The Expression Of Renal And Urinary DcR2 In I/R Injury And Its Relationship With Chronic Progression

BackgroundAcute kidney injury(AKI)is a group of common clinical critically ill disease caused by a variety of reasons.The incidence and mortality of AKI is increasing year by year.The incidence in the general population is about 0.25%,hospitalized patients up to 18%,while in the intensive care unit is as high as 30%-60%.AKI has a mortality rate of about 8.8% in the general population and 50-80% in critically ill patients.However,Follow-up studies have shown that about 33.6% of severe AKI patients progressed to CKD or even ESRD after 14 months of discharge.However,the mechanism for the progress of AKI for CKD is still unclear.Renal tubular cells are rich in mitochondria and metabolically active.Renal tubular cells is susceptible to ischemia,hypoxia and other stress factors.Renal tubular cells are rich in mitochondria and metabolically active.Renal tubular cells is susceptible to ischemia,hypoxia and other stress factors.The study found that renal tubular epithelial cells in the occurrence of ischemia and reperfusioncan be observed stress aging within a few hours.Thus,aging,as one of the important biological events in cells after ischemia-reperfusion,has received extensive attention in the role of AKI-CKD progression.Because of the characteristics of apoptotic resistance in senescent cells,senescent renal tubular epithelial cells that developed after renal ischemia and reperfusion continue to accumulate and by secreting a large number of proinflammatory factors,promoting fibrosis factor,chemokines,growth factors,matrix and protease and other senescence associated secretory phenotype(SASP).The release of the above factors aggravate the local inflammatory response,and promote the progress of renal interstitial fibrosis.At the same time,the aging cells also play an apoptotic resistance so that it is not easy to be removed and thus remain in the lesion.The damage effect can not be terminated and extended and amplified.In summary,the repaired of renal tissue is not completely,the normal function of the kidney is affected,leading to deterioration of renal function,and gradually progress for chronic kidney disease and even end-stage renal disease.DcR2 is one of the receptors of tumor necrosis factor-related apoptosis inducing ligand(TRAIL),which has the function of apoptosis and resistance.Studies have shown that,after the increase in DcR2 expression,cells in prostate cancer,cervical cancer,colorectal cancer and other tumor can express resistance to apoptosis,but by DcR2 low expression apoptosis is increased.At the same time,DcR2 can also be used as a marker of cell senescence,DcR2 is high expressed in the aging of tumor cells and fibroblasts and in vitro high glucose cultured HK-2 cells.It has been found that DcR2 may play an important role in the progression of fibrosis by interfering with the degree of hepatic fibrosis after DcR2 expression.All in all,DcR2 may play an important role in the progression of the disease.In addition,as a transmembrane receptor,the extracellular segment can be cut down by protese to form a biologically functional soluble molecules,and it can be detected in the serum.However,the relationship between DcR2 expression in renal tissue and urine and the degree of chronic progression in ischemia-reperfusion rats has not been reported.This sudy can be stated from three aspacts: first of all the DcR2 expression levels in the ischemia-reperfusion rats kidney tissue and urine and its correlation analysis with renal function and chronic injury scores;Followed by DcR2 expression in ischemia-reperfusion in rat kidney tissue and co-expression with renal interstitial fibrosis markers?-SMA and Collagen IV.Finally,co-expression of DcR2 and SA-?