal fibrosis in CKD.

Effects of Curcumin on High Glucose-Induced Epithelial-to-Mesenchymal Transition in Renal Tubular Epithelial Cells Through the TLR4-NF-κB Signaling Pathway.

ObjectiveDiabetic kidney disease (DKD) is a microvascular complication in diabetes mellitus, while tubuloepithelial to mesenchymal transition (EMT) of mature tubular epithelial cells is a key point in the early development and progression of renal interstitial fibrosis. The present study aimed to investigate the protective effects of Curcumin on EMT and fibrosis in cultured normal rat kidney tubular epithelial cell line (NRK-52E).MethodsBy using immunofluorescence staining and Western blot protocols, in vitro experiments were designed to analyze EMT markers, including collagen I and E-cadherin in high glucose (HG) exposed NRK-52E cells and to detect the expression levels of phosphorylated-NF-κB, TLR4 and reactive oxygen species (ROS) after Curcumin pre-treatment. With co-treatment with TAK242, these molecules in the TLR4-NF-κB signaling pathway were further evaluated.ResultsCurcumin decreased the HG-induced EMT levels and ROS production in NRK-52E cells. Furthermore, Curcumin was found to inhibit the TLR4-NF-κB signaling activation in HG-induced EMT of NRK-52E cells.ConclusionThe present study provides evidence suggesting a novel mechanism that Curcumin exerts the anti-fibrosis effects via inhibiting activation of the TLR4-NF-κB signal pathway and consequently protecting the HG-induced EMT in renal tubular epithelial cells. Thereby, TLR4-NF-κB may be a useful target for therapeutic intervention in DKD.

Nucleoporin 160 (NUP160) inhibition alleviates diabetic nephropathy by activating autophagy

ABSTRACT Diabetic nephropathy (DN) is the leading cause of end-stage renal disease worldwide. Autophagy was reported to be related to the pathogenesis of DN. This research investigated the function of the Nucleoporin 160 (Nup160) gene in regulating autophagy in DN. A mouse model of DN was established through an intraperitoneal injection of streptozotocin (STZ). Normal rat kidney tubular epithelial cells (NRK-52E) were treated with high glucose to induce DN in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot, immunofluorescence assays were conducted to measure the expression of NUP160, autophagy-associated proteins, and inflammatory cytokines in vitro and in vivo. Pathological changes of kidney and liver tissues were analyzed using hematoxylin and eosin (H&E), Masson and periodic acid-silver (PAS) staining. The body weight, blood glucose, renal and lipid profiles of DN mice were examined. In this study, DN mice showed serious pathological injury. NUP160 expression was upregulated, autophagy was inhibited, and inflammatory response was increased in DN mice. Depletion of NUP160 restored autophagy and inhibited inflammation and fibrosis in high glucose (HG)-treated NRK-52E cells and STZ-induced DN mice by downregulating the expression of p62 and Collagen IV (Col-Ⅳ), increasing the ratio of LC3II/LC3I, and inactivating nuclear factor (NF)-κB signaling. Moreover, NUP160 knockdown could ameliorate pathological damage and glucose tolerance in DN mice. Overall, this study is the first to demonstrate the key role of NUP160 silencing in promoting autophagy against diabetic injury in DN.

DNase I Induces Other Endonucleases in Kidney Tubular Epithelial Cells by Its DNA-Degrading Activity.

Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I.

SLC30A7 has anti-oxidant stress effects in high glucose-induced apoptosis via the NFE2L2/HMOX1 signal transduction pathway

AimsApoptosis and oxidant stress are known to be involved in the pathogenesis of diabetic kidney disease (DKD). We have previously reported that zinc transporter 7 in SLC30 family (SLC30A7) inhibits apoptosis in rat peritoneal mesothelial cells under high glucose (HG) conditions. In the current study, we aimed to investigate whether SLC30A7 had effect for anti-oxidant stress in renal tubular epithelial cells under HG. MethodsSLC30A7 in HG-induced apoptosis in a normal rat kidney tubular epithelial cell line (NRK-52E cells)/kidneys of STZ-induced diabetic mice was examined and the activity of nuclear factor erythroid 2-related factor 2 (NFE2L2) was further analyzed by using real time RT-PCR, siRNA and Western blot protocols. ResultsSLC30A7 was found to be up-regulated, while NFE2L2 was activated in kidneys of STZ-induced diabetic mice and HG-induced apoptosis of NRK-52E cells. Knock-down of SLC30A7 with siRNA protocol resulted in lower intracellular free zinc levels in the cells and decreased zinc distribution in the Golgi apparatus. Furthermore, knock-down of NFE2L2 down-regulated its target HMOX1 gene expression, decreased SLC30A7 activity but increased HG-induced apoptosis. ConclusionThe current study provides new evidence that SLC30A7 has anti-oxidant stress effects in HG-induced apoptosis via the NFE2L2/HMOX1 signal transduction pathway.

