Murine Renal Tubular Epithelial Cells Research Articles

#2222 Suppression of miR-199a-3p ameliorates TGF-β-induced EMT in renal tubular cells

Abstract Background and Aims Various kidney conditions, such as acute kidney injury and fibrosis, cause dysregulation of miRNAs. TGF-β is one of the most potent inducer of epithelial-to-mesenchymal transition (EMT) and fibrosis. We recently reported increased miR-199 expression in TGF-β-induced kidney fibrosis mouse model (doi: 10.3390/ijms242115520). However, the exact role of miR-199 in fibrosis and EMT development is still unknown. Method Primary tubular cells were isolated from a 4-week-old C57BL6 mouse and kept in DMEM/F12 medium containing 2% serum, supplemented with insulin/transferrin/selenium. Cells were treated for 24 hours with 30 nM miR-199-3p miRNA inhibitor (anti-miR), with or without TGF-β (10 ng/ml). The expression of profibrotic genes, as well as miR-199a-3p, was assessed by qPCR. Western blot was used to evaluate the protein expression. Results are presented as mean fold expression relative to calibrator. Statistical significance was assessed using the Kruskal-Wallis test, and the level of significance was set to p < 0.05. Results The miR-199a-3p expression in the anti-miR transfected control and TGF-β groups decreased by 93.5% and 85.5%, respectively, as compared to non-transfected groups (p < 0.05). Morphologically, cells transfected with miR-199a-3p and TGF-β1 showed no difference from cells exposed to only TGF-β1 but appeared more elongated compared to the control groups. However, the suppression of miR-199a-3p alleviated TGF-β1-induced fibrotic cell responses as shown by downregulated fibronectin mRNA (Fn1) and protein expression by 30.2% (p < 0.05) and by 4.3%, respectively. This was accompanied by reduced Acta2 (α-SMA) and Tgfb1 mRNA expressions by 30.2% and 17.5%, respectively (p < 0.05). Conclusion Our findings suggest that in murine renal tubular epithelial cells, miR-199a-3p suppresses TGF-β1-induced EMT by inhibiting FN1, Acta2, and Tgfb1 both at mRNA and protein levels. Further studies are needed to elucidate the underlying molecular interactions.

XBP1 modulates endoplasmic reticulum and mitochondria crosstalk via regulating NLRP3 in renal ischemia/reperfusion injury

The functional status of mitochondria and the endoplasmic reticulum are central to renal ischemia/reperfusion injury (IRI). X-box binding protein 1 (XBP1) is an important transcription factor in endoplasmic reticulum stress. NLR family pyrin domain containing-3 (NLRP3) inflammatory bodies are closely related to renal IRI. In vivo and in vitro, we examined the molecular mechanisms and functions of XBP1-NLRP3 signaling in renal IRI, which influences ER-mitochondrial crosstalk. In this study, mice were subjected to 45 min of unilateral renal warm ischemia, the other kidney resected, and reperfusion was performed for 24 h in vivo. In vitro, murine renal tubular epithelial cells (TCMK-1) were exposed to hypoxia for 24 h and reoxygenation for 2 h. Tissue or cell damage was evaluated by measuring blood urea nitrogen and creatinine levels, histological staining, flow cytometry, terminal deoxynucleotidyl transferase-mediated nick-end labeling, diethylene glycol staining, and transmission electron microscopy (TEM). Western blotting, immunofluorescence staining, and ELISA were used to analyze protein expression. Whether XBP1 regulates the NLRP3 promoter was evaluated using a luciferase reporter assay. Kidney damage was reduced with decreasing blood urea nitrogen, creatinine, interleukin-1β, and interleukin-18 levels. XBP1 deficiency reduced tissue damage and cell apoptosis, protecting the mitochondria. Disruption of XBP1 was associated with reduced NLRP3 and cleaved caspase-1 levels and markedly improved survival. In vitro in TCMK-1 cells, XBP1 interference inhibited caspase-1-dependent mitochondrial damage and reduced the production of mitochondrial reactive oxygen species. The luciferase assay showed that spliced XBP1 isoforms enhanced the activity of the NLRP3 promoter. These findings reveal that XBP1 downregulation suppresses the expression of NLRP3, a potential regulator of endoplasmic reticulum mitochondrial crosstalk in nephritic injury and a potential therapeutic target in XBP1-mediated aseptic nephritis.

Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells.

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

Aldehyde dehydrogenase 2 protects against acute kidney injury by regulating autophagy via Beclin-1 pathway

The mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) catalyzes the detoxification of acetaldehyde and endogenous lipid aldehydes. Approximately 40% of East Asians, accounting for 8% of the human population, carry the E504K mutation in ALDH2 that leads to accumulation of toxic reactive aldehydes and increases the risk for cardiovascular disease, cancer, and Alzheimer disease, among others. However, the role of ALDH2 in acute kidney injury (AKI) remains poorly defined and is therefore the subject of the present study using various cellular and organismal sources. In murine models, in which AKI was induced by either the contrast agent iohexol or renal ischemia/reperfusion, KO, activation/overexpression of ALDH2 were associated with increased and decreased renal injury, respectively. In murine renal tubular epithelial cells (RTECs), ALDH2 upregulated Beclin-1 expression, promoted autophagy activation, and eliminated ROS. In vivo and in vitro, both 3-MA and Beclin-1 siRNAs inhibited autophagy and abolished ALDH2-mediated renoprotection. In mice with iohexol-induced AKI, ALDH2 knockdown in RTECs using AAV-shRNA impaired autophagy activation and aggravated renal injury. In human renal proximal tubular epithelial HK-2 cells exposed to iohexol, ALDH2 activation potentiated autophagy and attenuated apoptosis. In mice with AKI induced by renal ischemia/reperfusion, ALDH2 overexpression or pretreatment regulated autophagy mitigating apoptosis of RTECs and renal injury. In summary, our data collectively substantiate a critical role of ALDH2 in AKI via autophagy activation involving the Beclin-1 pathway.

IL-23 reshapes kidney resident cell metabolism and promotes local kidney inflammation.

Interstitial kidney inflammation is present in various nephritides in which serum interleukin 23 (IL-23) is elevated. Here we showed that murine and human renal tubular epithelial cells (TECs) expressing the IL-23 receptor (IL-23R) responded to IL-23 by inducing intracellular calcium flux, enhancing glycolysis, and upregulating calcium/calmodulin kinase IV (CaMK4), which resulted in suppression of the expression of the arginine-degrading enzyme arginase 1 (ARG1), thus increasing in situ levels of free L-arginine. Limited availability of arginine suppressed the ability of infiltrating T cells to proliferate and produce inflammatory cytokines. TECs from humans and mice with nephritis expressed increased levels of IL-23R and CaMK4 but reduced levels of ARG1. TEC-specific deletion of Il23r or Camk4 suppressed inflammation, whereas deletion of Arg1 exacerbated inflammation in different murine disease models. Finally, TEC-specific delivery of a CaMK4 inhibitor specifically curbed renal inflammation in lupus-prone mice without affecting systemic inflammation. Our data offer the first evidence to our knowledge of the immunosuppressive capacity of TECs through a mechanism that involves competitive uptake of arginine and signify the importance of modulation of an inflammatory cytokine in the function of nonlymphoid cells, which leads to the establishment of an inflammatory microenvironment. New approaches to treat kidney inflammation should consider restoring the immunosuppressive capacity of TECs.

Repurposing of metformin and colchicine reveals differential modulation of acute and chronic kidney injury

