Mouse Renal Tubular Epithelial Cells Research Articles

Tumor necrosis factor superfamily 14 is critical for the development of renal fibrosis.

Objective: Tumor necrosis factor superfamily protein 14 (TNFSF14) was recently identified as a risk factor in some fibrosis diseases. However, the role of TNFSF14 in renal fibrosis pathogenesis remains unknown.Results: It was found that TNFSF14 levels were significantly increased both in UUO-induced renal fibrotic mice and in patients with fibrotic nephropathy, compared with those in controls. Accordingly, Tnfsf14 deficiency led to a marked reduction in renal fibrosis lesions and inflammatory cytokines expression in the UUO mice. Furthermore, the levels of Sphk1, a critical molecule that causes fibrotic nephropathy, were remarkably reduced in Tnfsf14 KO mice with UUO surgery. In vitro recombinant TNFSF14 administration markedly up-regulated the expression of Sphk1 of primary mouse renal tubular epithelial cells (mTECs).Conclusion: TNFSF14 is a novel pro-fibrotic factor of renal fibrosis, for which TNFSF14 up-regulates Sphk1 expression, which may be the underlying mechanism of TNFSF14-mediated renal fibrosis.Methods: We investigated the effect of TNFSF14 on renal fibrosis and the relationship between TNFSF14 and pro-fibrotic factor sphingosine kinase 1 (Sphk1) by using the unilateral urethral obstruction (UUO)-induced mice renal fibrosis as a model and the specimen of patients with fibrosis nephropathy, by Masson trichrome staining, immunohistochemistry, qRT-PCR, and western blot analysis.

Long Non-coding RNA LRNA9884 Promotes Acute Kidney Injury via Regulating NF-kB-Mediated Transcriptional Activation of MIF.

Acute kidney injury (AKI) is one of the most common complications affecting hospitalized patients associated with an extremely high mortality rate. However, the underlying pathogenesis of AKI remains unclear that largely limits its effective management in clinic. Increasing evidence demonstrated the importance of long non-coding RNAs (lncRNAs) in the pathogenesis of AKI, because of their regulatory roles in transcription, translation, chromatin modification, and cellular organization. Here, we reported a new role of LRNA9884 in AKI. Using experimental cisplatin-induced AKI model, we found that LRNA9884 was markedly up-regulated in the nucleus of renal tubular epithelium in mice with AKI. We found that silencing of LRNA9884 effectively inhibited the production of inflammatory cytokines MCP-1, IL-6, and TNF-α in the mouse renal tubular epithelial cells (mTECs) under IL-1β stimulation in vitro. Mechanistically, LRNA9884 was involved into NF-κB-mediated inflammatory cytokines production especially on macrophage migration inhibitory factor (MIF). Collectedly, our study suggested LRNA9884 promoted MIF-triggered the production of inflammatory cytokines via NF-κB pathway after AKI injury. This study uncovered LRNA9884 has an adverse impact in AKI, and targeting LRNA9884 might represent a potential therapeutic target for AKI.

Inhibition of ROCK2 alleviates renal fibrosis and the metabolic disorders in the proximal tubular epithelial cells.

Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson's trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-β1 (TGF-β1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-β1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD.

Mistimed H2S upregulation, Nrf2 activation and antioxidant proteins levels in renal tubular epithelial cells subjected to anoxia and reoxygenation.

Ischemia-reperfusion (I-R) injury is involved in the pathogenesis of several human diseases. In the present study, the kinetics of the H2S producing enzymes-nuclear factor erythroid 2-like 2 (Nrf2)-antioxidant proteins axis under anoxia or reoxygenation was evaluated, as well as its effects on survival of mouse renal proximal tubular epithelial cells (RPTECs). In RPTECs subjected to anoxia and subsequent reoxygenation, reactive oxygen species (ROS) production, lipid peroxidation, ferroptotic cell death, the levels of the H2S producing enzymes and H2S, the expression of Nrf2 and its transcriptional targets superoxide dismutase-3, glutathione reductase, ferritin H and cystine-glutamate antiporter, as well as apoptosis, and the levels of p53, Bax and phosphorylated p53 were assessed. When needed, the H2S producing enzyme inhibitor aminooxyacetate, or the ferroptosis inhibitor α-tocopherol, were used. Reoxygenation induced ferroptosis, whereas anoxia activated the p53-Bax pathway and induced apoptosis. The H2S producing enzymes-Nrf2-antioxidant proteins axis was activated only during anoxia and not during reoxygenation, when cellular viability is threatened by ROS overproduction and the ensuing ferroptosis. The activation of the above axis during anoxia ameliorated the effects of the apoptotic p53-Bax pathway, but did not adequately protect against apoptosis. In conclusion, the H2S-Nrf2 axis is activated by anoxia, and although it reduces apoptosis, it does not completely prevent apoptotic cell death. Additionally, following reoxygenation, the above axis was not activated. This mistimed activation of the H2S producing enzymes-Nrf2-antioxidant proteins axis contributes to reoxygenation-induced cell death. Determining the exact molecular mechanisms involved in reoxygenation-induced cell death may assist in the development of clinically relevant interventions for preventing I-R injury.