Obeticholic acid ameliorates hepatorenal syndrome in ascitic cirrhotic rats by down-regulating the renal 8-iso-PGF2α-activated COX-TXA2 pathway.

The present study explores the potential of chronic treatment with the Foresaid X receptor (FXR) agonist obeticholic acid (OCA), which inhibits oxidative stress-related pathogenesis, in ascitic cirrhotic rats with hepatorenal syndrome (HRS) developed 6 weeks after bile duct ligation (BDL). Systemic, splanchnic, and renal hemodynamics and pathogenic cascades were measured in ascitic BDL and sham rats receiving 2-weeks of either vehicle or OCA treatments (sham-OCA and BDL-OCA groups), and NRK-52E cells, rat kidney tubular epithelial cells. Chronic OCA treatment significantly normalized portal hypertension, glomerular filtration rate, urine output, renal blood flow; decreased ascites, renal vascular resistance, serum creatinine, and the release of renal tubular damage markers, including urinary neutrophil gelatinase-associated lipocalin (uNGAL) and kidney injury moleculae-1 (uKim-1) in BDL-OCA rats. In the BDL group, inhibition of the renal oxidative stress (8-iso-PGF2α)-activated cyclooxygenase-thromboxane A2 [COX-TXA2] pathway, apoptosis, and tubular injury accompanied by a decrease in hyper-responsiveness to the vasoconstrictor 8-iso-PGF2α in perfused kidneys. In vitro experiments revealed that 8-iso-PGF2α induced oxidative stress, release of reactive oxygen species, and cell apoptosis, which were reversed by concomitant incubation with the FXR agonist. Through the inhibition of renal 8-iso-PGF2α production and the down-regulation of the COX-TXA2 pathway, our study suggests that chronic OCA treatment can ameliorate the HRS in ascitic cirrhotic rats. Thus, OCA is an agent with antioxidative stress, antivasoconstrictive, antiapoptotic properties which benefit ascitic, cirrhotic rats with systemic, hepatic, and renal abnormalities.

Effects of ZnT8 on epithelial-to-mesenchymal transition and tubulointerstitial fibrosis in diabetic kidney disease

Zinc transporter 8 (ZnT8) transports zinc ions for crystallization and storage of insulin in pancreatic beta-cells and ZnT8 dysfunction is involved in pathogenesis of diabetes. The current study aimed to investigate whether ZnT8 has effects in pathophysiology of diabetic kidney disease (DKD) by using animal models for diabetes, including STZ-induced diabetic, db/db, ZnT8-KO, ZnT8-KO-STZ and ZnT8-KO-db/db mice. Results demonstrated that urine albumin to creatinine ratio and epithelial-to-mesenchymal transition (EMT) were increased in kidneys of ZnT8-KO-STZ and ZnT8-KO-db/db mice compared with C57BL/6 J and ZnT8-KO mice, while serum TGF-β1, IL-6, and TNF-α levels were elevated in parallel. In kidneys of mice intercrossed between ZnT8-KO and STZ-induced diabetic or db/db mice, these three inflammatory factors, ACR and EMT were also found to be increased compared with C57BL/6J, db/db and ZnT8-KO mice. Furthermore, ZnT8 up-regulation by hZnT8-EGFP reduced the levels of high glucose (HG)-induced EMT and inflammatory factors in normal rat kidney tubular epithelial cell (NRK-52E cells). Expression of phosphorylated Smad2/Smad3 was up-regulated after HG stimulation and further enhanced by ZnT8 siRNA but down-regulated after hZnT8-EGFP gene transfection. The current study thus provides the first evidence that ZnT8 protects against EMT-tubulointerstitial fibrosis though the restrain of TGF-β1/Smads signaling activation in DKD.

