Fatty acid-binding protein 4 is a therapeutic target for septic acute kidney injury by regulating inflammatory response and cell apoptosis

Sepsis is a systemic inflammatory state in response to infection, and concomitant acute kidney injury (AKI) significantly increases morbidity and mortality. Growing evidence suggests that fatty acid-binding protein 4 (FABP4) is critically involved in kidney diseases, while its role in septic AKI remains unknown. Here, FABP4 was mainly upregulated in renal tubular epithelial cells (RTECs) following cecal ligation and puncture (CLP)- or lipopolysaccharide (LPS)-induced septic AKI. FABP4 inhibition by genetic deletion or BMS309403 treatment both attenuated kidney dysfunction and pathological injury in CLP- or LPS-treated mice. Notably, RTEC-specific deletion of FABP4 also showed similar renoprotective effects. Moreover, FABP4 inhibition alleviated inflammation and apoptosis in CLP-injured kidneys and LPS-stimulated mouse tubular epithelial cells. Mechanistically, TLR4 blockage improved sepsis-induced kidney injury, as well as suppressed c-Jun phosphorylation and FABP4 expression, where c-Jun knockdown also inhibited LPS-stimulated FABP4 level. Meanwhile, FABP4 inhibition reduced the elevated phosphorylated c-Jun, while the levels of TLR4 and MyD88 were uninfluenced. Collectively, the increased FABP4 in RTECs is dependent on TLR4/c-Jun signaling activation and contributes to kidney injury, by forming a positive feedback loop with c-Jun to aggravate inflammation and apoptosis in septic AKI. Thus, FABP4 may be a therapeutic target for septic AKI.Subject terms: Acute kidney injury, Chronic kidney disease

Upregulation of tubular FABP4 in septic AKI is dependent on TLR4/c-Jun signaling activation, and FABP4 mediates sepsis-induced RTEC injury, likely by forming a positive feedback loop with c-Jun to aggravate inflammation and apoptosis.     

Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression

Sepsis related acute kidney injury (AKI) is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC) from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS), a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO). Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3) and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.     

Tubular cell-derived exosomal miR-150-5p contributes to renal fibrosis following unilateral ischemia-reperfusion injury by activating fibroblast in vitro and in vivo

Unilateral ischemia reperfusion injury (UIRI) with longer ischemia time is associated with an increased risk of acute renal injury and chronic kidney disease. Exosomes can transport lipid, protein, mRNA, and miRNA to corresponding target cells and mediate intercellular information exchange. In this study, we aimed to investigate whether exosome-derived miRNA mediates epithelial-mesenchymal cell communication relevant to renal fibrosis after UIRI. The secretion of exosomes increased remarkably in the kidney after UIRI and in rat renal tubular epithelium cells (NRK-52E) after hypoxia treatment. The inhibition of exosome secretion by Rab27a knockout or GW4869 treatment ameliorates renal fibrosis following UIRI in vivo. Purified exosomes from NRK-52E cells after hypoxia treatment could activate rat kidney fibroblasts (NRK-49F). The inhibition of exosome secretion in hypoxic NRK-52E cells through Rab27a knockdown or GW4869 treatment abolished NRK-49F cell activation. Interestingly, exosomal miRNA array analysis revealed that miR-150-5p expression was increased after hypoxia compared with the control group. The inhibition of exosomal miR-150-5p abolished the ability of hypoxic NRK-52E cells to promote NRK-49F cell activation in vitro, injections of miR-150-5p enriched exosomes from hypoxic NRK-52E cells aggravated renal fibrosis following UIRI, and renal fibrosis after UIRI was alleviated by miR-150-5p-deficient exosome in vivo. Furthermore, tubular cell-derived exosomal miR-150-5p could negatively regulate the expression of suppressor of cytokine signaling 1 to activate fibroblast. Thus, our results suggest that the blockade of exosomal miR-150-5p mediated tubular epithelial cell-fibroblast communication may provide a novel therapeutic target to prevents UIRI progression to renal fibrosis.     

