Mouse Renal Proximal Tubular Cells Research Articles

Histone methyltransferase MLL1 drives renal tubular cell apoptosis by p53-dependent repression of E-cadherin during cisplatin-induced acute kidney injury

Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation, and senescence. The role of MLL1 in the pathogenesis of acute kidney injury (AKI) is unknown. In this study, we demonstrate that MLL1, WDR5, and trimethylated H3K4 (H3K4me3) were upregulated in renal tubular cells of cisplatin-induced AKI in mice, along with increased phosphorylation of p53 and decreased expression of E-cadherin. Administration of MM102, a selective MLL1/WDR5 complex inhibitor, improved renal function and attenuated tubular injury and apoptosis, while repressing MLL1, WDR5, and H3K4me3, dephosphorylating p53 and preserving E-cadherin. In cultured mouse renal proximal tubular cells (RPTCs) exposed to cisplatin, treatment with MM102 or transfection with siRNAs for either MLL1 or WDR5 also inhibited apoptosis and p53 phosphorylation while preserving E-cadherin expression; p53 inhibition with Pifithrin-α lowered cisplatin-induced apoptosis without affecting expression of MLL1, WDR5, and H3K4me3. Interestingly, silencing of E-cadherin offset MM102’s cytoprotective effects, but had no effect on p53 phosphorylation. These findings suggest that MLL1/WDR5 activates p53, which, in turn, represses E-cadherin, leading to apoptosis during cisplatin-induced AKI. Further studies showed that MM102 effectively inhibited cisplatin-triggered DNA damage response (DDR), as indicated by dephosphorylation of ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) proteins, dephosphorylation of checkpoint kinase 1 and 2 (Chk1 and Chk2); depression of γ-H2AX; and restrained cell cycle arrest, as evidenced by decreased expression of p21 and phospho-histone H3 at serine 10 in vitro and in vivo. Overall, we identify MLL1 as a novel DDR regulator that drives cisplatin-induced RPTC apoptosis and AKI by modulating the MLL1/WDR5-/ATR/ATM-Chk-p53-E-cadherin axis. Targeting the MLL1/WDR5 complex may have a therapeutic potential for the treatment of AKI.

MO067: Role of ANT2 in the Development of Obesity-Induced Chronic Kidney Disease

Abstract BACKGROUND AND AIMS Obesity, a widespread condition with an ever-increasing prevalence, imposes a great medical concern since it serves as a risk factor for a wide range of conditions, including the development of chronic kidney disease (CKD). Lipid accumulation and impaired fatty acid β-oxidation in the renal proximal tubular cells (RPTCs) as well as an overall mitochondrial dysfunction have been shown to induce kidney injury, inflammation and fibrosis, leading to the development of CKD. However, the molecular mechanisms linking mitochondrial dysfunction in RPTCs to obesity-induced CKD are not fully understood and require further examination. Adenine nucleotide translocases (ANTs), which transport ADP and ATP through the mitochondrial inner membrane, play an essential role in energy metabolism of eukaryotic cells. ANT2 (Slc25a5) mediates fatty acid-induced uncoupled respiration, and its complete deletion is embryonic lethal. Here we explore its specific role in RPTCs in the development of obesity-induced CKD. METHOD ANT2 protein levels were measured using western blot and immunohistochemistry analyses. RPTC-ANT2-/- animals were generated using the Cre/loxP system. The null mice and their wild-type littermate controls were fed with either a high-fat diet (HFD) or a standard diet (STD) for 24 weeks. Kidney function and lipid content were assessed by biochemical analysis. Histological techniques were used to visualize the morphological changes in the kidney as well as fibrogenesis. Kidney injury and inflammatory markers were measured via qPCR and ELISAs. Mitochondrial function was tested in primary mouse RPTCs extracted from the null and WT animals by measuring oxygen consumption rate via Seahorse as well as reactive oxygen species (ROS) production by DCFDA staining. RESULTS As evident from the double-stained immunofluorescence staining of mouse kidney, high levels of ANT2 were found in RPTCs. Interestingly, its protein expression was downregulated in HFD-fed WT mice. Nullification of ANT2 specifically in RPTCs prevented the obesity-induced kidney dysfunction as reflected by lowered albumin-to-creatinine ratio. This was associated with the improved renal morphology in HFD-fed RPTC-ANT2-/- mice, evident by the reduction in obesity-related increased glomerular and Bowman's space areas and mesangial expansion. Compared with their littermate controls, obese RPTC-ANT2-/- mice showed reduced renal KIM-1 and NGAL protein levels and downregulation in the expression levels of Mcp1 and Lcn2, markers of renal injury and inflammation, respectively. The null mice were also protected from obesity-induced fibrosis, measured by trichrome staining, as well as accumulation of triglycerides and cholesterol in the kidney. In addition, preserved mitochondrial function was found in primary mouse RPTCs lacking ANT2, measured by increased spare respiratory capacity and decreased ROS production. Lastly, downregulated Hif1α gene and protein expression levels were found in RPTCs and kidney tissue lacking ANT2. CONCLUSION Our findings introduce ANT2 in the RPTCs as a key player in the development of obesity-induced CKD. Deletion of RPTC-ANT2 protects the kidney from the deleterious effects of lipotoxicity. Therefore, targeted manipulation of renal mitochondrial metabolism, particularly via inhibiting ANT2, may represent a novel approach for the treatment of obesity-induced CKD.