Acute kidney injury (AKI) is a major health problem affecting millions of patients globally. There is no effective treatment for AKI and new therapies are urgently needed. Novel drug development, testing and progression to clinical trials is overwhelmingly expensive. Drug repurposing is a more cost-effective measure. We identified 2 commonly used drugs (colchicine and metformin) that alter inflammatory cell function and signalling pathways characteristic of AKI, and tested them in models of acute and chronic kidney injury to assess therapeutic benefit. We assessed the renoprotective effects of colchicine or metformin in C57BL/6 mice challenged with renal ischemia reperfusion injury (IRI), treated before or after injury. All animals underwent analysis of renal function and biomolecular phenotyping at 24 h, 48 h and 4 weeks after injury. Murine renal tubular epithelial cells were studied in response to in vitro mimics of IRI. Pre-emptive treatment with colchicine or metformin protected against AKI, with lower serum creatinine, improved histological changes and decreased TUNEL staining. Pro-inflammatory cytokine profile and multiple markers of oxidative stress were not substantially different between groups. Metformin augmented expression of multiple autophagic proteins which was reversed by the addition of hydroxychloroquine. Colchicine led to an increase in inflammatory cells within the renal parenchyma. Chronic exposure after acute injury to either therapeutic agent in the context of reduced renal mass did not mitigate the development of fibrosis, with colchicine significantly worsening an ischemic phenotype. These data indicate that colchicine and metformin affect acute and chronic kidney injury differently. This has significant implications for potential drug repurposing, as baseline renal disease must be considered when selecting medication.

Macrophage-secreted Lipocalin-2 Promotes Regeneration of Injured Primary Murine Renal Tubular Epithelial Cells.

Lipocalin-2 (Lcn-2) is rapidly upregulated in macrophages after renal tubular injury and acts as renoprotective and pro-regenerative agent. Lcn-2 possesses the ability to bind and transport iron with high affinity. Therefore, the present study focuses on the decisive role of the Lcn-2 iron-load for its pro-regenerative function. Primary mouse tubular epithelial cells were isolated from kidney tissue of wildtype mice and incubated with 5 µM Cisplatin for 24 h to induce injury. Bone marrow-derived macrophages of wildtype and Lcn-2−/− mice were isolated and polarized with IL-10 towards an anti-inflammatory, iron-release phenotype. Their supernatants as well as recombinant iron-loaded holo-Lcn-2 was used for stimulation of Cisplatin-injured tubular epithelial cells. Incubation of tubular epithelial cells with wildtype supernatants resulted in less damage and induced cellular proliferation, whereas in absence of Lcn-2 no protective effect was observed. Epithelial integrity as well as cellular proliferation showed a clear protection upon rescue experiments applying holo-Lcn-2. Notably, we detected a positive correlation between total iron amounts in tubular epithelial cells and cellular proliferation, which, in turn, reinforced the assumed link between availability of Lcn-2-bound iron and recovery. We hypothesize that macrophage-released Lcn-2-bound iron is provided to tubular epithelial cells during toxic cell damage, whereby injury is limited and recovery is favored.

Protective effects of Panax notoginseng saponins on PME-Induced nephrotoxicity in mice

Polymyxin E (PME) plays an important role in fighting against Gram-negative bacterial infections; however, it causes nephrotoxicity, which limits its clinical use. The aim of this study was to investigate the protective effects of a plant extract Panax notoginseng saponins (PNS) on PME-induced nephrotoxicity in mice. In vivo studies showed that PNS significantly reduced blood urea nitrogen (BUN), serum creatinine (CRE) and number of apoptotic cells in kidney, as well as renal histopathological damage which increased in the presence of PME, and suppressed PME-induced oxidative stress in kidney, as shown by the up-regulation of superoxide dismutase (SOD) and the down-regulation of malondialdehyde (MDA) levels. Furthermore, PNS inhibited the expression of Bax, while increased the expression of Bcl-2 compared to the PME-treated group. In vitro studies showed that PNS decreased intracellular reactive oxygen species (ROS) and MDA levels, increased glutathione (GSH) levels, and enhanced the activity of SOD and glutathione peroxidase (GSH-Px) in murine renal tubular epithelial cells (TCMK-1 cells). In addition, PNS enhanced cell viability and the expression of Bcl-2, restored the mitochondrial membrane potential, inhibited the expression of Bax, inhibited the activity of caspase-3 and caspase-9, and reduce apoptotic rate in PME-treated TCMK-1 cells. PNS could reduce PME-induced nephrotoxicity. The protective effects could result from inhibition of oxidative stress, and prevention of cell apoptosis via the mitochondrial pathway. These findings highlight the potential of PNS as a safe adjunct for ameliorating the nephrotoxicity.