SUN-042 PREGNANE X RECEPTOR (PXR) IS A NOVEL THERAPEUTIC TARGET FOR THE TREATMENT OF CISPLATIN-INDUCED NEPHROTOXICITY IN MICE

As a widely used chemotherapeutic agent for treating malignancies, the clinical application of cisplatin (CP) is limited by the toxicity to several vital human organs, especially nephrotoxicity. The pregnane X receptor (PXR) is a ligand-dependent transcriptional factor belonging to the nuclear receptor superfamily. PXR is highly expressed in the liver, intestine and kidney, where it plays an important role in xenobiotic and endobiotic metabolism. PXR is constitutively expressed in normal kidney with relatively high levels in the proximal tubular cells, which are initially affected in CP-induced nephrotoxicity. In this study, we aimed to investigate the role of PXR in CP-induced nephrotoxicity. In vivo, wild-type male C57BL/6 mice were orally pretreated with pregnenolone 16α-carbonitrile (PCN, 40mg/kg), a well-known mouse PXR agonist, for 3 days. After a single nephrotoxic dose of CP (20mg/kg) was administered at day 4, the mice were treated daily with PCN for another 3 days. Levels of blood urea nitrogen (BUN), serum creatinine (sCr), urinary N-acetyl-D-glucosaminidase (NAG) and urinary albumin were measured by commercial kits, high performance liquid chromatography (HPLC), fluorescence probe examination or enzyme linked immunosorbent assay (ELISA). Renal histopathologic features were evaluated by hematoxylin and eosin (H&E) staining on formalin-fixed kidney sections. Caspase 3 activity was measured in kidney homogenates, and TUNEL staining was performed on formalin-fixed sections. Expression levels of important genes involved in apoptosis, oxidative stress, and inflammation were investigated in kidney homogenates by immunoblot and real-time PCR. Whole transcriptome sequencing was applied to reveal the potential mechanisms of PXR protective effects to CP-induced nephrotoxicity. In vitro, PXR expression and activity were modulated by PCN, overexpression, or knockdown by shRNAs in mouse renal tubular epithelial (MCT) cells. A significant increase in BUN, sCr, urinary NAG and albumin levels induced by CP, was markedly prevented by PCN treatment. Histologically, PXR activation ameliorated CP-induced renal morphologic damage, especially in the proximal tubules, characterized by a large number of apoptotic cells. TUNEL-positive cells and renal activity of caspase 3 were both decreased in PCN treated mice. PXR activation abolished CP-induced p53 transcription and phosphorylation, and significantly reduced p53 protein levels. PXR activation also dramatically prevented oxidative and inflammatory stress in the kidneys of mice treated with CP. Whole transcriptome sequencing revealed that protective effects of PXR were associated with multiple crucial signaling pathways, including fatty acid metabolism and degradation, metabolism of xenobiotics by cytochrome P450, protein processing in endoplasmic reticulum, biosynthesis of unsaturated fatty acids, carbon metabolism, steroid hormone biosynthesis, PI3K-Akt signaling pathway, PPAR signaling pathway. Notably, we discovered that PXR protects against CP-induced nephrotoxicity by translational regulating multidrug and toxin extrusion 1 (MATE1), an important transporter mediating cellular excretion of CP, in the kidney. Our data provide evidence that PXR activation preserves renal function after CP treatment, and suggest the possibility of PXR as a novel therapeutic target for CP-induced nephrotoxicity.

