Renal Cell Apoptosis Research Articles

MicroRNA-486-5p is implicated in the cisplatin-induced apoptosis and acute inflammation response of renal tubular epithelial cells by targeting HAT1.

The proinflammatory property of cisplatin is potentially destructive andcontributes to the pathogenesis of acute kidney injury (AKI). The role and upstream regulatory mechanism of histone acetyltransferase 1 (HAT1) in acute kidney inflammation are still unknown. We performed RNA sequencing to filter differentially expressed microRNAs (miRNAs) in the kidney tissue of mice with AKI induced by cisplatin and ischemia-reperfusion. Here, we found that miR-486-5p was upregulated and that the expression of HAT1 was reduced in AKI mouse models and injured human renal proximal tubular epithelial cell (HK-2) model induced by cisplatin. miR-486-5p is implicated in cisplatin-induced kidney damage in vivo. Bioinformatics analysis predicted a potential binding site between miR-486-5p and HAT1. The Luciferase reporter assay and Western blot confirmed that miR-486-5p directly targeted the 3'-untranslated region of HAT1 mRNA and inhibited its expression in the cytoplasm of HK-2 cells. In the in vitro study, inhibiting miR-486-5p reduced apoptosis, and the expression of proinflammatory mediators was induced by cisplatin in HK-2 cells. Simultaneously, the downregulation of miR-486-5p inhibited the activation of the toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB). We further found that HAT1 could inhibit apoptosis and the activation of cisplatin on the TLR4/NF-κB pathway and that the upregulation of miR-486-5p reversed this effect. Therefore, the upregulation of miR-486-5p targeting HAT1 promoted the cisplatin-induced apoptosis and acute inflammation response of renal tubular epithelial cells by activating the TLR4/NF-κB pathway, providing a new basis to highlight the potential intervention of regulating the miR-486-5p/HAT1 axis.

BMP-7 ameliorates partial epithelial-mesenchymal transition by restoring SnoN protein level via Smad1/5 pathway in diabetic kidney disease

Tubulointerstitial fibrosis (TIF) is involved in the development of diabetic kidney disease (DKD). Transforming growth factor β1 (TGF-β1) is involved in the extensive fibrosis of renal tissue by facilitating the partial epithelial-mesenchymal transition (EMT), increasing the synthesis of extracellular matrix (ECM), inhibiting degradation, inducing apoptosis of renal parenchyma cells, and activating renal interstitial fibroblasts and inflammatory cells. Recent studies indicated that bone morphogenetic protein-7 (BMP-7) upregulated the expression of endogenous SnoN against renal TIF induced by TGF-β1 or hyperglycemia. Nevertheless, the mechanisms underlying the BMP-7-mediated restoration of SnoN protein level remains elusive. The present study demonstrated the increased expression of BMP-7 in diabetic mellitus (DM) mice by hydrodynamic tail vein injection of overexpressed BMP-7 plasmid, which attenuated the effects of DM on kidney in mice. Partial tubular EMT and the accumulation of Collagen-III were resisted in DM mice that received overexpressed BMP-7 plasmid. Similar in vivo results showed that BMP-7 was competent to alleviate NRK-52E cells undergoing partial EMT in a high-glucose milieu. Furthermore, exogenous BMP-7 activated the Smad1/5 pathway to promote gene transcription of SnoN and intervened ubiquitination of SnoN; both effects repaired the SnoN protein level in renal tubular cells and kidney tissues of DM mice. Therefore, these findings suggested that BMP-7 could upregulate SnoN mRNA and protein levels by activating the classical Smad1/5 pathway to refrain from the partial EMT of renal tubular epithelial cells and the deposition of ECM in DKD-induced renal fibrosis.