Inhibition of soluble epoxide hydrolase attenuates the kidney injury caused by ischemia/reperfusion in a murine model of acute kidney injury involved in GSK‐3β phosphorylation

Acute kidney injury (AKI) causes severe morbidity and mortality for which new therapeutic strategies are needed. Inhibition of soluble epoxide hydrolase (sEH) has been reported previously to attenuate the kidney injury in cisplatin‐caused AKI. However, the effect of sEH inhibition on ischemia/reperfusion (I/R)‐caused‐AKI remains unknown. In addition, the mechanism underlying the benefit of sEH inhibition to AKI has not been well understood. Here we report that epoxyeicosatrienoic acids (EETs) are the major mediators responsible for the beneficial effect of sEH inhibition in I/R‐caused AKI. Inhibition of sEH resulted in a significant increase in plasma level of 14(15)‐EET. Administration of both 14(15)‐EET alone and TPPU (a potent sEH inhibitor) significantly prolonged the survival of AKI mice. Co‐administration of 14(15)‐EET with TPPU enhanced such effect in survival. Administration of 14(15)‐EET with or without TPPU to AKI mice significantly attenuated the kidney injury, which was supported by the histological analysis of renal tissue, plasma levels of creatinine and urea nitrogen, and renal levels of NGAL. Furthermore, 14(15)‐EET significantly reversed the I/R‐caused reduction in glycogen synthase kinase 3β (GSK3β) phosphorylation in murine kidney, dose‐dependently inhibited the hypoxia/reoxygenation (H/R)‐caused apoptosis of murine renal tubular epithelial cells (mRTECs), and reversed the H/R‐caused reduction in GSK3β phosphorylation in mRTECs. Therefore, inhibition of sEH attenuates kidney injury via, at least partially, 14(15)‐EET modulating the apoptosis of RTECs and GSK3β phosphorylation in mRTECs. This study provides AKI patients with promising therapeutic strategies.Support or Funding InformationThis study was supported in part by NSFC grants 81470588 and 81100090, as well as NIEHS Grant (R01 ES02710), NIEHS Superfund Grant (P42 ES04699), NIH/NHLBI grant (R01 HL59699‐06A1), and a Translational Technology Grant from the UC Davis Medical Center. K.S.S.L. is supported by NIEHS Grant R00 ES024806.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epoxide metabolites of arachidonate and docosahexaenoate function conversely in acute kidney injury involved in GSK3β signaling

Acute kidney injury (AKI) causes severe morbidity and mortality for which new therapeutic strategies are needed. Docosahexaenoic acid (DHA), arachidonic acid (ARA), and their metabolites have various effects in kidney injury, but their molecular mechanisms are largely unknown. Here, we report that 14 (15)-epoxyeicosatrienoic acid [14 (15)-EET] and 19 (20)-epoxydocosapentaenoic acid [19 (20)-EDP], the major epoxide metabolites of ARA and DHA, respectively, have contradictory effects on kidney injury in a murine model of ischemia/reperfusion (I/R)-caused AKI. Specifically, 14 (15)-EET mitigated while 19 (20)-EDP exacerbated I/R kidney injury. Manipulation of the endogenous 19 (20)-EDP or 14 (15)-EET by alteration of their degradation or biosynthesis with selective inhibitors resulted in anticipated effects. These observations are supported by renal histological analysis, plasma levels of creatinine and urea nitrogen, and renal NGAL. The 14 (15)-EET significantly reversed the I/R-caused reduction in glycogen synthase kinase 3β (GSK3β) phosphorylation in murine kidney, dose-dependently inhibited the hypoxia/reoxygenation (H/R)-caused apoptosis of murine renal tubular epithelial cells (mRTECs), and reversed the H/R-caused reduction in GSK3β phosphorylation in mRTECs. In contrast, 19 (20)-EDP dose-dependently promoted H/R-caused apoptosis and worsened the reduction in GSK3β phosphorylation in mRTECs. In addition, 19 (20)-EDP was more metabolically stable than 14 (15)-EET in vivo and in vitro. Overall, these epoxide metabolites of ARA and DHA function conversely in I/R-AKI, possibly through their largely different metabolic stability and their opposite effects in modulation of H/R-caused RTEC apoptosis and GSK3β phosphorylation. This study provides AKI patients with promising therapeutic strategies and clinical cautions.