-gal and P16Ink4 a in renal tissue of ischemia-reperfusion rats.Methods1.Experimental Procedures for renal ischemic model80 male SD rats were randomly selected.Ischemia-reperfusion(I/R)kidney injury model was established by clamping the bilateral renal pedicle with micro arterial clip.I/R45 min ratswas clamped for 45 min,and the I/R60 min rats was clamped for 60 min,then release the bilateral arterial clip.Sham group,only separat the bilateral renal pedicle and do not use arterial clip,the other steps is similar to the I/R group.2.Detection of renal functionPreparation of I/R kidney injury model,abdominal aorta puncture to collect blood 1ml,metabolic cages to collect 24 hours urine at 1,3,7,21,35 d after operation.Levels of Serumcreatinine and urinary NAG/Cr(u NAG/Cr)were detected by biochemical analysis.3.Assessment of chronic kidney injuryPreparation of I/R kidney injury model,collect bilateral renal tissue,at 1,3,7,21,35 d after operation.The renal tissue was fixed using10% paraformaldehyde at room temperature,and paraffin embed after 24 hours.PAS staining and Masson staining were performed on renal tissue sections(thickness 2 ? m).The pathological changes of renal tissue in IRI rats were observed under high power microscope.The damage degree was observed according to the following method[8,9].Morphological changes of renal tissue were observed under microscope.Randomly selected 10 discontinuous areas,and calculate the rate of inflammatory cell infiltration,renal tubular atrophy and interstitial fibrosis: 0,no lesion;1,<25%;2,25 % ~ 50%;3,51%~ 75%;4,> 75%.4.Immunohistochemistry stainingThe expression of DcR2 in renal tissue was detected by immunohistochemistry after dewaxing and dewaxing.Expression of DcR2 was tested by using Immunohistochemistry;expression of urine DcR2/Cr level was measured by ELISA.The correlation between the expression level of DcR2 and the degree of renal function and tissue damage was analyzed using the spearman statistical method.Randomly selected 10 discontinuous areas,under 200 times microscope.Using double-blind method,by two physicians.The scores of renal DcR2 were scored according to the positive area: 0 points,none;1 point,<25%;2 points,25% ~ 50%;3 points,51% ~75%;4 points,?76% [10].5.Enzyme-linked immunosorbent assayCollect 24 hours urine by metabolic cage at 1,3,7,21,35 days after the construction of I/R rat kidney injury model.3000 rpm / min centrifuged 20-30 minutes,the upper serum.The levels of urinary DcR2(u DcR2)were measured by enzyme-linked immunosorbent assay(ELISA)and use urine creatinine correct the results.The results were expressed as u DcR2 / Cr.6.The relationship between the expression of renal and urinary DcR2 and renal function and the degree of chronic injury scoresThe expression of DcR2 in renal tissue and the correlation between u DcR2 levels and chronic renal function and renal tissue scoring were analyzed using spearman statistical analysis.7.Immunofluorescence staining(1)Co-expression of DcR2 and ?-SMA and Collagen IV was observed by immunofluorescence,analyze the relationship between the expression of DcR2 and the degree of renal interstitial fibrosis.Calculate the ratio of DcR2 and ?-SMA or Collagen IV positive areas to total areas,0 points: 0% to 5%;1 point: 6% to 25%;2 points: 26% to 50%;3 points: 51% to 75%;4points:76%.(2)The relationship between the expression of DcR2 and the degree of renal interstitial fibrosis in renal tissue of I/R rats was analyzed by spearman.8.The relationship between DcR2 expression and senescent phenotype(1)Immunohistochemistry and SA-?-Gal kit were used to detect the co-expression of DcR2 and SA-?-Gal in renal tissue.The results were observed under high magnification microscope and the percentage of renal tubules expressing DcR2 and SA-?-Gal double positive expression was counted.(2)Immunofluorescence was used to detect the expression of DcR2 and P16Ink4 a in renal tissue.The results were observed under laser confocal microscopy and the ratio of DcR2 and P16Ink4 a double positive expression of renal tubular epithelial cells was counted.Results1.Renal function of two groups of I/R rats.The levels of Scr and u NAG/Cr in I/R45 min and I/R60 min rats were significantly higher than those in Sham group.The level of Scr in I/R45 min rats gradually decreased after 1 day of reperfusion,then decreased to normal at 35 days(P> 0.05).The levels of Scr in I/R60 min rats were significantly higher than those in I/R45 min at each time point,and