Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity

One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity.     

改构型酸性成纤维细胞生长因子对顺铂肾损害保护作用的实验研究

目的 研究改构型酸性成纤维细胞生长因子(MaFGF)对顺铂(DDP)引起的体外培养的肾小管上皮细胞损害的保护作用。方法 将原代培养的肾小管上皮细胞接种于96孔培养板：(1)培养72h后加入一系列浓度的DDP,实验组在DDP作用12h后加入不同浓度MaFGF,再培养36h后用WST-8法检测细胞存活率。（2）以DDP建立损伤模型,并在加药后12小时加入一定量的MaFGF,观察MaFGF对肾小管上皮细胞的保护作用。结果 (1) MaFGF能使DDP对肾小管上皮细胞的半数抑制浓度（IC50）升高。（2）DDP组与对照组比较,各生化和酶学指标差异均有统计学意义;而MaFGF + DDP组与对照组比较,SOD、GSH-Px酶活性差异无统计学意义,MDA、NO升高差异仍有统计学意义。结论 MaFGF对DDP损伤的肾小管上皮细胞有明显的保护作用。     

Autophagy protects renal tubular cells against cyclosporine toxicity

A major side effect of the powerful immunosuppressive drug cyclosporine (CsA) is the development of a chronic nephrotoxicity whose mechanisms are not fully understood. Recent data suggest that tubular cells play a central role in the pathogenesis of chronic nephropathies. We have shown that CsA is responsible for endoplasmic reticulum (ER) stress in tubular cells. Autophagy has recently been described to be induced by ER stress and to alleviate its deleterious effects. In this study, we demonstrate that CsA induces autophagy in primary cultured human renal tubular cells through LC3II expression and autophagosomes visualization by electron microscopy. Autophagy is dependant on ER stress because various ER stress inducers activate autophagy, and salubrinal, an inhibitor of eIF2alpha dephosphorylation that protects cells against ER stress, inhibited LC3II expression. Furthermore, autophagy inhibition during CsA treatment with beclin1 siRNA significantly increases tubular cell death. Finally, immunohistochemical analysis of rat kidneys demonstrates a positive LC3 staining on injured tubular cells, suggesting that CsA induces autophagy in vivo. Taken together, these results demonstrate that CsA, through ER stress induction, activates autophagy as a protection against cell death.     

Autophagy protects renal tubular cells against cyclosporine toxicity

A major side effect of the powerful immunosuppressive drug cyclosporine (CsA) is the development of a chronic nephrotoxicity whose mechanisms are not fully understood. Recent data suggest that tubular cells play a central role in the pathogenesis of chronic nephropathies. We have shown that CsA is responsible for endoplasmic reticulum (ER) stress in tubular cells. Autophagy has recently been described to be induced by ER stress and to alleviate its deleterious effects. In this study, we demonstrate that CsA induces autophagy in primary cultured human renal tubular cells through LC3II expression and autophagosomes visualization by electron microscopy. Autophagy is dependant of ER stress because various ER stress inducers activate autophagy and salubrinal, an inhibitor of eIF2α dephosphorylation that protects cells against ER stress, inhibited LC3II expression. Furthermore, autophagy inhibition during CsA treatment with beclin1 siRNA significantly increases tubular cell death. Finally, immunohistochemical analysis of rat kidneys demonstrates a positive LC3 staining on injured tubular cells, suggesting that CsA induces autophagy in vivo. Taken together, these results demonstrate that CsA, through ER stress induction, activates autophagy as a protection against cell death.     

Glutamate dehydrogenase requirement for apoptosis induced by aristolochic acid in renal tubular epithelial cells

Ingestion of aristolochic acids (AA) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adduct formation, is well-documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. Epithelial cell death is a critical characteristic of these pathological conditions. To elucidate the mechanisms of AA-induced cytotoxicity, we explored AA-interacting proteins in tubular epithelial cells (TEC). We found that AA interacts with a mitochondrial enzyme glutamate dehydrogenase (GDH) and moderately inhibits its activity. We report that AA induces cell death in GDH-knockdown TEC preferentially via non-apoptotic means, whereas in GDH-positive cells, death was executed by both the non-apoptotic and apoptotic mechanisms. Apoptosis is an energy-reliant process and demands higher adenosine 5′-triphosphate (ATP) consumption than does the non-apoptotic cell death. We found that, after AAI treatment, the ATP depletion is more pronounced in GDH-knockdown cells. When we reduced ATP in TEC cells by inhibition of glycolysis and mitochondrial respiration, cell death mode switched from apoptosis and necrosis to necrosis only. In addition, in cells incubated at low glucose and no glutamine conditions, oxaloacetate and pyruvate reduced AAI-induced apoptosis our data suggest that AAI-GDH interactions in TEC are critical for the induction of apoptosis by direct inhibition of GDH activity. AA binding may also induce changes in GDH conformation and promote interactions with other molecules or impair signaling by GDH metabolic products, leading to apoptosis.     