Bone mesenchymal stem cells ameliorate ischemia/reperfusion-induced damage in renal epithelial cells via microRNA-223

BackgroundRecent studies have indicated that microRNA-223 (miR-223) plays a role in the tissue-protective effect of mesenchymal stem cells (MSCs). NLR family-pyrin domain containing 3 (NLRP3) was reported to affect a renal ischemia/reperfusion (I/R) injury by exerting a direct effect on the renal tubular epithelium. Therefore, we investigated how miR-223 and NLRP3 might function in kidneys exposed to conditions of ischemia and subsequent reperfusion.MethodsHypoxia/reoxygenation (H/R) murine renal tubular epithelial cells (RTECs) were cocultured with either MSCs or hypoxia-pretreated MSCs (htMSCs), after which the RTECs were examined for their viability and evidence of apoptosis. Next, miR-223 expression in the MSCs was downregulated to verify that MSCs protected RTECs via the transport of miR-223. Kidney I/R KM/NIH mouse models were created and used for in vivo studies.ResultsThe results showed that coculture with MSCs significantly increased the viability of RTECs and decreased their rates of apoptosis. The levels of hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF) in samples of coculture supernatants were higher than those in samples of non-coculture supernatants. A bioinformatics analysis revealed a targeting relationship between miR-223 and NLRP3. A dual luciferase assay showed that miR-223 inhibited NLRP3 expression. The htMSCs displayed a protective function associated with an upregulation of miR-223 as induced by Notch1 and the downregulation of NLRP3. Conversely, inhibition of miR-223 impeded the protective effect of MSCs. In the I/R mouse models, injection of either MSCs or htMSCs ameliorated the damage to kidney tissue, while suppression of miR-223 expression in MSCs reduced their protective effect on mouse kidneys.ConclusionsOur results demonstrate that miR-223 and NLRP3 play important roles in the treatment of renal tissue injuries with transplanted MSCs.

Cdc42-Interacting Protein 4 Represses E-Cadherin Expression by Promoting β-Catenin Translocation to the Nucleus in Murine Renal Tubular Epithelial Cells.

Renal fibrosis is an inevitable outcome of end-stage chronic kidney disease. During this process, epithelial cells lose E-cadherin expression. β-Catenin may act as a mediator by accumulation and translocation to the nucleus. Studies have suggested that CIP4, a Cdc42 effector protein, is associated with β-catenin. However, whether CIP4 contributes to E-cadherin loss in epithelial cells by regulating β-catenin translocation is unclear. In this study, we investigated the involvement of CIP4 in β-catenin translocation. Expression of CIP4 was upregulated in renal tissues of 5/6 nephrectomized rats and mainly distributed in renal tubular epithelia. In TGF-β1-treated NRK-52E cells, upregulation of CIP4 expression was accompanied by reduced expression of E-cadherin. CIP4 overexpression promoted the translocation of β-catenin to the nucleus, which was accompanied by reduced expression of E-cadherin even without TGF-β1 stimulation. In contrast, CIP4 depletion by using siRNA inhibited the translocation of β-catenin to the nucleus and reversed the decrease in expression of E-cadherin. The interaction between CIP4 and β-catenin was detected. We also show that β-catenin depletion could restore the expression of E-cadherin that was suppressed by CIP4 overexpression. In conclusion, these results suggest that CIP4 overexpression represses E-cadherin expression by promoting β-catenin translocation to the nucleus.