P53 inhibition attenuates cisplatin-induced acute kidney injury through microRNA-142-5p regulating SIRT7/NF-κB

Renal tubular epithelial cell apoptosis is the main mechanism of cisplatin-induced acute kidney injury. The role of microRNAs (miRNAs) in the apoptosis of renal tubular epithelial cells has been suggested, but the underlying mechanism has not been fully elucidated. We used microarray analysis to identify miR-142-5p involved in cisplatin-induced acute kidney injury. miR-142-5p was down-regulated in human renal tubular epithelial (HK-2) cells with cisplatin treatment. Notably, the overexpression of miR-142-5p attenuated the cisplatin-induced HK-2 cell apoptosis and inhibition of miR-142-5p aggravated cisplatin-induced HK-2 cell apoptosis. During cisplatin treatment, p53 was activated. The inhibition of p53 by pifithrin-α attenuated the cisplatin-induced kidney injury and up-regulated miR-142-5p expression. We also identified the Sirtuin7 (SIRT7) as a target of miR-142-5p. Furthermore, we demonstrated that the inhibition of SIRT7 prevented cisplatin-induced HK-2 cell apoptosis and decreased the expression of nuclear factor kappa B (NF-κB). Our data revealed that p53 inhibition could attenuate cisplatin-induced acute kidney injury by up-regulating miR-142-5p to repress SIRT7/NF-κB. These findings may provide a novel therapeutic target of cisplatin-induced acute kidney injury.

Knockout of farnesoid X receptor gene aggravates cisplatin-induced kidney injury.

Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand activated transcription factors and belongs to bile acid receptor. Studies have shown that the expression of FXR in renal tissue can reduce renal injury via regulation of glucose and lipid metabolism, inhibition of inflammatory response, reduction of oxidative stress and renal fibrosis. However, it is unclear whether FXR is involved in autophagy in renal diseases. This study aims to investigate the role of FXR in cisplatin-induced acute renal injury and whether its mechanism is related to autophagy regulation. Twelve male WT or FXR-KO mice at 12 weeks were randomly divided into a WT group, a WT+cisplatin group, a FXR-KO group, and a FXR-KO+cisplatin group, with 6 mice in each group. The WT+cisplatin group and the FXR-KO+cisplatin group were intraperitoneally injected with cisplatin (20 mg/kg), and the WT group and the FXR-KO group were intraperitoneally injected with equal volume of cisplatin solvent. Seventy-two hours later, the mice were killed and blood and renal tissue samples were collected. The levels of SCr and BUN were detected by immunoturbidimetry. After the staining, the pathological changes of renal tissue were observed under optical microscope. The protein levels of LC3 and p62 were detected by Western blotting and immunohistochemistry. The clearance of damaged mitochondria and the accumulation of lysosomal substrate were observed under electron microscope. The apoptosis of renal tubular epithelial cells was detected by TUNEL. Compared with the WT group or the FXR-KO group, both SCr and BUN levels in the WT+cisplatin group or the FXR-KO+cisplatin group were significantly increased (P<0.01 or P<0.001), and SCr and BUN levels in the FXR-KO+cisplatin group were significantly higher than those in the WT+cisplatin group (both P<0.05). Under the light microscope, there were no obvious pathological changes in the renal tissue of mice in the WT group and the FXR-KO group. Both the WT+cisplatin group and the FXR-KO+cisplatin group had vacuolar or granular degeneration of renal tubular epithelial cells, flat cells, lumen expansion, brush edge falling off, and even exposed basement membrane and tubular formation. The scores of renal tubular injury in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the score in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05). Under the transmission electron microscope, the mitochondria of mouse tubular epithelial cell in the WT+cisplatin group and the FXR-KO+cisplatin group was swollen, round, vacuolated, cristae broken or disappeared; the lysosome was uneven and high-density clumps, and the change was more obvious in the FXR-KO+cisplatin group. Western blotting showed that the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased in the WT+cisplatin group compared with the WT group and the FXR-KO+cisplatin group compared with FXR-KO group (P<0.05 or P<0.01); compared with the FXR-KO group, the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased significantly in the FXR-KO+cisplatin group (both P<0.05). Immunohistochemistry results showed that the expression of total LC3 and p62 in renal cortex of the WT+cisplatin group and the FXR-KO+cisplatin group was increased significantly, especially in the FXR-KO+cisplatin group. TUNEL results showed that the mice in the WT group and the FXR-KO group had negative staining or only a few apoptotic tubular epithelial cells, and the number of apoptotic cells in the WT+cisplatin group and the FXR-KO+cisplatin group were increased. The apoptosis rates of renal tubular epithelial cells in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the apoptosis rate in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05). Knockout of FXR gene aggravates cisplatin induced acute renal injury, and its mechanism may be related to inhibiting autophagy and promoting apoptosis.