MiR-150-5p protects against septic acute kidney injury via repressing the MEKK3/JNK pathway

BackgroundSeptic acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. MicroRNA is reportedly involved in sepsis-induced organ dysfunction, while the role of miR-150 in septic AKI remains ambiguous.MethodsQuantitative real-time PCR (qRT-PCR) was carried out to examine miR-150-5p expression in both septic AKI patients and volunteers without septic AKI. Lipopolysaccharide (LPS) was used to treat renal tubular epithelial cell line HK-2 and C57/BL6 mice to establish in vitro and in vivo sepsis-induced AKI models. Cell apoptosis was determined using TdT-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry. Cell viability was tested using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Renal pathological changes were examined via Hematoxylin-Eosin (H&E) staining, and renal function was measured via blood urea nitrogen (BUN) and creatinine (Cre) measurements. The MEKK3/JNK profile and oxidative stress markers (including COX2 and iNOS) were examined by immunoblot analysis, and the expression levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and oxidative stress markers (MDA, SOD, and CAT) were evaluated by ELISA.ResultsMiR-150-5p was down-regulated in the serum of patients with septic AKI (compared to healthy volunteers). Moreover, miR-150-5p levels were lower in LPS-treated HK-2 cell lines and in the septic AKI mouse model. Additionally, Stat-3 activation mediated the decrease of miR-150-5p. Functionally, miR-150-5p agomir attenuated LPS-induced apoptosis in HK-2 cells, in addition to renal inflammatory responses and oxidative stress. In contrast, inhibition of miR-150-5p aggravated LPS-induced apoptosis, inflammatory reactions and oxidative stress. Furthermore, miR-150-5p agomir decreased BUN and Scr levels in the septic AKI mice model repressed TNF-α, IL-6 and IL-1β, and up-regulated SOD and CAT down-regulated MDA in the kidney tissues. Moreover, miR-150-5p was identified as a target gene for Stat3, and the overexpression of Stat3 partially promoted the effect of down-regulating miR-150-5p on LPS-induced HK2 cell injury. Mechanistically, the MEKK3/JNK pathway was identified as a functional target of miR-150-5p, and the knockdown of MEKK3 showed protective effects against LPS mediated HK-2 cell apoptosis.ConclusionStat3-mediated miR-150-5p exerted protective effects in sepsis-induced acute kidney injury by regulating the MEKK3/JNK pathway.     

Mechanisms of HuR in regulation of epithelial cell apoptosis in rat ulcerative colitis

ObjectiveTo investigate the influence of HuR on the apoptosis rate of epithelial cells in rats with ulcerative colitis (UC) and its mechanism.MethodsUC cell models were established in LPS induced Caco-2 cells. After transfection of si-HuR, pcDNA3.1-HuR, pcDNA3.1-HMGB1, miR-29a-3p mimic or miR-29a-3p inhibitor and their negative controls, apoptosis rate and apoptosis-related proteins (Bcl-2, Bax and cleaved-caspase-3) were tested by flow cytometry, qRT-PCR and Western blot. Actinomycin D treatment was applied to verify the effect of HuR in Caco-2 cells. The binding of HMGB1 to HuR/miR-29a-3p was measured by RIP and dual luciferase reporter gene assays. Experimental UC rat models were established by rectum administration of TNBS/ethanol. The colonic weight/length ratio was calculated at the day 15. The morphology of colon tissues and the apoptosis of tissues were separately detected by H&E staining and TUNEL staining. qRT-PCR and Western blot were conducted to determine the levels of HuR, miR-29a-3p and HMGB1 in colon tissues.ResultsThe apoptosis of LPS-treated Caco-2 cells was inhibited following transfection of si-HuR or miR-29a-3p mimic while facilitated following transfection of pcDNA3.1-HMGB1 or miR-29a-3p inhibitor. RIP and dual luciferase reporter gene assays showed that both HuR and miR-29a-3p can bind HMGB1. Overexpression of HuR in Caco-2 cells results in less HMGB1 that can be bind to miR-29a-3p. The degradation rate of HMGB1 mRNA was increased after transfection of si-HuR in Caco-2 cells. Additionally, miR-29a-3p overexpression can abolish the increases of HMGB1 mRNA induced by HuR, therefore consequently suppress the HMGB1 mRNA that can be bind to HuR. Knockdown of HuR can alleviate TNBS-induced UC in rats and inhibit the apoptosis of colon tissues.ConclusionHuR competitively binds HMGB1 with miR-29a-3p to promote apoptosis of colonic epithelia in rats with UC.     