Cryptotanshinone inhibits hypoxia/reoxygenation-induced oxidative stress and apoptosis in renal tubular epithelial cells

Cryptotanshinone (CTS), an active component extracted from the root of Salvia miltiorrhiza Bunge , was reported to attenuate hepatic ischemia/reperfusion (I/R) injury. However, its protective effect against renal I/R injury remains unclear. In this study, the role of CTS in renal I/R injury in vitro and its possible mechanism were investigated. Our results showed that CTS improved cell viability in HK-2 cells exposed to hypoxia/reoxygenation (H/R). CTS also inhibited the H/R-mediated production of reactive oxygen species, as well as increased the activities of superoxide dismutase and catalase in H/R-stimulated HK-2 cells. In addition, CTS dramatically attenuated the induction of bax expression and caspase-3 activity and alleviated the reduction of bcl-2 expression in HK-2 cells cultured with H/R. Furthermore, CTS activated the levels of p-PI3K and p-Akt in H/R-injured HK-2 cells; meanwhile, the renal protective activity of CTS was inhibited by the inhibitor of the (phosphatidylinositol 3 kinase/protein kinase B) PI3K/Akt pathway (LY294002). These findings indicate that CTS can ameliorate renal I/R injury in vitro partly through regulating the PI3K/Akt pathway.     

Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK₁ cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.     

Partial attenuation of cytotoxicity and apoptosis by SOD1 in ischemic renal epithelial cells

Reactive oxygen species (ROS) contribute significantly to apoptosis in renal ischemia-reperfusion (IR) injury, however the exact mechanisms are not well understood. We used novel lentiviral vectors to over-express superoxide dismutase 1 (SOD1) in proximal tubular epithelial (LLC-PK₁) cells and determined effects of SOD1 following ATP depletion-recovery, used as a model to simulate renal IR. SOD1 over-expression partially protected against cytotoxicity (P < 0.001) and decreased superoxide (O₂ ^•−) in ATP depleted cells. The ATP depletion-mediated increase in nuclear fragmentation, an index of apoptosis and activation of caspase-3 was also partially blocked by SOD1 (P < 0.05). However, SOD1 over-expression was insufficient to completely attenuate caspase-3, indicating that ROS other than cytoplasmic O₂ ^•− are involved in ATP depletion mediated injury. To test the contribution of hydrogen peroxide, a subset of enhanced green fluorescent protein (EGFP) and SOD1 (serum free and injured) cells were treated with polyethylene glycol-catalase (PEG-catalase). As expected there was 50% reduction in cytotoxicity and caspase-3 in SOD1 cells compared to EGFP cells; catalase treatment decreased both indices by an additional 28% following ATP depletion. To test the role of mitochondrial derived superoxide, we also treated a subset of LLC-PK₁ cells with the mitochondrial antioxidant, MitoTEMPO. Treatment with MitoTEMPO also decreased ATP depletion induced cytotoxicity in LLC-PK₁ cells in a dose dependant manner. These studies indicate that both SOD1 dependent and independent pathways are integral in protection against ATP depletion-recovery mediated cytotoxicity and apoptosis, however more studies are needed to delineate the signaling mechanisms involved.     

Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK(1) cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.     

Polyamine depletion delays apoptosis of rat intestinal epithelial cells

The polyamines spermidine, spermine, and their precursorputrescine are essential for cell growth and the regulation of the cellcycle. Recent studies suggest that excessive accumulation of polyaminesfavors either malignant transformation or apoptosis, depending on thecell type and the stimulus. This study examines the involvement ofpolyamines in the induction of apoptosis by the DNA topoisomerase Iinhibitor, camptothecin. In IEC-6 cells, camptothecin induced apoptosiswithin 6 h, accompanied by detachment of cells. Detached cells showedDNA laddering and caspase 3 induction, characteristic features ofapoptosis. Depletion of putrescine, spermidine, and spermine byDL--difluoromethylornithine (DFMO), a specific inhibitorof ornithine decarboxylase (ODC) that is the first rate-limiting enzymefor polyamine biosynthesis, decreased the apoptotic index. Delayedapoptosis was accompanied by a decrease in caspase 3 activity inpolyamine-depleted cells. Addition of putrescine restored the inductionof apoptosis as indicated by an increase in the number of detachedcells and caspase 3 activity. Polyamine depletion did not change thelevel of caspase 3 protein. Inhibition of S-adenosylmethioninedecarboxylase by a specific inhibitor [diethylglyoxalbis-(guanylhydrazone); DEGBG] led to depletion of spermidine andspermine with a significant accumulation of putrescine and induction ofODC. The DEGBG-treated cells showed an increase in apoptosis,suggesting the importance of putrescine in the apoptotic process.Addition of putrescine to DFMO-treated cell extracts did not increasecaspase 3 activity. The above results indicate that polyamine depletiondelays the onset of apoptosis in IEC-6 cells and confers protectionagainst DNA damaging agents, suggesting that polyamines might beinvolved in the caspase activating signal cascade.