Degradation of different molecular weight fucoidans and their inhibition of TGF-β1 induced epithelial–mesenchymal transition in mouse renal tubular epithelial cells

In order to investigate the anti-fibrotic effect of different molecular weight (Mw) fucoidans on TGF-β1-induced mouse renal tubular epithelial cell (MTEC) mode. Oxidative degradation method was used to obtain fucoidans with different molecular weights and the reaction time, reaction temperature and the concentration of oxidants were investigated. Cell viability was detected by CCK-8, and EMT markers expression was detected by Western-bolt and Cell immunofluorescence assay. As a result, after chemical analysis of three independent batches of prepared samples, one batch of fucoidan sample (LHX 1–9) which chemical contents are similar but Mw ranging from 3.3 KDa to 49.3 KDa were selected to do further research. We found LHX1 (Mw = 3.3 KDa) and LHX 3–9 (Mw = 6.6 KDa, 8.3 KDa, 11.3 KDa, 14.9 KDa, 25.2 KDa, 35.4 KDa, 49.3 KDa) could resist the TGF-β1-induced depithelial–mesenchymal transition (EMT) by decreased expression of Fn and CTGF and maintained epithelial cell morphology in MTEC. However, the relationship between the Mw of fucoidans and their anti-EMT effect is not simply linear. Among the samples, LHX 1, 5 and 8 showed significant anti-EMT effects than others by de-regulated Fn and CTGF expression on MTEC cells.

Activation of GSDMD contributes to acute kidney injury induced by cisplatin.

Cisplatin is one of the most effective antitumor agents, but its clinical use is highly limited by its severe side effects, especially nephrotoxicity. Recently, the active form of gasdermin D (GSDMD), termed GSDMD-N, was identified to mediate pyroptotic inflammatory cell death in several diseases. However, the role of the GSDMD-N fragment in cisplatin-induced acute kidney injury (AKI) remains unclear. In the present study, we found that pyroptosis was induced by cisplatin in both mouse kidney tissues and renal tubular epithelial cells, accompanied by increased expression of the GSDMD-N fragment. In GSDMD knockout mice with cisplatin-induced AKI, we found that cisplatin-induced loss of renal function, renal tubular injury, and inflammation was significantly attenuated compared with wild-type mice. Furthermore, the GSDMD-N fragment was overexpressed by an established rapid plasmid tail vein injection approach to evaluate the role of this cleaved form of GSDMD in AKI. As expected, mice with GSDMD-N fragment overexpression in the kidney were more susceptible to cisplatin-induced AKI than control mice, as evidenced by further elevated serum levels of blood urea nitrogen and creatinine, aggravated renal pathology, increased expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and enhanced renal inflammatory cytokine secretion, which indicates a pathogenic role of GSDMD-N in cisplatin-induced AKI by triggering cell pyroptosis. Similar results were also observed in renal tubular epithelial cells overexpressing the GSDMD-N fragment. Thus these findings suggested that the activation of GSDMD contributes to cisplatin-induced AKI, possibly through triggering pyroptosis.

Melatonin Attenuates Cisplatin-Induced Acute Kidney Injury through Dual Suppression of Apoptosis and Necroptosis.

Melatonin is well known to modulate the sleep–wake cycle. Accumulating evidence suggests that melatonin also has favorable effects such as anti-oxidant and anti-inflammatory properties in numerous disease models. It has been reported that melatonin has therapeutic effects against cisplatin-induced acute kidney injury (AKI). However, mechanisms underlying the therapeutic action of melatonin on the renal side-effects of cisplatin therapy remain poorly understood. In this study, we showed that melatonin treatment significantly ameliorates cisplatin-induced acute renal failure and histopathological alterations. Increased expression of tubular injury markers was largely reduced by melatonin. Melatonin treatment inhibited caspase-3 activation and apoptotic cell death. Moreover, protein levels of key components of the molecular machinery for necroptosis were decreased by melatonin. Melatonin also attenuated nuclear factor-κB activation and suppressed expression of pro-inflammatory cytokines. Consistent with in vivo findings, melatonin dose-dependently decreased apoptosis and necroptosis in cisplatin-treated mouse renal tubular epithelial cells. Collectively, our findings suggest that melatonin ameliorates cisplatin-induced acute renal failure and structural damages through dual suppression of apoptosis and necroptosis. These results reveal a novel mechanism underlying the therapeutic effect of melatonin against cisplatin-induced AKI and strengthen the idea that melatonin might be a promising therapeutic agent for the renal side-effects of cisplatin therapy.

METTL3/m6A/miRNA-873-5p Attenuated Oxidative Stress and Apoptosis in Colistin-Induced Kidney Injury by Modulating Keap1/Nrf2 Pathway.