Suppression of HDAC2 by sodium butyrate alleviates apoptosis of kidney cells in db/db mice and HG‑induced NRK‑52E cells.

Butyrate is short-chain fatty acid, which is produced by intestinal microbiota metabolizing dietary fibers. Butyrate participates in various physiological processes predominantly by activating G-coupled-receptors, inhibiting histone deacetylases (HDACs) and serving as an energy substrate. Previous studies have shown that butyrate plays a protective role in diabetic nephropathy (DN); however, the exact mechanism remains unclear. The present study identified that providing sodium butyrate (NaBu) by gavage relieved renal damage and apoptosis in db/db mice, which is a widely used type 2 DN model. In vitro, NaBu suppressed high glucose (HG)-induced apoptosis in normal rat kidney tubular epithelial (NRK-52E) cells. Of the eleven HDACs (HDAC1-11) studied, only the mRNA expression of HDAC2 was attenuated by NaBu in NRK-52E cells under the HG condition. Overexpression of HDAC2 offset the anti-apoptotic effect of NaBu. NaBu also suppressed HG-induced oxidative stress. Additionally, H2O2 induced an upregulation of HDAC2 in NRK-52E cells, while NaBu inhibited this process. Mechanistically, NaBu acted as an antioxidant in HG-induced NRK-52E cells and suppressed HG-induced apoptosis of NRK-52E cells through inhibiting HDAC2 by virtue of its anti-oxidative property.

ROS induces epithelial-mesenchymal transition via the TGF-β1/PI3K/Akt/mTOR pathway in diabetic nephropathy.

Oxidative stress has been reported to serve an important role in the development and progression of diabetic nephropathy (DN). Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells promotes renal fibrosis in DN, while the mechanism of reactive oxygen species (ROS)-mediated EMT is not fully understood. The aim of the present study was to investigate the effect of high glucose-induced ROS on the activation of the transforming growth factor (TGF)-β1/phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in a normal rat kidney tubular epithelial cell line (NRK-52E) and rats with type 1 diabetes. In vitro, high glucose-stimulated ROS production resulted in increased TGF-β1 expression as well as an increase in the Akt and mTOR phosphorylation ratio, resulting in EMT. When cells were pre-treated with ROS inhibitors, changes in TGF-β1, Akt and mTOR were significantly ameliorated. In vivo, diabetic rats experienced a significant decline in renal function and severe renal fibrosis compared with control rats at 8 weeks following streptozocin injection. Levels of malondialdehyde and TGF-β1/PI3K/Akt/mTOR pathway activation were increased in the renal cortex of rats with diabetes compared with the control rats. Furthermore, renal fibrosis was further aggravated in DN compared with the control rats. The results of the present study suggest that ROS serves an important role in mediating high glucose-induced EMT and inhibits activation of the TGF-β1/PI3K/Akt/mTOR pathway. ROS may therefore have potential as a treatment approach to prevent renal fibrosis in DN.

Maternal exposure to di-n-butyl phthalate promotes Snail1-mediated epithelial-mesenchymal transition of renal tubular epithelial cells via upregulation of TGF-β1 during renal fibrosis in rat offspring

We previously demonstrated that maternal exposure to di-n-butyl phthalate (DBP) resulted in renal fibrosis in male offspring; however, the underlying mechanism governing this effect has not been thoroughly elucidated to date. We hypothesized that DBP exposure induces TGF-β expression and abnormal activation of epithelial-mesenchymal transition (EMT) in fibrotic kidneys. Pregnant rats received DBP orally at a dose of 850 mg/kg BW/day during gestational days 14–18. In the DBP-exposed group, immunohistochemistry (IHC) staining showed increased expression of TGF-β1 and EMT markers. In rat kidney tubular epithelial cells (NRK52E), ROS production increased expression levels of TGF-β1 and subsequently contributed to the induction of Snail1-mediated EMT. Notably, DBP exposure also promoted autophagy that downregulated TGF-β1. Taken together, our findings suggest that maternal exposure to DBP promotes EMT in tubular epithelial cells via upregulation of TGF-β1.

Crosstalk Between Connexin32 and Mitochondrial Apoptotic Signaling Pathway Plays a Pivotal Role in Renal Ischemia Reperfusion-Induced Acute Kidney Injury.

