P2X4and NALP3Expression Associated With Tubulointerstitial Inflammatory Injury In Diabetic Kidney Disease

Diabetic kidney disease（DKD） is one of the most typical complicationsof diabetes mellitus（DM）,the most single factor in the development of End-stage renal disease（ESRD）,has become a worldwide health problem.To date-,there are many researches about the mechanisms of DKD,mostly aroundmetabolic disorders and hemodynamic abnormalities link of DM,but in recentyears about the the DKD pathophysiological process has been elevated to thegenetic and molecular level,the evidence suggests that the traditional factorsis only part of the many complex reasons,there is growing evidence that immu-ne and inflammatory mechanisms play an very important role in occurre-nceand development in DKD.Interleukin-1（IL-1） family cytokines,including IL-1β,IL-18,IL-33,are im-portant pro-inflammatory cytokines,mainly mediate to infection,stress andimmune response caused by tissue damage.IL-1β can make pancreatic β cellapoptosis and injury,so it is considered to be the driving factors of DM occu-rred.Recent studies show that mononuclear macrophage infiltration increasedin kidney of DKD patients,and found activity of IL-1β,IL-18and other proin-flammatory cytokines significantly increased in renal interstitium and renaltubular epithelial cells,suggesting that these inflammatory cytokines is likelyinvolved in the occurrence and development of DKD,but the mechanism thathow these pro-inflammatory cytokines produce and activate is not clear.Currently considered that activation of IL-1family cytokines can play abiological effect,and this activation process must rely on the shearing of cas-pase-1to complete,the Cryopyrin （NALP3） is one of the NALP3inflammasome,is the most important part,belonging to the NOD like receptors（NLRs） family members,is responsible for identifying danger signals, activate casp-ase-1to the shearing precursors of IL-1family to be the activity proinflamma-tory cytokines such as IL-1β,IL-18,IL-33,etc,thereby causing a series of infla-mmatory responses.study found that high glucose induced the NALP3^-/-m-ouse islet β cells produce IL-1β significantly reduced,our previous work hasfound that high glucose can increase caspase-1activity of the renal tubular ep-ithelial cell,IL-1β production increased significantly,suggesting that the NAL-P3inflammasome likely involved in the high glucose-stimulated renal tubul-ar epithelial cells to produce IL-1β,thereby causing damage to renal interst-itial inflammation,but it is still unclear that how the renal tubular epithelialcells transmit stimulate signals caused by high glucose to the cytoplasm andthen activate the NALP3inflammasome,recent studies have shown that ROSand low potassium within the cell may be an important way of high glucoseactivate the pancreatic β cell.The ligand-activated cation channels （Purinergicreceptor X, P2Xs） are ion channel purinergic receptor which can lead to hypo-kalemia,generation of ROS and release of IL-1β,IL-18and other inflammato-ry cytokines in the cells,an important member P2X4expressed both in tubula-r epithelial cells and mesangial cells,and P2X4expression in mesangial cellsin high glucose load,has an important role in the occurrence of glomerularsclerosis.Based on the above,we believe that the NALP3activation mediate high gl-ucose to stimulate islet beta cells to produce and release Il-1β,P2X4activati-on may be mediate ROS production and potassium channels open, causing N--ALP3activation,while high glucose can increase mesangial cells P2X4expression and therefore speculate that high glucose may through theactivation of P2X4,to activate the NALP3inflamasome,regulating the produ-ction of IL-1β and IL-18,amplified inflammatory response waterfall causedby the high sugar,eventually leading to intertitial inflammation in diabetickidney disease.To this end,the subject disscus renal tubular epithelial cellsP2X4,NALP3,IL-1β,IL-18expression pattern and its variation in the DKD pat-ients,theoretical basis for further study of its mechanism,designed