Nephrotoxicity of colistin is the major factor limiting its clinical application. However, the exact mechanism of colistin-induced nephrotoxicity is still elusive. N6-Methyladenosine (m6A) modification has been implicated in many biological processes, however, its role in colistin-induced nephrotoxicity needs to be elucidated. Mouse renal tubular epithelial cells (mRTECs) were treated with 200 μM colistin with or without METTL3 overexpression. Cells injury, m6A assay, oxidative stress and apoptosis were examined. Levels of m6A are decreased after colistin treatment in mRTECs. METTL3 is the major factor involved in abnormal m6A modification. METTL3 overexpression plays a protective role against colistin-induced oxidative stress and apoptosis. Moreover, METTL3 interacts with the microprocessor protein DGCR8 and positively modulates miR-873-5p mature process in an m6A-dependent manner. Further experiments show that miR-873-5p could regulate Keap1-Nrf2 pathway against colistin-induced oxidative stress and apoptosis. These studies revealed an important role of METTL3/m6A in colistin-induced nephrotoxicity and provide a new insight on m6A modification in drug induced toxicity.

3-deazaneplanocin A protects against cisplatin-induced renal tubular cell apoptosis and acute kidney injury by restoration of E-cadherin expression

3-deazaneplanocin A (3-DZNeP) has been used as an inhibitor of enhancer of zeste homolog 2 (EZH2). Here, we explore the role and underlying mechanisms action of 3-DZNeP in abrogating cisplatin nephrotoxicity. Exposure of cultured mouse renal proximal tubular epithelial cells (mTECs) to cisplatin resulted in dose and time-dependent cleavage of caspase-3, decrease of cell viability, and increase of histone H3 lysine 27 trimethylation (H3K27me3), whereas expression levels of EZH2, a major methyltransferase of H3K27me3, were not affected. Treatment with 3-DZNeP significantly inhibited cisplatin-induced activation of caspase-3, apoptosis, loss of cell viability but did not alter levels of EZH2 and H3K27me3 in cultured mTECs. 3-DZNeP treatment did not affect activation of extracellular signal-regulated kinase (ERK) 1/2, p38 or c-Jun N-terminal kinases (JNK) 1/2, which contribute to renal epithelial cell death, but caused dose-dependent restoration of E-cadherin in mTECs exposed to cisplatin. Silencing of E-cadherin expression by siRNA abolished the cytoprotective effects of 3-DZNeP. In contrast, 3-DZNeP treatment potentiated the cytotoxic effect of cisplatin in H1299, a non-small cell lung cancer cell line that expresses lower E-cadherin levels. Finally, administration of 3-DZNeP attenuated renal dysfunction, morphological damage, and renal tubular cell death, which was accompanied by E-cadherin preservation, in a mouse model of cisplatin nephrotoxicity. Overall, these data indicate that 3-DZNeP suppresses cisplatin-induced tubular epithelial cell apoptosis and acute kidney injury via an E-cadherin-dependent mechanism, and suggest that combined application of 3-DZNeP with cisplatin would be a novel chemotherapeutic strategy that enhances the anti-tumor effect of cisplatin and reduces its nephrotoxicity.

Factors that May Protect the Native Hibernator Syrian Hamster Renal Tubular Epithelial Cells from Ferroptosis Due to Warm Anoxia-Reoxygenation.

Warm anoxia-reoxygenation induces ferroptotic cell death in mouse proximal renal tubular epithelial cells (RPTECs), whereas RPTECs of the native hibernator Syrian hamster resist cell death. Clarifying how hamster cells escape ferroptosis may reveal new molecular targets for preventing or ameliorating ischemia-reperfusion-induced human diseases or expanding the survival of organ transplants. Mouse or hamster RPTECs were subjected to anoxia and subsequent reoxygenation. Cell death was assessed with the lactated dehydrogenase (LDH) release assay and lipid peroxidation by measuring cellular malondialdehyde (MDA) fluorometrically. The effect of the ferroptosis inhibitor α-tocopherol on cell survival was assessed by the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay. The expression of the critical ferroptotic elements cystine-glutamate antiporter (xCT), ferritin, and glutathione peroxidase 4 (GPX4) was assessed by Western blot. Contrary to mouse RPTECs, hamster RPTECs resisted anoxia-reoxygenation-induced cell death and lipid peroxidation. In mouse RPTECs, α-tocopherol increased cell survival. Anoxia increased the levels of xCT, ferritin, and GPX4 in both cell types. During reoxygenation, at which reactive oxygen species overproduction occurs, xCT and ferritin decreased, whereas GPX4 increased in mouse RPTECs. In hamster RPTECs, reoxygenation raised xCT and ferritin, but lowered GPX4. Hamster RPTECs resist lipid peroxidation-induced cell death. From the three main evaluated components of the ferroptotic pathway, the increased expression of xCT and ferritin may contribute to the resistance of the hamster RPTECs to warm anoxia-reoxygenation.