Aims: Perioperative acute kidney injury (AKI) resulting from renal ischemia reperfusion (IR) is not conducive to the postoperative surgical recovery. Our previous study demonstrated that reactive oxygen species (ROS) transmitted by gap junction (GJ) composed of connexin32 (Cx32) contributed to AKI. However, the precise underlying pathophysiologic mechanisms were largely unknown. This study focuses on the underlying mechanisms related to ROS transmitted by Cx32 responsible for AKI aggravation. Results: In a set of in vivo studies, renal IR was found to cause severe impairment in renal tissues with massive ROS generation, which occurred contemporaneously with activation of NF-κB/p53/p53 upregulated modulator of apoptosis (PUMA)-mediated mitochondrial apoptosis pathways. Cx32 deficiency alleviated renal IR-induced AKI, and simultaneously attenuated ROS generation and distribution in renal tissues, which further inhibited NF-κB/p53/PUMA-mediated mitochondrial apoptotic pathways. Correspondingly, in a set of in vitro studies, hypoxia reoxygenation (HR)-induced cellular injury, and cell apoptosis in both human kidney tubular epithelial cells (HK-2s) and rat kidney tubular epithelial cells (NRK52Es) were significantly attenuated by Cx32 inhibitors or Cx32 gene knockdown. More importantly, Cx32 inhibition not only decreased ROS generation and distribution in human or rat kidney tubular epithelial cells but also inhibited its downstream NF-κB/p53/PUMA-mediated mitochondrial apoptotic pathway activation. Innovation and Conclusion: This is the first identification of the underlying mechanisms of IR-induced renal injury integrally which demonstrates the critical role played by Cx32 in IR-induced AKI. Moreover, GJ composed of Cx32 manipulates ROS generation and distribution between neighboring cells, and alters activation of NF-κB/p53/PUMA-mediated mitochondrial apoptotic pathways. Both inhibiting Cx32 function and scavenging ROS effectively reduce mitochondrial apoptosis and subsequently attenuate AKI, providing effective strategies for kidney protection.

Role of JAK3 in the Pathogenesis of Oxidative Stress-Induced Kidney Fibrosis

The Janus kinase (JAK) tyrosine kinase family and JAK/STAT signal transduction pathway may act in kidney fibrogenesis. JAK3 expression was investigated in in vitro and in vivo models of kidney fibrosis involving oxidative stress. There was a marked down-regulation of JAK3 mRNA in rat kidney tubular epithelial cells (NRK52E) and fibroblasts (NRK49F) exposed to 1.0 mM H2O2 for 18–20 h compared with controls, which correlated with increased apoptosis and decreased mitosis in both cell lines. However, JAK3 protein levels were not significantly different in control and H2O2-treated epithelial and fibroblast cultures. JAK3 activation (phospho-tyrosine) increased in NRK52E cells and decreased in NRK49F cells with oxidative stress. STAT3 phosphorylation decreased in both cell lines with oxidative stress compared with controls. JAK3 protein expression and localisation were investigated in kidneys using the unilateral ureteral obstruction (UUO) model (0–7 days, rats) of kidney fibrosis that involves oxidative stress. JAK3 protein expression did not differ between UUO and controls; however, JAK3 localisation increased temporally with UUO, with strong epithelial expression in mitotic cells compared with controls. Apoptotic tubular epithelium showed minimal JAK3. In summary, in vitro, decreased kidney JAK3 mRNA after oxidative stress was not seen translationally. Differences in the activation of the JAK3/STAT3 pathway may have different consequences for renal fibrosis. In vivo, changes in JAK3 protein localisation, and especially its co-localisation with mitotic cells, indicate that JAK3 protein may contribute to renal tubular epithelial cell proliferation after oxidative stress.

Ozone oxidative postconditioning inhibits oxidative stress and apoptosis in renal ischemia and reperfusion injury through inhibition of MAPK signaling pathway.