Nephroprotective potential of Panduratin A against colistin-induced renal injury via attenuating mitochondrial dysfunction and cell apoptosis

Colistin is a last-resort polypeptide antibiotic widely used to treat against multidrug-resistant Gram-negative bacterial infections. However, this treatment is associated with nephrotoxicity. The aim of this study was to examine the potential protective effect of panduratin A, a bioactive compound of Boesenbergia rotunda, on colistin-induced nephrotoxicity in both in vivo and in vitro models. Intraperitoneal injection of 15 mg/kg colistin for 7 days markedly promoted renal tubular degeneration, increased blood urea nitrogen (BUN) levels, and upregulated the expression of renal injury biomarker and apoptosis proteins. In addition, treatment with colistin increased oxidative stress and apoptosis in mice kidney tissues. Interestingly, these defects were attenuated when co-administered of colistin with panduratin A (2.5 or 25 mg/kg). The underlying mechanisms of panduratin A attenuating colistin toxicity was investigated in human renal proximal tubular cells (RPTEC/TERT1). The mechanisms by which colistin-triggered cytotoxicity was determined by analysis of cell death, reactive oxygen species (ROS) levels, mitochondria function as well as the expression of proteins related to apoptosis pathway. Colistin treatment (200 µg/ml) significantly increased cell apoptosis, elevated ROS production, reduced mitochondrial membrane potential, and decreased anti-apoptotic protein (Bcl-2) expression. These effects were notably suppressed by co-treatment with panduratin A (5 μM). Collectively, panduratin A exerts as a novel nephroprotective agent to protect against colistin-induced renal injury by attenuating mitochondrial damage and renal cell apoptosis.

Zinc Deficiency Induces Inflammation and Apoptosis via Oxidative Stress in the Kidneys of Mice.

Zinc (Zn) is an essential element that regulates not only cellular immunity but also antioxidant and anti-inflammatory agents. The present study investigated the effect of Zn deficiency on renal cell apoptosis and its mechanism. A Zn-deficient kidney model in mice was created by a Zn-deficient diet. Mice were fed diets with different Zn levels for 41days as follows: normal-Zn group (NG, 34mg Zn/kg), low-Zn group (LG, 2mg Zn/kg), and high-Zn group (HG, 100mg Zn/kg). H&E staining showed that inflammatory cells and many erythrocytes exuded in the renal tissue space of the low-Zn group, and TUNEL staining indicated massive death of kidney cells in the low-Zn group. In the low-Zn group, the levels of oxygen free radicals (ROS) were significantly increased, the antioxidants were significantly decreased, and the total antioxidant capacity was decreased. Moreover, RT-qPCR and ELISA results showed that inflammatory factors (TNF-α, IL-1β, and IL-6) were significantly increased in the low-Zn group. In addition, the levels of p-IκBα, p-NF-κB p65, p-ERK, p-JNK, and p-p38 were significantly increased in the low-Zn group, indicating that zinc deficiency activates NF-κB and MAPK signalling as well as increases its expression. RT-qPCR analysis of apoptosis-related genes, including Bcl-2 Bax, Caspa8, Caspa6, and Caspa3, demonstrated that the expression levels of proapoptotic genes in mouse kidneys were significantly increased. Importantly, the in vitro results were consistent with the in vivo results. Together, these data suggested that zinc deficiency induces renal oxidative stress to activate NF-κB and MAPK signalling, thereby inducing renal cell apoptosis.