Negative regulation of microRNA-709 in mitochondrial function participating in the renal tubular epithelial cell injury

Objective To investigate the role of microRNA-709 (miR-709)in murine renal tubular epithelial cells with Cisplatin treatment and its underlying mechanism. Methods The cells were cultured with Cisplatin in the concentrations (0, 1, 5, 10, 20 μmol/L) for 24 h in vitro, and underwent 10 μmol/L Cisplatin stimulation at diffe-rent time points (0, 2, 6, 12, 24 h). Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to investigate the level of miR-709.mPTC were divided into a negative control group, miR-709 mimic group, inhibitor negative control (INNC)group, miR-709 inhibitor group, INNC group and miR-709 inhibitor group were treated with Cisplatin treatment.Annexin V-FITC/PI double staining was applied to detect apoptosis.Caspase-3 activity was detected by spectrophotometry.The mRNA and protein levels of E-cadherin were detected by RT-PCR and Western blot.Mitochondrial membrane potential and mitochondrial superoxide (mitoSOX) generation were detected by flow cytometry.Mitochondrial DNA(mtDNA) copy number was verified by PCR. Results The level of miR-709 increased in Cisplatin 5 μmol/L stimulation group(F=22.17, P<0.05)and increased further 12 h later after cultured in 10 μmol/L Cisplatin compared with that of the control group(F=33.462, P<0.05). After overexpression of miR-709, apoptotic rate and Caspase-3 activity of the mPTC increased, while the expression of E-cadherin declined when compared with that in the negative control group (apoptotic rate: 1.54±0.20 vs 1.00±0.23; Caspase-3 activity: 1.27±0.08 vs 0.97±0.08; E-cadherin: 0.47±0.15 vs 1.00±0.10; t=-3.086, -5.882, 5.671, all P<0.05). In addition, the levels of JC-1 and mtDNA decreased while mitoSOX increased compared with that of the control group (JC-1: 0.80±0.04 vs 1.05±0.08; mtDNA: 0.58±0.15 vs 1.00±0.75; mitoSOX: 1.31±0.16 vs 1.00±0.05; t=4.687, 4.943, -3.694, all P<0.05). Downregulation of miR-709 attenuated the tubular cell injury and the mitochondrial dysfunction was induced by Cisplatin (apoptotic rate: 1.35±0.10 vs 1.86±0.44; Caspase-3 activity: 1.04±0.12 vs 1.30±0.09; E-cadherin: 0.86±0.08 vs 0.54±0.05; JC-1: 0.94±0.06 vs 0.75±0.05; mtDNA: 0.68±0.09 vs 0.27±0.12; mitoSOX: 0.91±0.09 vs 1.22±0.08; F=18.489, 20.932, 33.323, 21.726, 59.330, 23.813, all P<0.05). Conclusion Mitochondrial function negatively regulated by miR-709 may be involved in the renal tubular epithelial cell injury induced by Cisplatin. Key words: Cisplatin; Mitochondrial function; MicroRNA-709; Renal tubular epithelial cell injury

Regulation of TLR2 and NLRP3 in Primary Murine Renal Tubular Epithelial Cells

Pattern recognition receptors (PRRs) are now recognized to be key triggers of injury in a variety of renal diseases. Several families of these receptors are present in the kidney, and recent data suggest that they are differentially expressed and regulated in the kidney. This study evaluated the interaction between two distinct PRRs that are expressed in the kidney, i.e. TLR2 (Toll-like receptor 2) and the NLRP3 inflammasome. The regulation and activation of these receptors in primary renal tubular epithelial (RTE) cells from murine kidneys were evaluated. RTE cells were extracted from WT and NLRP3-mutant mice and treated ex vivo with ligands specific for TLR2 or NLRP3. We found that TLR2 upregulated NLRP3 as well as its substrate IL-1β, and that signaling through the NLRP3 inflammasome induced RTE cell necrosis. The results of this study suggest a previously unknown interaction between TLR2 and NLRP3 in primary RTE cells and highlight the importance of the cross talk that occurs in kidney-related PRRs. Understanding how PRRs are regulated is important for the design of rationale therapeutic strategies to modulate these receptors in renal disease.