BackgroundOzone has been used as a curative agent for a variety of different diseases for over 150 years. In our previous study, we found that ozone oxidative preconditioning could alleviate renal damage induced by ischemia and reperfusion injury (I/R). Although this method had obvious protective effects in the reduction of I/R, its clinical application remains limited because this treatment must be commenced prior to the ischemic period, which is not practical in the clinic.PurposeIn the present study, we investigated whether ozone oxidative postconditioning (OzoneOP) could attenuate renal I/R in vivo and in vitro, as well as the mechanisms underlying the effects of this treatment.MethodsSprague Dawley rats were subjected to right renal ischemia for 45 min and reperfusion for 24 h, or to sham operation with the left kidney removed, both with and without OzoneOP. In addition, normal rat kidney tubular epithelial cells (NRK-52E) were chosen to create a hypoxia–reoxygenation (H/R) model of 3 h hypoxia and 24 h reoxygenation processes, both with or without OzoneOP and mitogen-activated protein kinase (MAPK) inhibitors.ResultsOur results showed that OzoneOP significantly reversed apoptosis and the abnormal superoxide dismutase and malondialdehyde levels induced by I/R or H/R. OzoneOP also inhibited activation of the MAPK pathways both in vivo and in vitro, which resulted in significant protection against apoptosis and oxidative stress.ConclusionOur current data provide evidence that OzoneOP might serve as a potential therapy for renal I/R.

High concentration calcium induces the transdifferentiation of rat kidney tubular epithelial cells to osteoblast-like cells via inhibiting matrix Gla protein

High concentration calcium induces the transdifferentiation of rat kidney tubular epithelial cells to osteoblast-like cells via inhibiting matrix Gla protein

Protective Effect of Znt7 on High Glucose-Induced Epithelial-to-Mesenchymal Transition in Renal Tubular Epithelial Cells

Background/Aims: Evidence from our and other groups has demonstrated that zinc transporter 7 in SLC30 family (ZnT7) inhibited epithelial-to-mesenchymal transition (EMT) and apoptosis in rat peritoneal mesothelial cells (RPMCs) under high glucose (HG) concentration. In the present study, we investigated the effect of ZnT7 on EMT of renal tubular epithelial cells (RTECs) in an in vitro model of diabetic nephropathy (DN). Methods: A dual-fluorescent staining protocol was used for detection of ZnT7 in a normal rat kidney tubular epithelial cell line (NRK-52E cells). EMT was induced with HG (30 mM). NRK-52E cells were transfected with plasmids codifying for hZnT7-EGFP and interfering RNA for determination of the effect of ZnT7 over-expression and silencing, respectively. Expression of ZnT7, activation of the MAPK/ERK and TGF-β/Smad pathways were analyzed with by means of Western blot. Results: ZnT7 was localized in the perinuclear region and Golgi apparatus. In HG-induced EMT of NRK-52E cells, ZnT7 was up-regulated. Over-expression of ZnT7 led to inhibition of HG-induced EMT, while knock-down of ZnT7 increased EMT. Furthermore, knock-down of ZnT7 and increased HG-induced EMT was accompanied by activation of the MAPK/ERK and TGF-β/Smad pathways. Conclusion: The present study provides evidence that ZnT7 has a protective effect over EMT of RTECs in DN and suggests that the inhibition of HG-induced EMT may be achieved through the MAPK/ERK and TGF-β/Smad pathways. Thereby, ZnT7 could be a potential target for translation medicine and prevention program in DN.

Curcumin suppresses AGEs induced apoptosis in tubular epithelial cells via protective autophagy.

Renal tubular cell apoptosis and tubular dysfunction is an important process underlying diabetic nephropathy (DN). Understanding the mechanisms underlying renal tubular epithelial cell survival is important for the prevention of kidney damage associated with glucotoxicity. Curcumin has been demonstrated to possess potent anti-apoptotic properties. However, the roles of curcumin in renal epithelial cells are yet to be defined. The present study investigated advanced glycation or glycoxidation end-product (AGE)-induced toxicity in renal tubular epithelial cells via several complementary assays, including cell viability, cell apoptosis and cell autophagy in the NRK-52E rat kidney tubular epithelial cell line. The extent of apoptosis was significantly increased in the NRK-52E cells following treatment with AGEs. The results also indicated that curcumin reversed this effect by promoting autophagy through the phosphoinositide 3-kinase/AKT serine/threonine kinase signaling pathway. These conclusions suggested that curcumin exerts a renoprotective effect in the presence of AGEs, at least in part by activating autophagy in NRK-52E cells. Collectively, these findings indicate that curcumin not only exerts renoprotective effects, however may also act as a novel therapeutic strategy for the treatment of diabetic nephropathy.