RORA alleviates LPS-induced apoptosis of renal epithelial cells by promoting PGC-1α transcription

ObjectiveTo explore the effect of RORA on LPS-induced renal epithelial cell apoptosis and the underlying mechanism.MethodsLPS-treated HK-2 cells were established as a cellular model of acute kidney injury. The expression of RORA or/and PGC-1α in LPS-induced HK-2 cells was altered by transfection. qRT-PCR and Western blotting were used to detect the expression changes of RORA and PGC-1α. ELISA was performed to detect the expression of IL-1β and IL-6 and the activity of caspase-3. Western blotting was applied for visualization of cleaved caspase-3. CCK-8 and flow cytometry were used to assess cell proliferation and apoptosis. Dual-luciferase reporter and ChIP-qPCR were utilized to verify the binding of RORA to PGC-1α promoter.ResultsLPS treatment decreased the expression of RORA and PGC-1α and increased that of cleaved caspase-3 in HK-2 cells. Also, LPS treatment inhibited HK-2 cell proliferation and promoted HK-2 cell apoptosis and secretion of IL-1β and IL-6. Overexpression of RORA or PGC-1α eliminated the adverse effects of LPS treatment in HK-2 cells. RORA drove the transcription of PGC-1α by binding PGC-1α promoter. Knockdown of PGC-1α offset the reduction in HK-2 cell injury caused by overexpression of RORA.ConclusionRORA reduces LPS-induced apoptosis of renal epithelial cells by promoting PGC-1α transcription.

Chicoric Acid Attenuated Renal Tubular Injury in HFD-Induced Chronic Kidney Disease Mice through the Promotion of Mitophagy via the Nrf2/PINK/Parkin Pathway.

As the main factor in the pathogenesis of chronic kidney disease (CKD), the excessive apoptosis of renal tubular epithelial cells (RTECs) and its underlying mechanism of action are worth further investigation. Chicoric acid (CA), a major active constituent of the Uyghur folk medicine chicory, was recorded to possess a renal protective effect. The precise effect of CA on renal tubular injury in obesity-related CKD remains unknown. In the current study, CA was proven to ameliorate metabolic disorders including overweight, hyperglycemia, hyperlipidemia, and hyperuricemia in high fat diet (HFD)-fed mice. Furthermore, the reverse effect of CA on renal histological changes and functional damage was confirmed. In vitro, the alleviation of lipid accumulation and cell apoptosis was observed in palmitic acid (PA)-exposed HK2 cells. Treatment with CA reduced mitochondrial damage and oxidative stress in the renal tubule of HFD-fed mice and PA-treated HK2 cells. Finally, CA was observed to activate the Nrf2 pathway; increase PINK and Parkin expression; and regulate LC3, SQSTM1, Mfn2, and FIS1 expression; therefore, it would improve mitochondrial dynamics and mitophagy to alleviate mitochondrial damage in RTECs of obesity-related CKD. These results may provide fresh insights into the promotion of mitophagy in the prevention and alleviation of obesity-related CKD.

Thrombospondin 1 Promotes Endoplasmic Reticulum Stress and Apoptosis in HK-2 Cells by Upregulating ATF6-CHOP.

The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1 (TSP1) in human renal tubular epithelial cells (HK-2 cells). HK-2 cells were exposed to high concentrations of glucose (HG). The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis. The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry. Western blotting was used to detect the apoptosis- and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA. HG treatment induced high cell apoptosis, abundantly expressed TSP1 level and restrained viability in HK-2 cells. Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions. Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression. 4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers, such as PERK, ATF4, ATF6, p-eIF2α, IRE1, CHOP and XBP1, suggesting that the administration of 4-PBA was successful. Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis. TSP1 restrained cell proliferation, and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.

Effects of transforming growth factor beta-activated kinase 1 (TAK1) on apoptosis of HK-2 cells in the high glucose environment

