The Role Of NLRC5 In The Pathogenesis Of Acute Kidney Injury

Objective Acute kidney injury(AKI)is a common complication with a high morbidity and mortalityworldwide,which is often induced by ischemia/reperfusion injury(IRI),sepsis or toxins.Although the pathogenesis of AKI is multifactorial,mounting evidence has indicated thatinflammation is a hallmark of AKI.The inflammatory processes mainly consist of thestimulation of innate immune responses with subsequent cytokine release and crosstalk betweenrenal cells and immune cells,and the activation of adaptive immune responses with evidentparticipation of T cells in renal injury and repair.Among them,the activation of the innateimmune system is of importance for triggering the initial renal injury and mediating long-termstructural changes including interstitial fibrosis or repair.Pattern recognition receptors(PRRs)act as sensors of the innate immune system to initiate immune responses by the recognition ofpathogen-associated molecular patterns(PAMPs)or damage-associated molecular patterns(DAMPs).Inappropriate or chronic PRR activation results in excessive inflammation andexacerbates tissue damage.Recently NOD like receptors(NLRs),one family of intracellularPRRs,increasingly receive attention from a wide spectrum of biomedical fields in relation torenal function and disease.Although some of NLRs such as NOD2 and NLRP3 are found tobe widely distributed in various renal parenchymal cells and triggered innate immune responsesunder pathogenic conditions,the majority of PRRs are still unknown in the kidney.Therefore,it is necessary to explore the functional role of individual NLR in the kidney,which may provideunexpected opportunities to develop new therapies for renal disease by modulation of an innateimmune system.NLRC5 is a newly identified member of the NLR family that acts as a transcriptional activator ofMHC class I genes.In line with the function of several related NLR proteins in innate immuneresponses,emerging evidence has indicated that NLRC5 contributes to innate and adaptiveimmune responses beyond the regulation of MHC class I genes such as inflammatory and type Iinterferon responses.However,the role of NLRC5 in immune responses is not well understoodand remains controversial.NLRC5 was originally reported to inhibit NF-κB signaling by directinteraction between NLRC5 and IκB kinase α/β(IKKα/β)hat prevents the binding of(IKKy)to the IKK subunits,thereby inhibits their autophosphorylation and kinase activity.However,otherstudies reveal that reversible ubiquitination of NLRC5 has an important regulatory role in theactivation of NF-κB signaling.Therefore,a better understanding of the multiple rolesof NLRC5 will help to define its overall contribution to immune responses in differentphysiological and pathophysiological conditions.MethodsPart I The role of NLRC5 in the pathogenesis of renal ischemia/reperfusion injury1.1 The expression of NLRC5 in a mouse model of renal ischemia/reperfusion injury 1.1.1 Detecting the expression of NLRC5 in mouse tissue and kidney cell lines Reverse transcriptase-PCR(RT-PCR)analysis of the expression of NLRC5 inselected murine tissues including heart,liver,spleen,lung,kidney,brain,stomach,intestines and renal cells includingmurine podocytes(MPC),rat proximal tubule epithelial cells(NRK-52E)cells,rat glomerular mesangialcells(RMC),and rat glomerular endothelial cells(GENC).1.1.2 Detecting the expression of NLRC5 in renal ischemia/reperfusion injuryRenal I/R was performed in anesthetized(pentobarbital)Nlrc5-/-mice and age-matched C57BL/6 wild type(WT)mice by bilateral clamping of the renal pedicles for 30 min using microaneurysm clamps.Detected the expression of NLRC5 in the kidney with IRI by Western Blot(WB),Real-time RT-PCR and immunohistochemistry(IHC).1.1.3 Co-loclization of NLRC5 with renal tubular markersDouble immunofluorescence(IF)staining to detect the expression of NLRC5(green)in various renal tubular(red)1.1.4 Co-loclization of NLRC5 with immune cellsDouble immunofluorescence(IF)for NLRC5(green)and various immune cells(macrophages,neutrophils,dendritic cells,CD4+ T cells)markers(red).1.1.5 Different approaches to mimic hypoxia condition and detection of NLRC5Three approaches to mimic hypoxia condition including oxygen-glucose deprivation and chemical anoxia/recovery induced by incubating rat proximaltubule epithelial(NRK-52E)cells in glucose-free