the Scr level decreased gradually after 3 days of reperfusion.However,the function of 80%I/R60 min rats did not return to normal at 35 days.The variation of u NAG/Cr was the same as that of Scr.The renal function of I/R45 min rats gradually returned to normal after 1 day of reperfusion.u NAG/Cr was still higher than that of I/R60 min rats(P <0.05).2.Renal chronic scores of two groups of I/R rats.After I/R renal injury there were extensive renal tubular necrosis and loss of acute pathological damage.Compared with the Sham group(0 points),the renal tissue of the I/R45 min rats returned to normal at 35 day,and the chronic score of the renal tissue was(3.67 ± 0.58).Compared with I/R45 min,the degree of pathological damage in I/R60 min rats was significantly increased.In I/R60 min rats more than 30% of regional renal tubular atrophy,renal interstitial fibrosis and other chronic performance can be observed at 35 day.Renal tissue chronic score was(8.33±1.29)points.3.Expression of DcR2 of two groups of I/R rats.DcR2 was only expressed in renal tubular epithelial cells,and the expression of DcR2 was low in normal renal tissue.The expression of DcR2 in I/R45 min rats reached the peak at 1 day after reperfusion,and then decreased gradually.There was no significant difference between the expression of DcR2 and the sham(P> 0.05).The expression of DcR2 in I/R60 min rats was significantly higher than that in I / R45 min,and reached the peak at 3days after reperfusion.However,there was still a large amount of DcR2 expression at 35 days,which was statistically significant(P <0.05).DcR2 overexpression was mainly in the poor repair areas,and low expression in good repair areas.4.Urinary DcR2 of two groups of I/R rats.Compare with Sham group,the level of uDcR2 was significantly higher after ischemia-reperfusion.The levels of u DcR2/Cr in the I/R45 min group were significantly increased and reached the peak level at 1 day after reperfusion,and the level of u DcR2/Cr decreased gradually with the reperfusion time.The level of u DcR2/Cr decreased to normal at 35 days after reperfusion(P>0.05).The level of u DcR2 reached the peak at 3 day after reperfusion in the I/R60 min rats,and the level of u DcR2 decreased gradually with the prolongation of reperfusion time(P <0.05).5.Correlation between the expression of DcR2 and renal function and chronic kidney scores.The expression of DcR2 in I/R45 min and I/R60 min rats kidney was positively correlated to Scr,u NAG/Cr and renal tissue chronic scores.In I/R45 min groups the correlation coefficient were 0.943,0.913 and 0.829(P<0.05);In I/R60 min groups the correlation coefficients were 0.857,0.909 and 0.851(P <0.05).The levels of u DcR2/Cr in I/R45 min and I/R60 min rats were positively correlated with the level of Scr,u NAG/Cr and chronic renal tissue scores,Correlation coefficient in I/R45 min rats were 0.943,0.943,0.886(P<0.05);The correlation coefficients in I/R60 min rats were 0.920,0.847 and 0.827(P<0.05).6.The relationship between the expression of DcR2 and the degree of renal interstitial fibrosis in I/R60 min rats.By immunofluorescence staining,renal tubulointerstitial fibrosis markers ?-SMA and Collagen IV were observed in DcR2 positive renal tubules at 21 and 35 days in I/R60 min rats.The correlation coefficients were 0.885,0.806(P <0.05).Suggesting that DcR2 is associated with chronic progression in I/R injury.7.The relationship between DcR2 expression and senescent phenotype in I/R60 min rats.Immunohistochemical staining showed that SA-?-gal and P16Ink4 a increased in the I/R60 min rats,and the expression of SA-?-gal and P16Ink4 a was higher in 85% in DcR2 positive renal tubules.Suggesting that DcR2 positive tubular epithelial cells have a senescent phenotype.ConclusionThe expression of DcR2 in renal tissue was significantly correlated with renal tissue and renal tubular senescence in the I/R model,indicating that DcR2 may play an important role in the tissue repair.At the same time,u DcR2/Cr levels were significantly higher in the repair period,indicating that u DcR2/Cr may be a potential prognostic factor for assessing renal ischemia-reperfusion injury.