Protective effects of Klotho protein on acute kidney injury in septic mice and its mechanism

To investigate the protective effects of Klotho protein, a kind of single-pass transmembrane protein, on acute kidney injury (AKI) in septic mice and its mechanism. Sixty SPF healthy male C57BL/6 mice (6-8 weeks) were randomly divided into sham operation group (Sham group), sepsis model group (CLP group) and Klotho protein injection group (CLP+KL group), with 20 in each group. The septic AKI mice model was established by cecal ligation and puncture (CLP); Sham group had the same procedure except that the cecal was not ligated. The CLP+KL group was received Klotho protein (0.02 mg/kg) by intraperitoneal consecutive injection for 4 days after operation; Sham group and CLP group were injected with the same amount of saline. Blood samples were obtained at 24 hours after operation, the levels of serum creatinine (SCr) and urea nitrogen (BUN) were measured by sarcosine oxidase and urease method. The mice were sacrificed under anesthesia at 5 days after operation to harvest renal tissues, and the pathological damage of the kidney was evaluated by hematoxylin-eosin (HE) staining. The ultrastructure of mitochondria in mouse renal tubular epithelial cells was observed under transmission electron microscope. The levels of reduced glutathione hormone (GSH), malondialdehyde (MDA) and nitric oxide synthase (NOS) in mitochondrion were determined by micro-enzyme method, thiobarbituric acid method, colorimetry method, respectively. The protein expressions of Klotho, Bcl-2 and cytochrome C (Cyt C) were detected by Western Blot. The pathological structure of the kidneys in the Sham group was clear and intact. Compared with the Sham group, the renal tissue edema of the mice in the CLP group was significant, and the transparent tube type was observed in the small lumen, and the interstitial inflammatory cells infiltrated; the levels of SCr and BUN were significantly increased [SCr (μmol/L): 182.60±6.97 vs. 47.20±5.37, BUN (mmol/L): 53.70±5.12 vs. 18.70±2.62, both P < 0.01]; the mitochondria were swollen and deformed, the sputum structure was destroyed, the matrix density was decreased, the outer membrane was lost, and the levels of MDA, GSH and NOS were significantly increased [MDA (μmol/g): 1.172±0.046 vs. 0.746±0.094, GSH (μmol/g): 5.765±0.059 vs. 4.223±0.072, NOS (kU/g): 0.91±0.05 vs. 0.68±0.03, all P < 0.01]; the protein expressions of Klotho and Bcl-2 in renal tissue were decreased, and the protein expression of Cyt C was increased (Klotho/β-actin: 0.188±0.020 vs. 0.538±0.024, Bcl-2/β-actin: 0.311±0.010 vs. 0.391±0.015, Cyt C/β-actin: 0.226±0.010 vs. 0.135±0.006, all P < 0.01). Comparing with the CLP group, the glomerular and tubular tissue epithelial edema and the small lumen in the CLP+KL group were reduced; the levels of SCr and BUN were significantly decreased [SCr (μmol/L): 85.70±7.23 vs. 182.60±6.97, BUN (mmol/L): 35.30±3.50 vs. 53.70±5.12, both P < 0.01]; the mitochondrial structure was relatively intact; the levels of MDA, GSH and NOS were significantly decreased [MDA (μmol/g): 0.958±0.072 vs. 1.172±0.046, GSH (μmol/g): 4.756±0.107 vs. 5.765±0.059, NOS (kU/g): 0.79±0.02 vs. 0.91±0.05, all P < 0.01]; the protein expressions of Klotho, Bcl-2 were significantly increased, but the protein expression of Cyt C was significantly decreased (Klotho/β-actin: 0.336±0.011 vs. 0.188±0.020, Bcl-2/β-actin: 0.474±0.017 vs. 0.311±0.010, Cyt C/β-actin: 0.168±0.006 vs. 0.226±0.010, all P < 0.01). Klotho protein has significant protective effects on AKI in septic mice, and its mechanism is related to maintaining mitochondrial structural integrity and oxidative stress response.

TRIM29 Is a New Gene That Regulates IRS-1 to Induce Insulin Resistant in Diabetes