5-Aminolevulinic Acid Protects against Cisplatin-Induced Nephrotoxicity without Compromising the Anticancer Efficiency of Cisplatin in Rats In Vitro and In Vivo

Background/Aims

Nephrotoxicity is a frequent and major limitation in cisplatin (CDDP)-based chemotherapy. 5-Aminolevulinic acid (ALA) is widely distributed in animal cells, and it is a precursor of tetrapyrole compounds such as heme that is fundamentally important in aerobic energy metabolism. The aim of this study is to evaluate the protective role of ALA in CDDP-induced acute kidney injury (AKI).

Method

We used CDDP-induced AKI rat model and cultured renal tubular cells (NRK-52E). We divided four groups of rats: control, CDDP only, CDDP + ALA(post);(ALA 10 mg/kg + Fe in drinking water) after CDDP, CDDP + ALA(pre & post).

Result

CDDP increased Cr up to 6.5 mg/dl, BUN up to 230 mg/dl, and ALA significantly reduced these changes. ALA ameliorates CDDP-induced morphological renal damages, and reduced tubular apoptosis evaluated by TUNEL staining and cleaved caspase 3. Protein and mRNA levels of ATP5α, complex(COX) IV, UCP2, PGC-1α in renal tissue were significantly decreased by CDDP, and ALA ameliorates reduction of these enzymes. In contrast, Heme Oxigenase (HO)-1 level is induced by CDDP treatment, and ALA treatment further up-regulates HO-1 levels. In NRK-52E cells, the CDDP-induced reduction of protein and mRNA levels of mitochondrial enzymes was significantly recovered by ALA + Fe. CDDP-induced apoptosis were ameliorated by ALA + Fe treatment. Furthermore, we evaluated the size of transplantated bladder carcinoma to the rat skin, and ALA did not change the anti cancer effects of CDDP.

Conclusion

These data suggested that the protective role of ALA in cisplatin-induced AKI is via protection of mitochondrial viability and prevents tubular apoptosis. Also there are no significant effects of ALA on anticancer efficiency of CDDP in rats. Thus, ALA has the potential to prevent CDDP nephrotoxicity without compromising its anticancer efficacy.     

Study on therapeutic action of bone marrow derived mesenchymal stem cell combined with vitamin E against acute kidney injury in rats

AimsThe study aims to investigate the effect to treat acute kidney injury (AKI) with bone marrow derived mesenchymal stem cells (BMSCs) combined with vitamin E and to develop a new treatment mode for AKI preclinical study.Main methodsBMSCs were separated from rat bone marrow. Gentamicin was used as a damage factor in the culture of renal tubular epithelial cells (RTECs) in vitro. After co-cultured with BMSCs and vitamin E, cell proliferation of each group was detected with CCK-8. In vivo, BMSCs (3.3 × 10⁶ cells/kg) combined with vitamin E (80 mg/kg) were administered in AKI rats induced by gentamicin intravenously. The pathological changes, biochemical parameters and apoptosis genes after treatment were investigated furthermore.Key findingsIn co-cultured system, proliferating ability of RTECs was improved by BMSCs or vitamin E, especially for the combined group (P < 0.05). The treated rats in combined group presented the lowest serum creatinine and the highest urea nitrogen compared to non-treated rats. The improvement in renal pathological changes was followed by less necrosis, degeneration and expansion of renal tubule. Under transmission electron microscope, unclear cell structure and reduction of endoplasmic reticulum in the cytoplasm of RTECs were ameliorated with the treatment. Most apoptosis genes were up-regulated in model group while down-regulated with the therapy. Further analysis showed that the two treatments may act independently with each other.SignificanceOur data demonstrated that both BMSC and vitamin E hold therapeutic action to AKI induced by gentamicin. Especially, the combined treatment is better than BMSC or vitamin E alone.     