NOD2 promotes renal injury by exacerbating inflammation and podocyte insulin resistance in diabetic nephropathy

An increasing number of clinical and animal model studies indicate that activation of the innate immune system and inflammatory mechanisms are important in the pathogenesis of diabetic nephropathy. Nucleotide-binding oligomerization domain containing 2 (NOD2), a member of the NOD-like receptor family, plays an important role in innate immune response. Here we explore the contribution of NOD2 to the pathogenesis of diabetic nephropathy and found that it was upregulated in kidney biopsies from diabetic patients and high-fat diet/streptozotocin-induced diabetic mice. Further, NOD2 deficiency ameliorated renal injury in diabetic mice. In vitro, NOD2 induced proinflammatory response and impaired insulin signaling and insulin-induced glucose uptake in podocytes. Moreover, podocytes treated with high glucose, advanced glycation end-products, tumor necrosis factor-α, or transforming growth factor-β (common detrimental factors in diabetic nephropathy) significantly increased NOD2 expression. NOD2 knockout diabetic mice were protected from the hyperglycemia-induced reduction in nephrin expression. Further, knockdown of NOD2 expression attenuated high glucose-induced nephrin downregulation in vitro, supporting an essential role of NOD2 in mediating hyperglycemia-induced podocyte dysfunction. Thus, NOD2 is one of the critical components of a signal transduction pathway that links renal injury to inflammation and podocyte insulin resistance in diabetic nephropathy.

DNAM-1 - a New Player in Renal Allograft Rejection

Despite effective treatment protocols to prevent rejection many renal allografts are lost due to the toxicity of immunosuppressants. Thus, more specific and less toxic immunosuppression is needed. DNAM-1 (CD226) on T cells has been shown to play an important role for allogeneic graft-versus-host responses. DNAM-1 has two ligands: the adhesion molecules CD155 and CD112. Both are expressed on dendritic, epithelial, and endothelial cells. The importance of DNAM-1and its ligands for renal allograft rejection is unknown. Primary cultures of murine renal tubular epithelial cells (rTECs) were prestimulated with IFN-β and IFN-γ to induce high surface expression of MHC. Surface expression of CD155 and CD112 was tested by FACS. Responder T cells were stimulated in vitro with allogeneic fully MHC-mismatched splenocytes. From these stimulation cocultures we measured proliferation (thymidine incorporation), cytokine production (ELISA) and cytotoxicity against IFN-stimulated rTECs from WT or CD155−/− mice (51Cr-release-assay). To test for a role of this pathway in vivo, we performed fully MHC-mismatched skin and kidney grafts from WT and CD155−/− donors. CD155 and CD112 were both highly expressed on rTECs and could be further increased by IFN-stimulation. Also, in acutely rejected murine renal allografts an upregulation of both molecules was detected. However, when CD155 was missing on targets in an allospecific chromium-release-assay, no difference in cytotoxicity was measured compared to WT targets. Also, allospecific T cell proliferation and IFN-γ secretion were not altered, when stimulator cells lacked CD155. When testing for the role of this pathway for allograft rejection in vivo, no difference between skin graft survival of WT and CD155−/−was observed. Renal allografts from CD155−/− donors show similar amounts of infiltrates and interestingly higher incidence of infarcts compared to WT donors. The reason for this is under investigation. In contrast to these results, allospecific T cell proliferation was be reduced with the use of a blocking antibody against DNAM-1. Also allospecific cytotoxicity was reduced, when DNAM-1 was blocked during stimulation. Experiments blocking DNAM1 or CD112 in vivo are ongoing. Thus, DNAM-1 is an important costimulatory receptor for alloreactive T cells. However, the important ligand might be CD112 rather than CD155. Thus, blocking DNAM-1 or CD112 might be a therapeutically interesting option to prevent renal allograft rejection.