Insulin resistance is a major risk for development of type 2 diabetes. Several genes involved in regulation of insulin receptor substrate 1 (IRS1) and lead to chronic diabetes. Our RNA sequence data from kidney of diabetic mice show a new candidate gene involved in the regulation of IRS1. New candidate gene, Tripartite motif protein 29 (TRIM29), acts as an E3 ligase targeting insulin receptor (IR) and insulin receptor substrate 1 (IRS1) for ubiquitin-dependent degradation, resulting in insulin resistance. RNA seq data show that TRIM29 is higher 2.5 and 3.6 fold in kidney cortex of diabetic mice (db/db) at age of 10 and 12 months compared to wild type mice, respectively. Mouse renal proximal tubular epithelial cells treated with high glucose (25mM) for 48 hours showed significant increased in TRIM29 and decreased in IRS-1 compared to cells grown in normal glucose (5mM). Pre-transfected cells with siRNA against TRIM29 resulted in significant increased IRS-1 and cell proliferative marker, cyclin D1 expression. Downregulation of tuberin by siRNA significantly decreased TRIM29 expression and increased cyclin D1 expression. In addition, downregulation of AMPK by DN-AMPK and compound C resulted in significant decreased TRIM29 compared to cells transfected with control-siRNA. Kidney cortex of db/db mice and WT mice at age 10 and 12 months were analyzed by Western blot showed that TRIM29 is significantly increased and IRS-1 decreased compared to wild type mice. Increase TRIM29 in kidney diabetic mice was associated with increased in cyclin D1. Here, we identified a new candidate gene, TRIM29 that involves in the regulation IRS-1 pathways to increase cell proliferation and enhance insulin resistance diabetes. The new gene, TRIM29, may be used as a new candidate target to treat insulin resistant in diabetes. Disclosure S.L. Habib: None.

The In Vitro Differentiation of GDNF Gene-Engineered Amniotic Fluid-Derived Stem Cells into Renal Tubular Epithelial-Like Cells.

Amniotic fluid is an alternative source of stem cells, and human amniotic fluid-derived stem cells (AFSCs) obtained from a small amount of amniotic fluid collected during the second trimester represent a novel source for use in regenerative medicine. These AFSCs are characterized by lower diversity, a higher proliferation rate, and a wider differentiation capability than adult mesenchymal stem cells. AFSCs are selected based on the cell surface marker c-kit receptor (CD117) using immunomagnetic sorting. Glial cell line-derived neurotrophic factor (GDNF) is expressed during early kidney development and regulates the proliferation and differentiation of stem cells in vitro. In this study, c-kit-sorted AFSCs were induced toward osteogenic or adipogenic differentiation. AFSCs engineered via the insertion of GDNF were cocultured with mouse renal tubular epithelial cells (mRTECs), which were preconditioned by hypoxia-reoxygenation in vitro. After coculture for 8 days, AFSCs differentiation into epithelial-like cells was evaluated by performing immunofluorescence, flow cytometry, and quantitative real-time polymerase chain reaction to identify cells expressing the renal epithelial markers, cytokeratin 18 (CK18), E-cadherin, aquaporin-1 (AQP1), and paired box 2 gene (Pax2). The GDNF gene enhanced AFSCs differentiation into RTECs. AFSCs possess self-renewal ability and multiple differentiation potential and thus represent a new source of stem cells.

Impact of epigallocatechin‑3‑gallate on expression of nuclear factor erythroid 2‑related factor 2 and γ‑glutamyl cysteine synthetase genes in oxidative stress‑induced mouse renal tubular epithelial cells

The aim of the present study was to investigate the antioxidant response mechanism of epigallocatechin‑3‑gallate (EGCG) in H2O2‑induced mouse renal tubular epithelial cells (MRTECs). The cultured MRTECs were divided into normal, H2O2 (control) and EGCG treatment groups. The MTT assay was used to assess cell viability, and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), immunocytochemical and western blot analyses were performed to detect the expression of nuclear factor erythroid 2‑related factor 2 (Nrf2) and γ‑glutamyl cysteine synthetase (γ‑GCS). EGCG was able to mitigate H2O2‑mediated cell damage. The RT‑qPCR results demonstrated that EGCG was able to upregulate the gene expression of Nrf2 and γ‑GCS in MRTECs in a dose‑dependent manner. The immunocytochemistry and western blot analyses demonstrated that EGCG was able to increase the protein expression of Nrf2 and γ‑GCS in MRTECs in a dose‑dependent manner. Oxidative stress may lead to a decrease in the viability of MRTECs, while EGCG was able to promote the expression of Nrf2 and γ‑GCS in MRTECs, thereby improving the antioxidant capacity of the cells and promoting the repair of oxidative stress injury.