ABSTRACT To observe the role of transforming growth factor beta-activated kinase 1 (TAK1)/p38 MAPK/TGF-β1 signal pathway plays in oxidative stress and apoptosis in human renal tubular epithelial cells (HK-2) under high glucose induction. HK-2 cells were cultured in high glucose medium with and without TAK1 inhibitor 5Z-7-oxozeaenol. TUNEL and flow cytometry were used to detect cell apoptosis. The protein expression of TAK1, TGF-β1, Bax and Bcl-2 was detected by immunofluorescence. Meanwhile, flow cytometry was used to detect the production of reactive oxygen species (ROS), and MitoSOX staining was performed to detect the production of mitochondrial ROS. Moreover, real-time quantitative PCR and Western blotting was used to measure the expression of TAK1, TGF-β1, NOX1, NOX4 and HO-1, Bax, Bcl-2, p38MAPK, p-p38MAPK and TGF-β1. Results showed that high glucose up-regulated the protein expression of p-TAK1, p-p38 MAPK and TGF-β1, which induced the aggravation of oxidative stress by promoting the production of ROS, thus promote the apoptosis in HK-2 cells. However, addition of 5z −7-oxozeaenol in HK-2 cells reversed all the above functions induced by high glucose. Another experimental result also showed that SB203580, a p38MAPK inhibitor can down-regulated TGF-β1 expression and reduce ROS production, thus alleviate cell apoptosis in TAK1 overexpression group. In summary, high glucose intervention could activate TAK1 and promote apoptosis in HK-2 cells. Inhibition of TAK1 expression could block p38 MAPK/TGF-β1 signaling pathway and reduce ROS production and oxidative stress, which may be one of the signal pathways of TAK1 to reduce apoptosis of HK-2 cells induced by high glucose. Abbreviations: DN, Diabetic nephropathy; TAK1, transforming growth factor β-activated kinase-1; TGF-β, transforming growth factor-β; NG, normal glucose; HG, high glucose; p38 MAPK, p38 mitogen-activated protein kinase; ROS, reactive oxygen species.

Rehmannioside A protects against high glucose-induced apoptosis and oxidative stress of renal tubular epithelial cells by inhibiting the MAPK pathway

Purpose: To investigate the effects and mechanism of rehmannioside A (ReA) on diabetic nephropathy (DN) progression.&#x0D; Methods: Various concentrations of glucose and ReA were added to HK2 cells, and cell viability was analyzed using the 3-(4,5)-dimethylthiahiazo(-2)-3,5 diphenytetrazoliumromide (MTT) assay. Cell apoptosis, caspase 3 activity, and expression levels of BAX, Bcl-2, and cleaved poly (ADP-ribose) polymerase were evaluated to assess the effect of ReA on cell apoptosis. The effect of ReA on oxidative stress was also evaluated by assessing superoxide dismutase, catalase, and malondialdehyde levels. Lactate dehydrogenase release and reactive oxygen species levels were also measured. Finally, activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 was determined by immunoblot technique.&#x0D; Results: ReA significantly enhanced the survival of HK2 cells induced with high glucose (HG). In addition, ReA suppressed apoptosis and inhibited oxidative stress of HK2 cells induced with HG (p &lt; 0.05). ReA protected against HG-induced apoptosis and oxidative stress of renal tubular epithelial cells by inhibiting the MAPK pathway (p &lt; 0.05).&#x0D; Conclusion: ReA is a potential as a therapeutic agent for DN; however, in vivo and clinical investigations are required to validate this assertion.

Augmenter of liver regeneration protects the kidney against ischemia-reperfusion injury by inhibiting necroptosis

ABSTRACT Necroptosis plays an important role in the pathogenesis of acute kidney injury (AKI), and necroptosis-related interventions may therefore be an important measure for the treatment of AKI. Our previous study has shown that augmenter of liver regeneration (ALR) inhibits renal tubular epithelial cell apoptosis and regulates autophagy; however, the influence of ALR on necroptosis remains unclear. In this study, we investigated the effect of ALR on necroptosis caused by ischemia-reperfusion and the underlying mechanism. In vivo experiments indicated that kidney-specific knockout of ALR aggravated the renal dysfunction and pathological damage induced by ischemia-reperfusion. Simultaneously, the expression of renal necroptosis-associated protein receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), and mixed-lineage kinase domain-like protein (MLKL) significantly increased. In vitro experiments indicated that overexpression of ALR decreased the expression of hypoxia-reoxygenation-induced kidney injury molecules, the inflammation-associated factor tumor necrosis factor-alpha (TNF-α), and monocyte chemotactic protein. Additionally, the expression of RIP1, RIP3, and MLKL, which are elevated after hypoxia and reoxygenation, was also inhibited by ALR overexpression. Both in vivo and in vitro results indicated that ALR has a protective effect against acute kidney injury caused by ischemia-reperfusion, and the RIP1/RIP3/MLKL pathway should be further verified as a probable necroptosis regulating mechanism.