Asymmetric Total Synthesis of Toxicodenane A by Samarium-Iodide-Induced Barbier-Type Cyclization and Its Cell-Protective Effect against Lipotoxicity.

The asymmetric total synthesis of toxicodenane A, a sesquiterpenoid expected to be promising for diabetic nephropathy, was achieved. In the synthesis, a samarium iodide (SmI2)-induced Barbier-type cyclization and a regio- and stereoselective allylic oxidation followed by a dehydration cyclization were employed as key steps. Furthermore, the first asymmetric syntheses of both enantiomers were accomplished using the previously mentioned synthetic strategy. Finally, the synthetic compounds significantly inhibited lipotoxicity-mediated inflammatory and fibrotic responses in mouse renal proximal tubular cells.

Irisin is induced in renal ischemia-reperfusion to protect against tubular cell injury via suppressing p53

Renal ischemia-reperfusion is a major cause of acute kidney injury, a disease currently without effective treatments. Irisin was initially identified as an important factor produced by muscles to mediate the health benefits of exercise, and recent work has further suggested its protective effect against lung and liver injury. However, the role of Irisin in kidney diseases, including renal ischemia-reperfusion injury (IRI), remains unknown. In the present study, we found that the Irisin precursor, fibronectin type III domain-containing protein 5 (Fndc5), was induced in renal tubules in a mouse model of renal IRI and in cultured mouse renal proximal tubular cells subjected ATP depletion injury. Functionally, silencing Fndc5 in cultured proximal tubular cells increased the sensitivity to ATP depletion-induced apoptosis, whereas both Fndc5 overexpression and supplementation of recombinant Irisin alleviated ATP depletion-induced apoptosis. In vivo, administration of recombinant Irisin dramatically attenuated kidney dysfunction, tissue damage, tubular cell apoptosis, and inflammation during renal IRI in mice. Mechanistically, Irisin suppressed the activation of p53 in renal IRI, a critical factor in tubular cell death. Together, these results indicate that Irisin is induced in renal IRI as a protective mechanism for renal tubular cells, suggesting the therapeutic potential of recombinant Irisin in renal IRI and related kidney diseases.

Genome-wide profiling to analyze the effects of FXR activation on mouse renal proximal tubular cells.

To assess the effect of farnesoid X receptor (FXR), a bile acid nuclear receptor, on renal proximal tubular cells, primary cultured mouse kidney proximal tubular cells were treated with GW4064 (a FXR agonist) or DMSO (as controls) overnight. Analysis of gene expression in the proximal tubular cells by whole genome microarrays indicated that FXR activation induced genes involved in fatty acid degradation and oxidation reduction. Among them, genes involved in glutathione metabolism were mostly induced. Here we describe in details the contents and quality controls for the gene expression and related results associated with the data uploaded to Gene Expression Omnibus (accession number GSE70296).