Leucine rich α-2 glycoprotein is a potential urinary biomarker for renal tubular injury

Recent evidence suggests that renal tubular injury plays a key role in deterioration of renal function in both chronic kidney disease (CKD) and acute kidney injury (AKI). Since commonly used biochemical indicators such as GFR, serum creatinine, blood urea nitrogen and creatinine clearance are inappropriate for detecting alteration in renal tubules, biomarkers reflecting renal tubular injury have been extensively explored.Our research group identified leucine rich α-2 glycoprotein (LRG) as a novel serum biomarker for various inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. In inflammatory diseases, LRG expression is up-regulated at the site of inflammation, in accordance with the induction of LRG in many cell types by various inflammatory cytokines. Recently, urinary LRG was reported as a possible biomarker for several renal diseases, but the mechanism of LRG excretion in urine is still unclear. In this study, by analyzing a mouse albumin (ALB) overload model that is commonly used to study proteinuria-induced renal tubular injury, we provided evidence that urinary LRG is produced in renal tubular epithelial cells by interleukin-1β (IL-1β) that is released during proteinuria-induced renal damage. In this model, urinary LRG became detectable after ALB overload. In kidney, mRNA expression of LRG together with that of NACHT LRR and PYD domains-containing protein 3 (NLRP3) and IL-1β was significantly up-regulated in ALB-overloaded mice, compared to PBS-treated mice. By pathological analysis of kidney, LRG was detected in the injured proximal tubules, distal tubules and collecting ducts in ALB-overloaded mice. Accordingly, in vitro stimulation of mouse renal cortical tubular epithelial cells with excessive ALB led to LRG mRNA up-regulation and its protein secretion, which was effectively blocked by IL-1 receptor antagonist. These results suggest that urinary LRG could be applied to a biomarker detecting renal tubular injury in various renal diseases.

Activation of Ca2+-sensing receptor as a protective pathway to reduce Cadmium-induced cytotoxicity in renal proximal tubular cells

Cadmium (Cd), as an extremely toxic metal could accumulate in kidney and induce renal injury. Previous studies have proved that Cd impact on renal cell proliferation, autophagy and apoptosis, but the detoxification drugs and the functional mechanism are still in study. In this study, we used mouse renal tubular epithelial cells (mRTECs) to clarify Cd-induced toxicity and signaling pathways. Moreover, we proposed to elucidate the prevent effect of activation of Ca2+ sensing receptor (CaSR) by Calcimimetic (R-467) on Cd-induced cytotoxicity and underlying mechanisms. Cd induced intracellular Ca2+ elevation through phospholipase C-inositol 1, 4, 5-trisphosphate (PLC) followed stimulating p38 mitogen-activated protein kinases (MAPK) activation and suppressing extracellular signal-regulated kinase (ERK) activation, which leaded to increase apoptotic cell death and inhibit cell proliferation. Cd induced p38 activation also contribute to autophagic flux inhibition that aggravated Cd induced apoptosis. R-467 reinstated Cd-induced elevation of intracellular Ca2+ and apoptosis, and it also increased cell proliferation and restored autophagic flux by switching p38 to ERK pathway. The identification of the activation of CaSR-mediated protective pathway in renal cells sheds light on a possible cellular protective mechanism against Cd-induced kidney injury.

Klotho Reduces Necroptosis by Targeting Oxidative Stress Involved in Renal Ischemic-Reperfusion Injury

Background/Aims: Klotho is a multifunctional protein expressed predominantly in kidney tubular epithelium. Here, we investigated the protective effects of Klotho on necroptosis in renal ischemic-reperfusion injury (IRI) and the role of oxidative stress in this process. Methods: Mice were subjected to bilateral renal pedicle clamping. Mouse renal tubular epithelial (TCMK-1) cells were exposed to hypoxia/reoxygenation (H/R) or H2O2. Kidney samples from acute kidney injury (AKI) patients and controls were examined by immunofluorescence. Klotho protein and N-acetyl-L-cysteine (NAC) were used to define their roles in mediating necroptosis. Necroptosis was assessed by TUNEL staining, immunoblotting, and real-time PCR. Oxidative stress was studied via ELISA, immunoblotting, colorimetric, and thiobarbituric acid reactive substances assays. Results: Renal IRI induced Klotho deficiency in the serum and kidney, but an increase in the urine. The levels of the necroptotic markers receptor-interacting protein kinase (RIP) 1, RIP3, IL-1β, and TUNEL-positive cells increased after IRI; all increases were ameliorated by Klotho. In TCMK-1 cells, Klotho and NAC attenuated the elevation in RIP1, RIP3, and LDH release induced by H/R or H2O2. Moreover, Klotho decreased the levels of oxidative stress biomarkers and elevated superoxide dismutase 2 expression in both in vivo and in vitro experiments. Studies in human samples further confirmed the Klotho deficiency and increased formation of RIP3 puncta in AKI kidneys. Conclusion: Klotho protects tubular epithelial cells from IRI and its anti-necroptotic role may be associated with oxidative stress inhibition.

Protective effect of GDNF-engineered amniotic fluid-derived stem cells on the renal ischaemia reperfusion injury in vitro.