TFPI2 suppresses the interaction of TGF-β2 pathway regulators to promote endothelial–mesenchymal transition in diabetic nephropathy

Endothelial–mesenchymal transition (EndMT) is an important source of myofibroblasts, but also contributes to the progression of diabetic nephropathy (DN). By several differential gene expression analyses from the Gene Expression Omnibus (GEO) database, the tissue factor pathway inhibitor 2 (TFPI2) gene, known as a tumor suppressor, was shown to be dysregulated in DN; however, the potential role and regulatory mechanism of TFPI2 in DN are unclear. Here, we found abnormal upregulation of TFPI2 in the renal cortex of diabetic mice, accompanied by impaired renal function. We also injected a single dose of adeno-associated virus (AAV)2 carrying shRNA targeting TFPI2 intravenously into these mice and found that knockdown of TFPI2 improved renal function and reduced renal fibrosis and cell apoptosis in experimental DN. Furthermore, hyperglycemia-induced EndMT was inhibited in the absence of TFPI2, as evidenced by increased expression of endothelial markers (VE-cadherin and CD31) and decreased expression of mesenchymal markers (α-SMA, desmin, and FSP-1). To further explore the mechanism in vitro, human renal glomerular endothelial cells (hRGECs) were incubated in the presence of high glucose or transforming growth factor beta (TGF-β)2. TFPI2 deficiency inhibited high glucose-induced cell apoptosis and TGF-β2-induced EndMT in hRGECs, while overexpression of TFPI2 had the opposite effects. Importantly, TGF-β2 is a crucial driver of EndMT, and we found that TFPI2 promoted TGF-β2/Smad signaling activation by interferring the interaction of TGF-β pathway regulators (SMURF2 with SMAD7). Our results show that TFPI2 regulates EndMT and the TGF-β2 signaling pathway and is a potential promoter of DN pathogenesis.

Ruscogenin protects against cisplatin-induced apoptosis, inflammation, and oxidative stress of renal tubular epithelial cells

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To determine the potential effect of ruscogenin in cisplatin-induced nephrotoxicity.&#x0D; Methods: Rat renal tubular epithelial cells (NRK-52E) were treated with 50 μM cisplatin to establish an in vitro cell model of nephrotoxicity. Cytotoxicity was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, flow cytometry, and western blot. Different concentrations of ruscogenin (2.5, 5, and 10 μM) were incubated with cisplatin-treated NRK-52E cells. Alterations in the nod-like receptor family, the pyrin domain-containing protein (NLRP3) inflammasome, toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB), and nuclear factor erythropoietin-2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) components were determined using western blot. Flow cytometry was also used to investigate the levels of reactive oxygen species (ROS).&#x0D; Results: Ruscogenin significantly increased cell viability (p &lt; 0.01) and suppressed apoptosis of NRK- 52E cells (p &lt; 0.01), attenuating cisplatin-induced cytotoxicity. The NLRP3 inflammasome was activated in cisplatin-treated NRK-52E cells with enhanced NLRP3, interleukin 1 beta, and cleaved caspase-1; however, ruscogenin significantly decreased the expression of NLRP3 inflammasome components (p &lt; 0.01). Ruscogenin attenuated cisplatin-induced expression of TLR4, myeloid differentiation primary response 88, and NF-κB. Further, cisplatin induction enhanced ROS formation, with increased malondialdehyde and decreased glutathione reductase and catalase levels. Ruscogenin attenuated cisplatin-induced ROS accumulation in NRK-52E cells through up-regulation of Nrf2 and HO-1.&#x0D; Conclusion: Ruscogenin protects against cisplatin-induced apoptosis, inflammation, and oxidative stress in renal tubular epithelial cells via suppression of TLR4/NF-κB activation and promotion of Nrf2/HO-1 activation. Therefore, ruscogenin provides a potential therapeutic strategy for mitigating cisplatin-induced nephrotoxicity.&#x0D; &#x0D; &#x0D; &#x0D;

POS-082 RacGAP1 ameliorates acute kidney injury by promoting proliferation and suppressing apoptosis of renal tubular cells

Acute kidney injury (AKI) remains correlated with high mortality. Novel therapeutic strategies are urgently needed for AKI patients. Rac GTPase-activating protein 1 (RacGAP1) regulates the activity of RhoGTPase and acts as a predictive biomarker in several types of malignant tumor but the role of RacGAP1 in AKI has not been revealed.