to provid -e new scientific evidence in revealing the diabetic nephropathy tubuloint erstitial inflammation mechanisms.Methods1.SubjectsAccording to the1999World Health Organization （WHO） and International Diabetes Federation （IDF） Promulgated diagnostic criteria for diabetes,and established Mogensen diagnostic criteria of diabetic kidney disease,toreceive September2010to October2011,the Third Military Medical University,Daping Hospital,by renal biopsy confirmed the diagnosis with2type DKD30patients （urine protein>500mg/d or of SCr>120μmol/L）,12male cases-,18females,from40to68old years,（average47.93±7.05years）;control group derived from kidney transplant or renal hamartoma resection of the pathological diagnosis of normal renal tissue（urine protein <500mg/d or SCr <120μmol/L）,10cases,6males,4females, from40to65old years,（average50.60±9.40years）,the cases were excluded from the sugar to adjust abnormal,excluded from smoking, hypertension.Collecting specimens to obtain the informed consent of patients themse-lves and their families,selected patients were excluded from the acute compli-cations of diabetes,urinary tract infection,severe heart,liver,renal insufficien-cy,as well as rheumatism,Cancer and other systemic diseases.2.Test index and methodsPre-collected control group blood and urine samples and collected the DKD patients blood and urine samples the day before biopsy,conventional bioc-hemical methods to detect serum total cholesterol,triglycerides,Low density l–ipoprotein（LDL）,blood glucose,24hour urinary protein,serum crea tinine（SCr）,and mearsuring systsystolic blood pressure（SBP）,diastolic blood pressure（DBP）,calculate body mass index （BMI）,calculate Estimated glomerul-arfiltration rate （eGFR）,accordance with the count on the Cockcroft-Gault form-ula:eGFR（mL/min/1.73m2）=（140-age）×BW（kg）/[72×Scr（mg/dl）]×0.85（if femal）.Immunohistochemistry detect control group and the DKD groups P2X4, NALP3, IL-1β and IL-18expression variation of renal tubular epithelial cells;confocal laser detecte P2X4,NALP3,IL-1β and IL-18expression variation;Enzyme inked immunosorbent assay （ELISA） was detected in both groupsurine of patients with IL-1β,IL-18level.Results:1.General compared with the control group,24hour microalbunminuria,-blood sugar,SBP,DBP,total cholesterol,LDL,Triglycerid and BMI was signifi-cantly higher in DKD patients,eGFR was significantly lower in DKD patien-ts （P<0.05）.2.Immunohistochemical results showed that in control group,the expres-sion of P2X4, NALP3were very low,IL-1β,IL-18basicaly did not express,while the expression of P2X4,NALP3,IL-1β and IL-18in DKD group signi-ficant increased（P<0.05）;correlation analysis showed that:DKD patients ren-al tubular epithelial cells,the expression of P2X4,NALP3,IL-1β,IL-18weresignificantly positively correlated with renal interstitial inflammation（r=0.82,-r=0.66,r=0.55,r=0.83,P<0.01）,renal interstitial fibrosis（r=0.80,r=0.65,r=0.5,r=0.81,P<0.01）,tubular atrophy（r=0.80,r=0.63,r=0.56,r=0.82,P<0.01）,significantly negative correlation with eGFR（r=-0.58,r=-0.73,r=-0.65,r=-0.58,P<0.01）.3.ELISA kits detect DKD Group patients urinary IL-1β,IL-18levels,wassignificantly higher than control group（P <0.05）.4.Confocal test results show that in DKD group,P2X4and IL-1β,IL-18coexpress in the renal tubular epithelial cells,NALP3and IL-1β,IL-18coexpre-ss in renal tubular epithelial cells,P2X4and NALP3coexpress in renal tubula-r epithelial cells.Conclusions:1.The relationship between renal tubular epithelial cells P2X4,NALP3,IL-1β,IL-18expressionin of DKD patients and renal interstitial damage andrenal function changes showed that renal interstitial inflammation responsemay be involved in the decline of kidney function,leading to DKD developme-nt of ESRD. 2. The coexpression among renal tubular epithelial cells P2X4,NALP3,IL-1β,IL-18of DKD patients prompted that inflammatory activation of tubulo-interstitial may be mediated by P2X4activating NALP3caused,which furthercauses the kidney chronic direction development,is likely to be the main mechanism of the DKD development.