The complement inhibitor Crry is critical for the prevention of complement activation on murine renal tubular epithelial cells

The complement inhibitor Crry is critical for the prevention of complement activation on murine renal tubular epithelial cells

Nod1 and Nod2 Are Expressed in Human and Murine Renal Tubular Epithelial Cells and Participate in Renal Ischemia Reperfusion Injury

Nucleotide-binding oligomerization domain (Nod) 1 and Nod2 are members of a family of intracellular innate sensors that participate in innate immune responses to pathogens and molecules released during the course of tissue injury, including injury induced by ischemia. Ischemic injury to the kidney is characterized by renal tubular epithelial apoptosis and inflammation. Among the best studied intracellular innate immune receptors known to contribute to apoptosis and inflammation are Nod1 and Nod2. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in Nod1, Nod2, Nod(1 x 2), and in their downstream signaling molecule receptor-interacting protein 2. We found that Nod1 and Nod2 were present in renal tubular epithelial cells in both mouse and human kidneys and that the absence of these receptors in mice resulted in protection from kidney ischemia reperfusion injury. Significant protection from kidney injury was seen with a deficiency of Nod2 and receptor-interacting protein 2, and the simultaneous deficiency of Nod1 and Nod2 provided even greater protection. We conclude that the intracellular sensors Nod1 and Nod2 play an important role in the pathogenesis of acute ischemic injury of the kidney, although possibly through different mechanisms.

High Glucose, High Insulin, and Their Combination Rapidly Induce Laminin-β1 Synthesis by Regulation of mRNA Translation in Renal Epithelial Cells

Laminin is a glycoprotein that contributes to renal extracellular matrix expansion in diabetes. We investigated regulation of laminin-beta1 synthesis in murine renal proximal tubular epithelial cells by 30 mmol/l glucose (high glucose), 1 nmol/l insulin (high insulin), and their combination (high glucose+high insulin), simulating conditions observed during progression of type 2 diabetes. Compared with 5 mmol/l glucose and no insulin (control), high glucose alone, high insulin alone, or high glucose+high insulin together increased laminin-beta1 chain protein synthesis within 5 min, lasting for up to 60 min with no change in laminin-beta1 mRNA levels. Cycloheximide, but not actinomycin-D, abrogated increased laminin-beta1 synthesis. High glucose, high insulin, and high glucose+high insulin stimulated phosphorylation of 4E-BP1, a repressor binding protein for eukaryotic initiation factor 4E (eIF4E), that was dependent on activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin. High glucose, high insulin, and high glucose+high insulin also promoted release of eIF4E from 4E-BP1, phosphorylation of eIF4E, and increase in eIF4E association with eIF4G, critical events in the initiation phase of mRNA translation. High glucose, high insulin, and high glucose+high insulin increased Erk phosphorylation, which is an upstream regulator of eIF4E phosphorylation, and PD098059, which is a MEK inhibitor that blocks Erk activation, abolished laminin-beta1 synthesis. This is the first demonstration of rapid increment in laminin-beta1 synthesis by regulation of its mRNA translation by cells exposed to high glucose, high insulin, or high glucose+high insulin.

Nitric Oxide Is an Important Mediator of Renal Tubular Epithelial Cell Death in Vitro and in Murine Experimental Hydronephrosis

Macrophages play a pivotal role in tissue injury and fibrosis during renal inflammation. Although macrophages may induce apoptosis of renal tubular epithelial cells, the mechanisms involved are unclear. We used a microscopically quantifiable co-culture assay to dissect the cytotoxic interaction between murine bone marrow-derived macrophages and Madin-Darby canine kidney cells and primary murine renal tubular epithelial cells. The induction of tubular cell apoptosis by cytokine-activated macrophages was reduced by inhibitors of nitric oxide synthase whereas tubular cell proliferation was unaffected. Furthermore, cytokine-activated macrophages derived from mice targeted for the deletion of inducible nitric oxide synthase were noncytotoxic. We then examined the role of nitric oxide in vivo by inhibiting inducible nitric oxide synthase in the model of murine experimental hydronephrosis. l-N(6)-(1-iminoethyl)-lysine was administered in the drinking water between days 5 and 7 after ureteric obstruction. Macrophage infiltration was comparable between groups, but treatment significantly inhibited tubular cell apoptosis at day 7. Tubular cell proliferation was unaffected. Inducible nitric oxide synthase blockade also reduced interstitial cell apoptosis and increased collagen III deposition. These data indicate that nitric oxide is a key mediator of macrophage-directed tubular cell apoptosis in vitro and in vivo and also modulates tubulointerstitial fibrosis.