The impact of Caspase-1 deletion on apoptosis and acute kidney injury in a murine transplant model

BackgroundCaspase-1 knockout mice (Casp1KO) are protected from Acute Kidney Injury (AKI) after warm ischemia/reperfusion injury in non-transplant models. Since Caspase-1 plays a central role as an inflammatory response initiator, we hypothesized that Casp1KO mice would be protected from AKI following transplant.MethodsRenal tubular cells (RTECs) were subjected to cold storage and rewarming (CS/REW). C57Bl/6 J wild type or Casp1KO kidneys were subjected to CI for 30 min and then transplanted into wild type recipients (CI + Txp). The recipients underwent bilateral native nephrectomy at the time of transplant. Serum creatinine (sCr) was measured 24 h after native nephrectomy to assess transplant function.ResultsWe found that RTECs subjected to CS/REW had significantly increased expression of the Caspase-1 and inflammasome protein NLRP1. Wild type kidneys subjected to CI + Txp into wild type recipients also demonstrated significantly increased Caspase-1 and NLRP1 protein expression compared to kidneys transplanted from Casp1KO donors into wild type recipients. Caspase-1 deletion results in significantly decreased RTEC apoptosis in transplanted Casp1KO vs WT kidneys. Surprisingly, however, renal function, ATN scores including brush border injury, cast formation and tubular simplification were similar in both groups and not significantly different.ConclusionsOur data suggest that other triggers of inflammation and programmed necrosis may need to be inhibited in addition to attenuating Caspase-1 to fully prevent AKI after kidney transplant. Importantly, requirements may be distinct for AKI induced by transplantation as opposed to other transient models such as the clamp model of AKI.     

Cul4a attenuates LPS-induced acute kidney injury via blocking NF-kB signaling pathway in sepsis

BackgroundAcute kidney injury (AKI) is a common disease that can develop into end-stage kidney disease. Sepsis is one of the main causes of AKI. Currently, there is no satisfactory way to treat septic AKI. Therefore, we have shown the protective function of Cul4a in septic AKI and its molecular mechanism.MethodsThe cellular and animal models of septic AKI were established by using lipopolysaccharide (LPS). Western blot (WB) was employed to analyze Cul4a expression. RT-qPCR was employed to test the expression of Cul4a, SOD1, SOD2, GPX1, CAT, IL-6, TNF-a, Bcl-2, IL1b, Bax and KIM-1 mRNA. ELISA was performed to detect the contents of inflammatory factors and LDH. CCK-8 was utilized to detect cell viability. Flow cytometry was utilized to analyze the apoptosis. DHE-ROS kit was used to detect the content of ROS.ResultsCul4a was down-regulated in cellular and animal models of septic AKI. Oxidative stress is obviously induced by LPS, as well as apoptosis and inflammation. However, these can be significantly inhibited by up-regulating Cul4a. Moreover, LPS induced the activation of the NF-kB pathway, which could also be inhibited by overexpression of Cul4a.ConclusionsCul4awas found to be a protective factor in septic AKI, which could inhibit LPS-induced oxidative stress, apoptosis and inflammation of HK-2 cells by inhibiting the NF-kB pathway.     

Arbutin attenuates LPS-induced acute kidney injury by inhibiting inflammation and apoptosis via the PI3K/Akt/Nrf2 pathway

BackgroundArbutin (Ar) has anti-oxidative and anti-inflammatory activities. However, the effects of Ar on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) are not clear.PurposeThis study aimed to investigate the effects of Ar on LPS-induced AKI in rats.MethodsThe possible data regarding the effects of Ar on AKI were collected by network pharmacology research. Histological changes in the kidney and the levels of blood urea nitrogen, serum creatinine, and kidney injury molecule 1 were measured to assess the effects of Ar on renal function in LPS-induced AKI. The levels of inflammatory were detected by live small-animal imaging, cytometric bead array and enzyme linked immunosorbent assay. The levels of reactive oxygen species and apoptosis of primary kidney cells were detected by flow cytometry. The oxidative stress-related markers were detected by the cuvette assay. The TLR4/NF-κB and PI3K/Akt/Nrf2 levels and apoptosis were detected by Western blot analysis. The effects of GDC-0068 (GDC, Akt inhibitor) on Ar interposed on LPS-induced NRK-52e cell apoptosis were investigated by flow cytometry.ResultsThe data collected by network pharmacology suggested that Ar might inhibit AKI by exerting an anti-inflammatory effect and regulating the Akt signaling pathway. The experimental results showed that Ar markedly improved renal function, and attenuated inflammation and cell apoptosis via regulating PI3K/Akt/Nrf2 pathway following LPS challenge in vivo, which blocked by GDC effectively in vitro.ConclusionIn a word, this study demonstrated that Ar attenuated LPS-induced AKI by inhibiting inflammation and apoptosis via the PI3K/Akt/Nrf2 pathway.     

Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione

The tripeptide GSH is important in maintenance of renal redox status and defense against reactive electrophiles and oxidants. Previous studies showed that GSH is transported across the basolateral plasma membrane (BLM) into the renal proximal tubule by both sodium-coupled and sodium-independent pathways. Substrate specificity and inhibitor studies suggested the function of several carriers, including organic anion transporter 3 (Oat3). To test the hypothesis that rat Oat3 can function in renal GSH transport, the cDNA for rat Oat3 was expressed as a His6-tagged protein in E. coli, purified from inclusion bodies and by Ni2+-affinity chromatography, and reconstituted into proteoliposomes. cDNA-expressed and reconstituted Oat3 transported both GSH and p-aminohippurate (PAH) in exchange for 2-oxoglutarate (2-OG) and 2-OG and PAH in exchange for GSH, and PAH uptake was inhibited by both probenecid and furosemide, consistent with function of Oat3. mRNA expression of Oat3 and several other potential carriers was detected by RT-PCR in rat kidney cortex but was absent from NRK-52E cells, a rat proximal tubular cell line. Basolateral uptake of GSH in NRK-52E cells showed little PAH- or 2-OG-stimulated uptake. We conclude that Oat3 can function in GSH uptake and that NRK-52E cells possess a low background rate of GSH uptake, making these cells a good model for overexpression of specific, putative GSH carriers.     

HuR Regulates Alternative Splicing of the TRA2β Gene in Human Colon Cancer Cells under Oxidative Stress

Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2β gene encodes splicing factor transformer 2β (Tra2β) and generates 5 mRNA isoforms (TRA2β1 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells to sodium arsenite stimulated checkpoint kinase 2 (Chk2)- and mitogen-activated protein kinase p38 (p38^MAPK)-mediated phosphorylation of HuR at positions S88 and T118. This induced an association between HuR and the 39-nucleotide (nt) proximal region of TRA2β exon 2, generating a TRA2β4 mRNA that includes exon 2, which has multiple premature stop codons. HuR knockdown or Chk2/p38^MAPK double knockdown inhibited the arsenite-stimulated production of TRA2β4 and increased Tra2β protein, facilitating Tra2β-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38^MAPK double knockdown were also confirmed using a TRA2β minigene spanning exons 1 to 4, and the effects disappeared when the 39-nt region was deleted from the minigene. In endogenous HuR knockdown cells, the overexpression of a HuR mutant that could not be phosphorylated (with changes of serine to alanine at position 88 [S88A], S100A, and T118A) blocked the associated TRA2β4 interaction and TRA2β4 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA2β4-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress.     

Mitochondrial Protein Phosphatase 2A Regulates Cell Death Induced by Simulated Ischemia in Kidney NRK-52E Cells

Acute renal failure can occur after an ischemic injury and results in significant mortality. The stress-signaling pathways that are activated during renal ischemia are unknown. PP2A has emerged as an important regulator of cell death. To study the role of PP2A in ischemia-induced cell death, we used an in vitro model of simulated ischemia. In the present study, simulated ischemia in rat renal tubule epithelial NRK-52E cells (a) results in cell death that involves both necrosis and apoptosis, (b) activates PP2A, and (c) up-regulates the PP2A B56 α regulatory subunit. Previous data have shown that PKC α negatively regulates B56 α protein expression. Consistent with this finding, simulated ischemia suppressed PKC α and up-regulated B56 α. Treatment of NRK-52E cells with ceramide suppressed PKC α and activated PP2A in a manner that mimicked simulated ischemia. A role for PP2A in simulated ischemia-induced cell death is likely since inhibition of PP2A protected NRK-52E cells. In addition, overexpression of exogenous B56 α but not B55 in NRK-52E cells enhanced simulated ischemia-induced cell death. These findings suggest that activation of a PP2A isoform that contains the B56 α regulatory subunit is required for ischemia-induced cell death in kidney epithelial proximal tubule cells.