     

Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells

Background

Human rhinoviruses (RV), the most common triggers of acute asthma exacerbations, are considered not cytotoxic to the bronchial epithelium. Recent observations, however, have questioned this knowledge. The aim of this study was to evaluate the ability of RV to induce epithelial cytotoxicity and affect epithelial repair in-vitro.

Methods

Monolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA) by flow cytometry.

Results

RV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation.

Conclusion

RV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling.     

PSTPIP2 inhibits cisplatin-induced acute kidney injury by suppressing apoptosis of renal tubular epithelial cells

Cisplatin (CP) is an effective chemotherapeutic agent widely used in the treatment of various solid tumours. However, CP nephrotoxicity is an important limitation for CP use; currently, there is no method to ameliorate cisplatin-induced acute kidney injury (AKI). Recently, we identified a specific role of proline–serine–threonine phosphatase-interacting protein 2 (PSTPIP2) in cisplatin-induced AKI. PSTPIP2 was reported to play an important role in a variety of diseases. However, the functions of PSTPIP2 in experimental models of cisplatin-induced AKI have not been extensively studied. The present study demonstrated that cisplatin downregulated the expression of PSTPIP2 in the kidney tissue. Administration of AAV-PSTPIP2 or epithelial cell-specific overexpression of PSTPIP2 reduced cisplatin-induced kidney dysfunction and inhibited apoptosis of renal tubular epithelial cells. Small interfering RNA-based knockdown of PSTPIP2 expression abolished PSTPIP2 regulation of epithelial cell apoptosis in vitro. Histone acetylation may impact gene expression at the epigenetic level, and histone deacetylase (HDAC) inhibitors were reported to prevent cisplatin-induced nephrotoxicity. The UCSC database was used to predict that acetylation of histone H3 at lysine 27 (H3K27ac) induces binding to the PSTPIP2 promoter, and this prediction was validated by a ChIP assay. Interestingly, an HDAC-specific inhibitor (TSA) was sufficient to potently upregulate PSTPIP2 in epithelial cells. Histone acetylation-mediated silencing of PSTPIP2 may contribute to cisplatin nephrotoxicity. PSTPIP2 may serve as a potential therapeutic target in the prevention of cisplatin nephrotoxicity.Subject terms: Cancer, Cancer prevention     

Mercury-induced Ca2+ increase and cytotoxicity in renal tubular cells

The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 microM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+ -induced [Ca2+]i increase by 27%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+ -free medium, the Hg2+ -induced [Ca2+]i increase was nearly abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+ -induced Ca2+ release was not altered by inhibition of phospholipase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 microM Hg2+ did not alter cell proliferation rate and mitochondrial activity, but 10 microM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.     

Ox-LDL aggravates contrast-induced injury of renal tubular epithelial cells

Hypercholesterolemia can aggravate contrast-induced acute kidney injury, and the exacerbation of renal tubular epithelial cell (RTEC) injury is a major cause. However, the exact mechanisms remain obscure. Mitophagy, a type of autophagy, selectively eliminates damaged mitochondria and reduces mitochondrial oxidative stress, which is strongly implicated in cell homeostasis and acute kidney injury. Oxidized low-density lipoprotein (Ox-LDL) is accumulated in hypercholesterolemia and has a cytotoxic effect. This study aimed to determine whether and how ox-LDL exacerbates contrast-induced injury in RTECs and to further explore whether PINK1/Parkin-dependent mitophagy is involved in this process. Iohexol and ox-LDL were used alone or in combination to treat HK-2 cells. Rapamycin pretreatment was utilized to enhance mitophagy. Cell viability, apoptosis, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) were detected by cell counting kit-8, TUNEL staining, JC-1 kit and MitoSOX fluorescence, respectively. The expression of mitophagy-related proteins (including PINK1, Parkin, and so on) and cleaved caspase-3 was confirmed by western blot. Colocalization of MitoTracker-labeled mitochondria and LysoTracker-labeled lysosomes was observed by fluorescence microscopy to evaluate mitophagy. The results of our study showed that ox-LDL aggravated MMP decline, mtROS release and apoptosis in iohexol-treated HK-2 cells, accompanied by a further increased autophagy level. Enhancement of PINK1/Parkin-dependent mitophagy by rapamycin alleviated apoptosis and mitochondrial injury in HK-2 cells in response to iohexol under ox-LDL condition. Therefore, our findings indicate that ox-LDL aggravates contrast-induced injury of RTECs by increasing mitochondrial damage and mitochondrial oxidative stress, which may be associated with the relative insufficiency of PINK1/Parkin-dependent mitophagy.     