medium with antimycin A(AA)/2-deoxyglucose(2-DG)for 60min and recovery in glucose-replete complete growth medium,and CoCl2 treatment for 12h.Detect the expression of NLRC5 in NRK-52E cells under these conditions by WB.1.1.6 The expression of NLRC5 in the kidney from patients with AKI IHC for the NLRC5 in the kidney from patients with biopsy-provenacutetubular necrosis(ATN).1.2 The role of NLRC5 in a mouse model of renal ischemia/reperfusion injury 1.2.1 Assessment of renal function in WT and Nlrc5-/-miceSerum creatinine was measured by an Agilent 1100 HPLC system.Blood urea nitrogen was measured by a Cobas 8000 modular analyzer.1.2.2 Histology examination of WT and Nlrc5-/-miceWT and Nlrc5-/-mice Formalin-fixed kidney sections were stained with hematoxylin and eosin(HE)to detect the renal morphological injury.IHC was carried out to detect the Kidney injury molecule 1(KIM-1).Terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)was used to detect cell death.Detect relative caspase-3 activity in the kidney from different groups of mice by Caspase 3 Activity Assay Kit.1.2.3 Effect of NLRC5 deficiency in kidney inflammatory responseDetect the cytokines and chemokines in kidney tissue homogenate by Real-time RT-PCR.IHC was carried out to detect the markers of neutrophil,macrophage,dendritic cells and CD4+ T cells(Ly6B,CD68,CD11c and CD4).1.2.4 Effect of NLRC5 deficiency in hypoxia-induced inflammatory response and apoptosis in NRK-52E cellsSilence NLRC5 by siRNA-NLRC5 transfection.Real-time RT-PCR was used to assess the level of proinflammatory mediators.Apoptosis of NRK-52E cells with administration of siRNA-NLRC5 in the treatment of oxygen-glucose deprivation was assessed by flow cytometry.Part II NLRC5 exacerbates renal injury in renal ischemia/reperfusion via mediating CEACAM1 signaling2.1 Effect of NLRC5 in the expression CEACAM1By Agilent Whole Mouse Genome Oligo Microarray for global gene expression analysis,we observed the profound changes of CEACAM1.WB,Real-time RT-PCR and IHC was used to detect the expression of CEACAM1 in kidney from WT and Nlrc5-/-mice with ischemia reperfusion injury.2.2 Effect of NLRC5 in CEACAM1-mediated signalingWB was carried out to detect the activation of MAPK/ERK and PI3K/Akt in the kidney from WT and Nlrc5-/-mice.WB was carried out to detect the activation of MAPK/ERK and PI3K/Akt in the NRK-52E cells with administration of si-RNA-NLRC5 and siRNA-CEACAMl in the treatment of oxygen-glucose deprivation.2.3 The effect of NLRC5 in activation and proliferation of CD4+ T cells.Flow cytometry(FC)analysis of the one experiment showing the percentages of CD45+CD4+ T cells in the kidney and spleen at 6h after reperfusion.Flow cytometry(FC)analysis of the one experiment showing the activation of CD45+CD4+ T cells in the spleen at 6h after reperfusion.Single-cell suspensions were prepared from the spleen.Splenic CD4+ T cells were isolated from mice by magnetic immunobeads.2×105 freshly isolated T cells were incubated in 100 μl complete RPMI-1640 with 1 mg/ml plate-bound anti-CD3,1 mg/ml soluble anti-CD28 37℃ and rIL-2 in a 5%CO2 humidified atmosphere incubator for 72 h.Flow cytometry(FC)analysis activation of isolated CD45+CD4+ T cells.2.5×105 cells/mL were incubatedwith 0.25M carboxyfluorescein succinimidyl ester(CFSE)/mL for 2 to3 minutes,and proliferation index were determined using FlowJo software.2.4 The effect of CEACAM1 in activation and proliferation of CD4+ T cells.Recombinant lentivirus vectors pGLV3 harboring a short-hairpin RNA sequence targeting CEACAM1 were constructed(pGLV3-shRNA-CEACAM1)for gene silencing of CEACAM1 in CD4+ T cells isolated form the spleen from Nlrc5-/-mice.FC analysis activation of CD45+CD4+ T cells and the percentages of CD4+ IFN-γ+ T cells treated with anti-CD3/CD28.CFSE detects the proliferation of CD4+T cells.2.5 The importance of NLRC5 in renal parenchymal cells and leukocytesWe detected the importance of NLRC5 by renal parenchymal cells or bone marrow(BM)-derived cells in the pathogenesis of renal IRI by generating BM chimeric mices subjected to renal ischemia.We detected the renal function by serum creatinine,HE and IHC for KIM 1 expression.Part Ⅲ The role of NLRC5 in the pathogensis of AKI induced by cisplatin3.1 The role of NLRC5 in cisplatin-induced AKI3.1.1 Establishing mouse model of cisplatin-induced AKI and detection of NLRC5C57BL/6 wild type(WT)male mice and