RAS Blockade Inhibits Bcl‐2 Modifying Factor (Bmf) Expression and Prevents Hypertension and Tubular Apoptosis in Diabetic Akita Mouse Kidneys

BackgroundWe reported that pro‐apoptotic protein Bmf is highly expressed in diabetic mouse renal proximal tubular cells (RPTCs) and induces RPTC apoptosis in vitro (Diabetes 2012). The regulation of Bmf expression in RPTCs, however, is not well understood. We investigated whether renin‐angiotensin system (RAS) blockade could prevent Bmf expression, hypertension and RPTC apoptosis in Akita mice (a model of type 1 diabetes) in vivo and in vitro.MethodsAdult male Akita mice (11 weeks) were treated ± RAS blockers (losartan, 30 mg.Kg−1.day−1 and perindopril, 4 mg.Kg− 1.day−1 in drinking water) until 16 weeks. Controls were untreated non‐Akita littermates. Plasma glucose, systolic blood pressure (SBP) and albuminuria were monitored weekly. Kidneys were processed for histology and apoptosis studies (TUNEL assay). RPTC gene and protein expression were quantified by real time‐qPCR and Western blotting, respectively. We also examined the effect of angiotensin II (Ang II) on Bmf gene expression in cultured rat immortalized RPTCs.ResultsAkita mice developed hyperglycemia and significantly higher SBPs, kidney/body weight ratios and albuminuria, cf. to non‐Akita littermates. Kidneys from Akita mice displayed renal hypertrophy and tubular apoptosis cf. to non‐Akita littermates. Increased expression of Agt, Bmf and active caspase‐3 were evident in RPTCs of Akita mice. Treatment with RAS blockers inhibited Agt and Bmf expression, prevented hypertension and albuminuria and attenuated tubular apoptosis in Akita mice. Finally, Ang II stimulated Bmf mRNA expression in RPTCs and its effect was abolished in the presence of losartan.ConclusionAng II up‐regulates Bmf expression and RAS blockade effectively inhibits Bmf expression and prevents hypertension and tubular apoptosis in diabetic kidneys.Supported by Canadian Institutes of Health Research.

Abstract 385: TRPA1 Mediates Angiotensinogen Augmentation in Renal Proximal Tubular Cells

Overactivity of the intrarenal renin-angiotensin system contributes to the development of hypertension and renal injury. In this process, regulation of angiotensinogen (AGT) in renal proximal tubular cells (RPTC) is a key factor. Oxidative stress stimulates AGT expression accompanied by activation of p38 MAPK in RPTC. Transient receptor potential A1 (TRPA1) is known to be a sensor activated by a variety of noxious stimuli including hydrogen peroxide. Activated TRPA1 induces calcium influx across the plasma membrane leading to activation of AGT-inducible signal transducers. However, the presence and function of TRPA1 in the kidney have not been delineated. Therefore, this study was performed to demonstrate expression of intrarenal TRPA1 and its role in AGT augmentation in RPTC. Expression of TRPA1 in mouse kidney and cultured mouse RPTC were determined by RT-PCR, in situ hybridization, western blot analysis and immunocytochemistry. The RPTC were treated with 100 μM H2O2 for 1 hr with or without 10μM HC030031, a TRPA1 specific antagonist. EGTA was used to the culture medium for depletion of extracellular calcium. Thereafter, AGT expression levels were evaluated by real-time RT-PCR, and TRPA1 expression levels and p38 MAPK activity were determined by western blot analyses. TRPA1 expression was observed in tubules of renal cortex, and the cultured PRTC expressed TRPA1 mRNA and protein. H2O2 increased AGT expression (1.54 ± 0.08, ratio to control) in RPTC. The AGT augmentation was suppressed by HC030031 (1.19 ± 0.05, ratio to control). Calcium depletion also resulted in attenuation of the AGT augmentation induced by H2O2. Although H2O2 induced phosphorylation of p38 MAPK, HC030031 did not inhibit the p38 MAPK activation. TRPA1 expression was increased by H2O2 under the experimental condition (1.90 ± 0.06, ratio to control). These results suggest that TRPA1 is expressed in RPTC, which contributes to H2O2-induced AGT augmentation via calcium-dependent but p38 MAPK-independent pathways. Furthermore, increase in TRPA1 expression by H2O2 likely facilitates the stimulation of AGT expression. Therefore, TRPA1 may play an important role in the progression of oxidative stress-associated renal injury and hypertension via the stimulation of AGT expression in RPTC.