Albumin handling by renal tubular epithelial cells in a microfluidic bioreactor

Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS-PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS-PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS-PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process.     

Oxidant-mediated apoptosis in proximal tubular epithelial cells following ATP depletion and recovery

Oxidant-mediated apoptosis has been implicated in renal injury due to ischemia reperfusion (IR); however, the apoptotic signaling pathways following IR have been incompletely defined. The purpose of this study was to examine the role of oxidants on cell death in a model of in vitro simulated IR injury in renal proximal tubular epithelial cells by analyzing the effects of a cell-permeable superoxide dismutase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTmPyP). Renal proximal tubular epithelial cells were ATP depleted for 2, 4, or 6 h, followed by 2 h of recovery. We found that MnTmPyP was effective in attenuating cytotoxicity (P<0.001) and decreasing steady-state oxidant levels (P<0.001) and apoptotic cell death (P<0.001) following ATP depletion-recovery. MnTmPyP treatment prevented the early cytosolic release of cytochrome c and increased Bcl-2 protein levels following short durations of ATP depletion-recovery. After longer periods of ATP depletion-recovery, we observed a significant increase in TNF-alpha protein levels (P<0.001) and caspase-8 activation (P<0.001), both of which were decreased (P<0.001) by treatment with MnTmPyP. Our results suggest that oxidant mediated apoptosis via the mitochondrial pathway during the early phase of ATP depletion and by activation of the receptor-mediated apoptotic pathway following longer durations of injury.     

JNK在血糖波动的糖尿病大鼠肾小管上皮细胞凋亡中的作用

目的:探讨血糖波动的糖尿病大鼠发生肾小管上皮细胞凋亡的信号转导机制。方法:健康SD大鼠随机分为正常对照组(A)、糖尿病稳定高血糖组(B)和糖尿病波动高血糖组(C),采用链脲佐菌素(STZ)65 mg/kg腹腔注射诱发糖尿病,血糖波动组每天定时腹腔注射速效胰岛素,并错时给予葡萄糖,造成一天中血糖浓度大幅度波动模型。制模12周后,采用比色法检测肾组织匀浆中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,免疫组化法和Western blot检测肾脏Nox4和JNK蛋白表达,原位缺口末端标记法(TUNEL)检测肾脏细胞凋亡。结果:与A组比较,B组和C组肾组织MDA含量增加、SOD活性下降,肾小管上皮细胞Nox4表达增加,肾小管磷酸化JNK(P-JNK)蛋白表达上调,细胞凋亡率明显增加,且C组的以上变化均较B组更加明显(P<0.05,P<0.01)。结论:波动性高血糖较稳定性高血糖更易促进糖尿病肾小管上皮细胞凋亡,其机制与JNK信号转导通路激活有关。     

miR-191 secreted by platelet-derived microvesicles induced apoptosis of renal tubular epithelial cells and participated in renal ischemia-reperfusion injury via inhibiting CBS

In this study, we aimed to reveal the role of miR-191 in apoptosis of renal tubular epithelial cells and in the involvement of renal ischemia-reperfusion injury. Renal transplantation rat model was established. miR-191 and Cystathionine-β-synthase (CBS) were measured by qRT-PCR and Western blot. The regulation of miR-191 on CBS was detected by luciferase reporter assay. We found miR-191 expression in platelets and platelet microvesicles (P-MVs) of patients and model rats was significantly upregulated than that of health and normal rats. Also, mRNA and protein levels of CBS in renal tissues of patients were significantly downregulated than that of health and normal rats. We also found that P-MVs could transfer miR-191 to HK-2 cells. Luciferase reporter assay showed that CBS was a direct target of miR-191. In addition, we proved that P-MVs-secreted miR-191 inhibited CBS expression in HK-2 cells, and P-MVs-secreted miR-191 promoted HK-2 cell apoptosis via CBS. Finally, we verified the trends of CBS expressions, HK-2 cell apoptosis and apoptosis-related proteins in vivo were similar as the trends in vitro. Therefore, CBS was a direct target of miR-191, and miR-191 could transfer to HK-2 cells via P-MVs to decrease the expression of CBS, thus to promote cell apoptosis and renal IR injury.