Amniotic fluid-derived stem cells (AFSCs) possessing multilineage differentiation potential are proposed as a novel and accessible source for cell-based therapy and tissue regeneration. Glial-derived neurotrophic factor (GDNF) has been hypothesized to promote the therapeutic effect of AFSCs on markedly ameliorating renal dysfunction. The aim of this study was to investigate whether AFSCs equipped with GDNF (GDNF-AFSCs) had capabilities of attenuating mouse renal tubular epithelial cells (mRTECs) apoptosis and evaluate its potential mechanisms. A hypoxia-reoxygenation (H/R) model of the mRTECs was established. Injured mRTECs were co-cultured with GDNF-AFSCs in a transwell system. The mRNA expressions of hepatocytes growth factor (HGF) and fibroblast growth factor (bFGF) were detected by qRT-PCR. Changes of intracelluar reactive oxygen species (ROS) and the level of superoxide dismutase (SOD) and malondialdehyde (MDA) were examined. The expressions of nitrotyrosine, Gp91-phox, Bax, and Bcl-2 were determined by Western blotting. Cell apoptosis was assayed by flow cytometry, and caspase-3 activity was monitored by caspase-3 activity assay kit. Our study revealed that expression of growth factors was increased and oxidative stress was dramatically counteracted in GDNF-AFSCs-treated group. Furthermore, apoptosis induced by H/R injury was inhibited in mRTECs by GDNF-AFSCs. These data indicated that GDNF-AFSCs are beneficial to repairing damaged mRTECs significantly in vitro, which suggests GDNF-AFSCs provide new hopes of innovative interventions in different kidney disease.

Effects of B cells activating factor on growth characteristics of mouse renal tubular epithelial cells

Objective To investigate the expression of B cell activating factor (BAFF) and BAFF receptor (BAFF-R) on primary mouse renal tubular epithelial cells (RTECs), and the effect on growth characteristics of RTECs. Methods Primary mouse RTECs were isolated by enzyme digestion method, and cultured in the conditioned medium. After stimulated by 500 U/ml Interferon-γ (IFN-γ) for 48 h, the expression of BAFF and BAFF-R on RTECs were detected by Flow Cytometry (FCM). After treated with recombinant mouse BAFF and/or blockade B cell activating factor receptor-Fc fusion protein (BAFF-R-Fc) for 48 h, the transport ability of fluorescein sodium and cytokeratin-18 (CK-18) expression of RTECs were detected, respectively; The proliferation ability and the apoptosis rates of RTECs were tested by 3- (4, 5-dimenthylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (MTS) and FCM method, respectively; Data were analyzed by SPSS 17.0, and P<0.05 was considered to be statistically significant. Results BAFF-R expression on RTECs in the IFN-γ treated group significantly up-regulated, compared with control group (4 166.250±913.914 vs. 3 602.750±875.584, t=4.124, P=0.026). Fluorescein sodium transport experiment showed the average absorbance of BAFF stimulation group was significantly lower than BAFF-R-Fc+ BAFF group (0.011±0.003 vs. 0.020±0.007, t=-3.437, P=0.041), and control group (0.011±0.003 vs. 0.016±0.006, t=-3.572, P=0.038); Immunofluorescence results showed the average absorbance of BAFF stimulation group was significantly lower than that of BAFF-R-Fc+ BAFF group (0.032±0.005 vs. 0.043±0.007, t=-8.006, P=0.015), and control group (0.032±0.005 vs. 0.041±0.008, t=-4.603, P=0.044); Cell proliferation assay results showed that, A value of 5 ng/ml and 20 ng/ml BAFF group were significantly higher than that of control group (1.532±0.058 vs. 1.473±0.045; 1.509±0.056 vs. 1.473±0.045, t=5.636, P=0.030; t=5.765, P=0.029, respectively), and A value of 5 ng/ml BAFF group were significantly higher than that of BAFF-R-Fc+ BAFF group (1.532±0.058 vs. 1.477±0.050, t=5.085, P=0.037); Apoptosis test results indicated that the apoptosis rate of BAFF-treated group was significantly lower than that of BAFF-R-Fc+ BAFF group [(39.850±8.544)% vs. (42.950±8.330)%, t=-3.399, P=0.019], and control group [(39.850±8.544)% vs. (44.467±7.642)%, t=-3.020, P=0.029]. Conclusion BAFF signaling could promote the proliferation ability of RTECs, but down-regulate the epithelial cell characteristics and Ion transport ability of RTECs. Key words: Renal tubular epithelial cell; B cell activating factor; Cytokeratin-18; Ion transport; Proliferation