age-matched NLRC5 deficient male mice(B6(Cg)-Nlrc5tm1,1Aidi/J)were selected to establish animal model by intraperitoneal injection of cisplatin.WB was used to assess the level of NLRC5 in kidney from a mouse model of cisplatin-induced AKI and in NRK-52E cells under the stimuation of cisplatin.3.1.2 Effect of NLRC5 deficiency in cisplatin-induced AKIHigh-performance liquid chromatography(HPLC)was performed to measure serum creatinine.Formalin-fixed kidney sections were stained with hematoxylin and eosin(HE).Terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)was used to detect cell death.3.1.3 NLRC5 regulated the expression of CEACAM1 in cisplatin-induced AKIWB and Real-time RT-PCR was used to detect the expression of CEACAM1 in kidney from WT and Nlrc5-/-mice with cisplatin-induced AKI.ResultsPart Ⅰ The role of NLRC5 in the pathogenesis of renal ischemia/reperfusion injury1.1 The expression of NLRC5 was increased in a mouse model of renal ischemia/reperfusion injury1.1.1 The expression of NLRC5 in mouse tissue and kidney cell linesRT-PCR analysis and WB result indicated that the high expression of NLRC5 inspleen,stomach and kidney.In renal cells,NLRC5 is highly expressed in NRK-52E,.1.1.2 NLRC5 was significantly increased in the kidney from a mouse model of renal IRIReal-time RT-PCR,WB and IHC analyses showed that the levels of NLRC5 were time-dependently increased in the kidney after 30 min of renal ischemia followed by different time points of reperfusion by real time.1.1.3 NLRC5 was mainly expressed in proximal tubules and distal tubules in cortex and outer medulla in kidneyIF analyses showed that NLRC5 was mainly expressed in proximal tubules and distal tubules in cortex and outer medulla in kidney.1.1.4 NLRC5 was expressed in the immune cells of renal with IRIIF analyses showed that NLRC5 was expressed in neutrophils,macrophage,CD4+ T cells and dendritic cells of kidney with IRI.1.1.5 NLRC5 was highly expressed in human kidneys from the patients with ATNIHC analyses showed that was highly expressed in human kidneys from the patients with ATN,especially expressed in renal tubular cells.1.1.6 All the approaches of mimicing hypoxia condition could increase the level of NLRC5 in NRK-52E cellsWB analyses showed that NLRC5 levels were reduced in NRK-52E cells under these mimic hypoxia conditions.1.2 NLRC5 deficiency protects against renal ischemia/reperfusion injury in mice1.2.1 NLRC5 deficiency ameliorated renal injury and inflammatory responses after ischemia/reperfusion(I/R)Compared with WT mice,Nlrc5-/-mice after I/R displayed lower level of serum creatinine and blood urea nitrogen,more minor morphological injury and the decreased cell death.NLRC5 deficiency reduced the levels of pro-inflammatory mediators by real-time RT-PCR.Consistently,neutrophil,macrophages,dendritic cells and CD4+ T cells accumulation was further decreased in the kidney from ischemic Nlrc5-/-mice,indicating that loss of NLRC5 signaling exacerbates renal inflammation.1.2.2 NLRC5 decreased hypoxia-induced inflammatory responses and apoptosis in proximal tubule epithelial cellsIn vitro,we employed different approaches to mimic hypoxia condition,all of them increased NLRC5 levels.Silencing of NLRC5 inhibited oxygen-glucose deprivation-enhanced the production of pro-inflammatory mediators,as well as cell apoptosis indicated by FC.Part Ⅱ NLRC5 exacerbates renal injury in renal ischemia/reperfusion via mediating CEACAM1 signaling2.1 NLRC5 negatively regulated the expression of CEACAM1By Agilent Whole Mouse Genome Oligo Microarray for global gene expression analysis,we observed the profound changes of CEACAM1,an important regulator of immune responses,in the kidney from Nlrc5-/-mice.It was found that IR-reduced CEACAM1 expression was recovered by NLRC5 deficiency in the kidney from ischemic mice,which was further confirmed by IHC,mRNA and Western blot analyses.2.2 NLRC5 negatively regulated CEACAMl-mediated signalingIn vitro,We found that hypoxia reduced CEACAM1 expression in a time-dependent manner.We investigated whether NLRC5 regulates ERK1/2 or Akt signaling pathway by CEACAM1.Compared with controls,ERK1/2 activation was increased under hypoxia condition,which was further induced by NLRC5 deficiency.Knockdown of CEACAM1 counteracted the effect of NLRC5 deficiency on ERK1/2 activation in NRK-52E cells.NLRC5-mediated ERK1/2 activation was also observed in the