Tetratricopeptide repeat domain 36 protects renal tubular cells from cisplatin-induced apoptosis potentially via maintaining mitochondrial homeostasis

The apoptosis of proximal tubule epithelial cells (PTECs) is a critical event of acute kidney injury (AKI). Tetratricopeptide repeat domain 36 (TTC36) with three tetratricopeptide repeats is evolutionarily conserved across mammals, which functions as a chaperone for heat shock protein 70. We have revealed that TTC36 is specifically expressed in PTECs in our previous work. There are few studies about the role of TTC36 in AKI. In this study, we observed that TTC36 was obviously down-regulated in a mouse model of acute kidney injury established by ischemia/reperfusion and its expression was negatively related to the degree of kidney injury. In addition, TTC36 protected HK2 cells against cisplatin-induced apoptosis. Moreover, we discovered the mechanism that TTC36 mitigated cisplatin-triggered mitochondrial disorder via partially sustaining the membrane potential of mitochondria and mitochondrial autophagy-related gene expression. Collectively, these results suggested that TTC36 plays a protective role in the cisplatin-induced apoptosis of renal tubular cells through maintaining the mitochondrial potential and mitochondrial autophagy-related genes’ expression to some extent. These observations highlight the essential role of TTC36 in regulating PTEC apoptosis and imply TTC36/mitochondrial homeostasis axis as a potential target for the therapeutic intervention in AKI.

Calpain Inhibitor Calpeptin Alleviates Ischemia/Reperfusion-Induced Acute Kidney Injury via Suppressing AIM2 Inflammasome and Upregulating Klotho Protein.

Renal ischemia/reperfusion injury is a major contributor of acute kidney injury (AKI), leading to renal cell necrosis, apoptosis, and inflammation. Calpains, a family of Ca2+-dependent cysteine proteases, play a pivotal role in the pathogenesis of renal diseases. Several studies have reported calpain inhibitors showing remarkable reno-protective effects against proteinuria and α-klotho deficiency-induced renal aging symptoms, particularly against glomerulus injury. However, little is known about the role of the calpain inhibitor calpeptin in acute kidney injury. The present study aims to investigate the potential mechanism of downregulation of Calpain 1 and 2 activity by calpeptin in the ischemia/reperfusion (IR)-induced AKI model. Firstly, we observed that the contents of Calpain 1 and 2 were significantly increased in the renal biopsy of clinical AKI patients, especially in the diseased tubules space. To investigate the impacts of calpain activity inhibition, we further pretreated with calpeptin in both the IR mouse model and in the HK-2 cells hypoxia model. We found that the calpain inhibitor calpeptin improved renal functional deterioration, attenuated pathological structure damage, and decreased tubular cell apoptosis in the IR injury-induced AKI mice model. Mechanistically, calpeptin significantly suppressed the AIM2 (absent in melanoma 2) and NLRP3 (NOD-like receptor protein 3) inflammasome signaling pathways and increased Klotho protein levels. Furthermore, immunofluorescence assays demonstrated that the application of calpeptin effectively inhibited Calpain 1 activation and gasdermin D (GSDMD) cleavage in the renal tubules of IR mice. Taken together, our both in vivo and in vitro experiments suggest that calpeptin conveyed reno-protection in AKI might be mediated by the inhibition of AIM2 inflammasome activation and upregulation of Klotho protein. As such, we provide new evidence that Calpain 1 and 2 activation may be closely associated with the pathogenesis of clinical AKI. The calpain-mediated AIM2 inflammasome signaling pathway and distinct interaction between calpain and Klotho may provide a potential novel preventative and therapeutic target for acute kidney injury.

Tetramethylpyrazine: An Active Ingredient of Chinese Herbal Medicine With Therapeutic Potential in Acute Kidney Injury and Renal Fibrosis.