Transglutaminase-1 protects renal epithelial cells from hydrogen peroxide-induced apoptosis through activation of STAT3 and AKT signaling pathways

Our recent studies showed that transglutaminase-1 (TGase-1) is uniquely expressed in mouse renal proximal tubular cells (RPTC) and mediates cell proliferation. In this study, we investigated the role of TGase-1 in cell survival and the survival signaling pathways regulated by TGase-1 in RPTC following oxidant injury. Exposure of RPTC to hydrogen peroxide (H2O2) resulted in apoptosis and an increase in TGase activity. Inhibition of TGase activity with monodansylcadervine (MDC), a TGase inhibitor, or knockdown of TGase-1 with small interference (si)RNA enhanced apoptosis and decreased cell survival in H2O2-treated RPTC. Conversely, overexpression of TGase-1 rendered RPTC more resistant to H2O2 toxicity and MDC treatment blocked this response. Concurrent with RPTC apoptosis, phosphorylation of AKT, signal transducer and activator of transcription-3 (STAT3), and glucogen synthase kinase-3beta (GSK-3beta) were observed. Pretreatment of cells with MDC or TGase-1 siRNA inhibited phosphorylation of all these molecules. Inhibition of either the AKT or STAT3 pathway potentiated H2O2-induced cell death and increased GSK-3beta activity by dephosphorylation at serine 9. Furthermore, treatment with GSK-3beta inhibitors reduced H2O2-induced apoptosis and abolished the death-promoting effect of AKT and STAT3 inhibition. Therefore, we have identified TGase-1 as a novel survival factor in renal epithelial cells and it contributes to cell survival through activation of the AKT and STAT3 signaling pathways following oxidant injury.

Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.

Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells.

The lack of cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) in mice increases renal proximal tubular cell death and enhances sensitivity to acute renal failure produced by the chemotherapeutic agent cisplatin. We used primary cultures of mouse renal proximal tubular cells (MPTC) grown in optimized culture conditions to investigate the cellular basis for increased apoptosis in p21 knockout mice. Cisplatin (15 microM) activated caspase-3 but not caspase-8 or caspase-9 and produced phosphatidylserine externalization, chromatin condensation, and nuclear fragmentation in wild-type [p21(+/+)] MPTC. Caspase-3 activation and apoptosis were accelerated in cisplatin-treated MPTC lacking p21 [p21(-/-) MPTC]. In contrast to p21(+/+) MPTC, cisplatin activated caspase-9 but not caspase-8 in p21(-/-) MPTC before caspase-3 activation. The caspase-3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone (DEVD-fmk) inhibited caspase-3 activity but did not abolish apoptosis in p21(+/+) and p21(-/-) MPTC. General caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone (ZVAD-fmk) inhibited caspase activity and decreased chromatin condensation by 51% in p21(-/-) but not in p21(+/+) MPTC. However, cisplatin-induced phosphatidylserine externalization was not inhibited by ZVAD-fmk in p21(-/-) MPTC. We conclude that 1) in the presence of p21, cisplatin activates caspase-3 through a mechanism independent of caspase-8 or caspase-9; 2) in the absence of p21, caspase-9 activation precedes caspase-3 activation; 3) the lack of p21 accelerates caspase-3 activation and cisplatin-induced MPTC apoptosis; and 4) MPTC apoptosis is caspase independent in the presence of p21 but partially dependent on caspases in the absence of p21.

Lysophosphatidic acid: a novel growth and survival factor for renal proximal tubular cells.

Lysophosphatidic acid (LPA) is the smallest and structurally simplest of all glycerophospholipids. LPA is a normal constituent of serum and binds with high affinity to albumin while retaining its biological activity. The effects of LPA are pleiotropic and range from mitogenesis to stress fiber formation. In this report, we demonstrate two novel functions for LPA. LPA acts as a survival factor to inhibit apoptosis of primary cultures of mouse renal proximal tubular (MPT) cells. LPA also acts as a potent mitogen for MPT cells. The ability of LPA to act as both a survival factor and a mitogen is mediated by the lipid kinase phosphatidylinositol 3-kinase (PI3K), since these activities were completely blocked by wortmannin or LY-294002, two structurally dissimilar inhibitors of PI3K. The identification of LPA as a proliferative and anti-apoptotic factor suggests a potential role for this lipid mediator during the injury and/or recovery phases following tubular damage.