kidney from mice with IRI.Similarly,we found that the activation of Akt pathway was also associated with NLRC5-mediated CEACAM1 signal pathway in NRK-52E cells,although we failed to detect the changes of Akt activity in the kidney from NLRC5-/-mice with IRI.2.3 NLRC5 deficiency reduced the activation and proliferation of CD4+ T cellsWe found that NLRC5 deficiency significantly decreased CD4+ T cell infiltration in the kidney and spleen after IRI.As well as in the blood,CD4+ T cells showed a tendency to decrease the percentages in the kidney and spleen from Nlrc5-/-mice under basal levels compared with WT mice,although there was no statistical difference.Gating the CD4+ T cell population,the portions of activated CD4+CD69+ T cells were much less in the kidney from Nlrc5-/-mice than WT mice after renal IRI.In consistent with the in vivo results,NLRC5 deficiency markedlydecreased CD4+CD69+ T cells when CD4+ T cells isolated from the spleen of NLRC5-/-mice and stimulated by anti-CD3/CD28.NLRC5 deficiency could also inhibit the proliferation of CD4+ T cells by using the CFSE tracking assay.2.4 Knockdown of CEACAM1 counteracted the effect of NLRC5 deficiency on CD4+ T cellsFlow cytometry analyses confirmed the knockdown of CEACAM1 counteracted the effeet of NLRC5 on the activation of CD4+ T cells as evidenced by the percentages of CD4+CD69+ T cells and INF-y releasing CD4+ T cells.CFSE tracking assay further demonstrated the knockdown of CEACAM1 counteracted the effect of NLRC5 on the proliferation of CD4+ T cells.2.5 NLRC5 signaling in renal parenchymal cells primarily contributed to renal IRI in miceBy generating BM chimeric mice,WTnal IRI in mideveloped renal injury after renal IRI as measured by serum creatinine and tubular damage,whereas Nlrc5-/-→Nlrc5-/-chimeras had relatively little renal dysfunction.Meanwhile,creatinine levels and tubular injury scores were similar to what was observed in wild-type and NLRC5-deficient mice,excluding an effect of the BM transplant procedure per se on the response to renal ischemia.Of note,WT→ Mlrc5-/-chimeras,which lacked renal parenchymal NLRC5 significantly ameliorated renal IRI to the similar degree as Nlrc5-/-→Nlrc5-/-mice,while Nlrc5-/-→ O-chimeras(mice lacking myeloid NLRC5)had only partial protection as assessed by serum creatinine and tubular damage.These results suggested that functional NLRC5 on renal parenchymal cells made the more significant contribution to renal damage although leukocytes are clearly also important in IRI.Part III The role of NLRC5 in the pathogensis of AKI induced by cisplatin3.1 NLRC5 was significantly up-regulated in cisplatin-induced AKI WB indicated that NLRC5 was increased in the kidney from mice afer cisplatin injection.In vitro,NLRC5 was up-regulated in NRK-52E cells with cisplatin treatment.3.2 NLRC5 deficency protected against cisplatin-induced AKI Compared with WT mice,Nlrc5-/-mice with cisplatin injection indicated lower elevation of serum creatinine and blood urea nitrogen and less severe morphological injury.Real-time RT-PCR,TUNEL and FC results demonstated that NLRC5 deficiency ameliorated inflammation and cell death of kidney with cisplatin injection,indicating that NLRC5 signaling exacerbates AKI induced by cisplatin.3.3 NLRC5 negatively regulated the expression of CEACAM1WB indicated that cisplatin-reduced CEACAM1 expression was recovered by NLRC5 deficiency in the kidney with cisplatin treatment.Research conclusions and innovation1.In this study,we first confirmed the expression of NLRC5 in renal parenchymal cells.The expression of NLRC5 were significantly increased in the kidney with AKI.We found that gene silencing of NLRC5 decreased hypoxia-induced production of proinflammatory mediators and apoptosis in proximal tubule epithelial cells.NLRC5 negatively regulated the expression of CEACAM1 and CEACAM1-mediated signaling.In renal parenchymal cells,NLRC5 deficency activated ERK1/2 and Akt signaling by regulated CEACAM1.2.We further identified that NLRC5 has an important role in the regulation of immunity,we also discovered a previously unknown function of NLRC5 in mediating CD4+ T lymphocyte activation by downregulation of CEACAM1.Our findings may have important therapeutic implications to reduce morbidity and mortality arising from AKI and will even provide unexpected opportunities for developing new therapies for various inflammation-related diseases.