As an increasing public health concern worldwide, acute kidney injury (AKI) is characterized by rapid deterioration of kidney function. Although continuous renal replacement therapy (CRRT) could be used to treat severe AKI, effective drug treatment methods for AKI are largely lacking. Tetramethylpyrazine (TMP) is an active ingredient of Chinese herb Ligusticum wallichii (Chuan Xiong) with antioxidant and anti-inflammatory functions. In recent years, more and more clinical and experimental studies suggest that TMP might effectively prevent AKI. The present article reviews the potential mechanisms of TMP against AKI. Through search and review, a total of 23 studies were finally included. Our results indicate that the undergoing mechanisms of TMP preventing AKI are mainly related to reducing oxidative stress injury, inhibiting inflammation, preventing apoptosis of intrinsic renal cells, and regulating autophagy. Meanwhile, given that AKI and chronic kidney disease (CKD) are very tightly linked by each other, and AKI is also an important inducement of CKD, we thus summarized the potential of TMP impeding the progression of CKD through anti-renal fibrosis.

Platelets Derived Transthyretin Participate in The Development of Sepsis Associated Acute Kidney Injury by Inducing Oxidative Stress and Apoptosis of Renal Tubular Epithelial Cells.

The pathophysiology of sepsis-associated acute kidney injury (S-AKI) is not well elucidated. Platelets have been reported to play a critical role in the pathogenesis of AKI, but the true mechanism remains unknown. Herein, we established a mouse model of S-AKI by cecal ligation and puncture (CLP). Ticagrelor was given 24 h before and after CLP by gastric gavage. Platelets were isolated and analyzed by the label-free proteome approach to identify platelet-derived damage-associated molecular patterns (DAMPs). Our results demonstrated that, among all differentially expressed proteins (DEPs), platelet-derived transthyretin (TTR) exerted effects in S-AKI. To examine the direct effects of platelet TTR on human renal proximal tubule epithelial (HK2) cells damage, platelets were co-cultured with HK2 cells. The results indicated that platelet TTR can cause reactive oxygen species production and apoptosis in HK2 cells. Further research found that platelet TTR can also result in increased levels of mRNA and protein for protein kinase B (AKT), phosphatidylinositol 3-kinase (PI3K), and extracellular regulated protein kinase (ERK), as analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. In conclusion, platelet-derived TTR may be one kind of DAMPs that plays an important role in the development of S-AKI.

The role of microRNA-155 in glomerular endothelial cell injury induced by high glucose

ObjectiveTo investigate the role of microRNA-155-5p on apoptosis and inflammatory response in human renal glomerular endothelial cells (HRGEC) cultured with high glucose.MethodsThe primary HRGEC were mainly studied, light microscopy was used to detect changes in cell morphology. Quantitative Real Time-Polymerase Chain Reaction, Western Blot, immunofluorescence were aimed to observe the mRNA and protein expression levels of target gene ETS-1, downstream factors VCAM-1, MCP-1 and cleaved caspase-3 in each group after high glucose treatment as well as transfection with miR-155 mimics or inhibitor.ResultsThe expression of inflammatory factors and apoptosis of HRGEC cells increased under high glucose treatment. Compared with normal-glucose treatment, the expression of microRNA-155 markedly increased in HRGECs treated with high-glucose, as well as the mRNA and protein levels of ETS-1, VCAM-1, MCP-1 and cleaved caspase-3. Overexpression of microRNA-155 remarkably downregulated mRNA and protein levels of ETS-1, VCAM-1, MCP-1 and cleaved caspase-3, whereas miRNA-155 knockdown upregulated their levels. In addition, HRGEC cells were transfected with miR-155 mimics and ETS-1 siRNA with high glucose stimulation. The expression of ETS-1 was positively correlated with the expression of downstream factors VCAM-1 and MCP-1. These results suggest that ETS-1 can mediate endothelial cell inflammation by regulating VCAM-1 and MCP-1.ConclusionMiR-155 can negatively regulate the expression of target gene ETS-1 and its downstream factors VCAM-1, MCP-1 and cleaved caspase-3, thus mediating the inflammatory response and apoptosis of HRGEC.