| Background and objectiveDiabetic nephropathy is one of the main reasons for end stage renal disease. About DN progressive mechanism has not been fully clarified, but the recent researches suggest that renal tubular interstitial lesion plays an important role and relates to prognosis of DN. Renal tubular epithelial cells(RTECs) senescence as the key events in cell biology in tubular interstitial lesion, which is widely attention and emphasis.RTECs senescence is different from the natural aging, which is independent of age and stress induced premature senescence(SIPS) in pathological microenvironment like sugar and lipid metabolism disorders, persistent oxygen stress, chronic inflammation, et al. The premature senescent cell performs cell cycle irreversible arrest and proliferation ability permanent loss, resulting in the decrease of ability of tissue regeneration and repair, couple with organ dysfunction. Senescent cells in addition to the specific molecular marker like SA- β- gal, p16 and p21, can also express senescence associated secretory phenotype(SASP) such as pro-inflammatory cytokines, inflammatory factors and so on. SASP can damage the surrounding normal cells and cause inflammation and fibrosis of local tissue cells. In addition, senescent cells can escape from removed by immune cells because of resistance to apoptosis. It can lead to senescent cells gather and the damage effects amplify, thus accelerating disease progression. Some studies show that the apoptosis resistance of senescent cells because of decoy receptor 2(DcR2). The high expression of DcR2 in senescent fibroblasts can escape from cell apoptosis, which can cause liver fibrosis and function damage. Prostate cancer and other tumor cells by DcR2 high expression level can resist to apoptosis, which can lead to malignant transformation of normal cells and metastasis of tumor cells. And the high expression of DcR2 in RTECs can inhibit apoptosis in DN rat model. About the correlation of DcR2 expression between kidney tissue damage and renal function, and the relationship of DcR2 with senescent RTECs is unclear.We will analysis the correlation of DcR2 and p16 expression between kidney tissue damage and renal function at first. Then we will study whether the DcR2 is related to senescent RTECs and whether these senescent cells perform resistance to apoptosis phenotypes. The aim of above researches is to clarify that DcR2 has an important role in the progress in DN, and DcR2 is closely related to the apoptosis resistance of senescent RTECs. It will help us look for key target molecules for early intervention of senescent renal tubule cells in DN, so it can improve the treatment and prognosis of DN through targeting removal of senescent cells.Methods1.clinical data :36 patients with type 2 diabetes mellitus and biopsy identified DN were recruited from Daping hospital affiliated to the third military medical university. According to the renal tubule interstitial lesion degree can be divided into early DN and progressive DN, 18 cases in each group. The control group was normal tissues from renal hamartoma or tumor resection by pathological examination. Acquisition all of patients with clinical data and related laboratory test results, and calculated estimated glomerular filtration(e GFR) according to CKD- EPI formula.2.The scoring and classification standard of renal tissue lesion: A new pathological scoring system and diagnosis criteria according to the journal of American society of nephrology in 2010. DN can be divided into early DN and progressive DN according to the different degrees of renal tubular atrophy and renal interstitial fibrosis, interstitial inflammation.3.Indicators and methods3.1 Immunohistochemical detected DcR2, p16 expression;3.2 Immunofluorescence detected DcR2, p16, FLIP, caspase-3 expression;3.3 Confocal microscope detected between DcR2 and p16, FLIP, caspase-3 expression.4.Scoring methodsThe results were assessed by two independent researchers with double blind method. Selected at random 10 fields and count related indicators positive expression in RTECs(nucleus expression) or renal tubule(cytoplasm). Then we calculated the positive expression percent of the total number of renal tubule cells. In confocal microscope, we counted DcR2 and p16, DcR2 and FLIP, DcR2 and caspase-3 double positive expression percent and of each indicator single positive number of total renal tubules.5.Statistical analysisThe data were statistically analyzed by SPSS18.0 statistical software. Measurement data performed assx ±. The clinical data of patients with DN and the positive expression percent of DcR2, p16 compared with control group, the normal distribution data using t test and the abnormal distribution data using rank sum test. The correlation between positive expression percent of DcR2, p16 and clinical data using Pearson correlation analysis, and the correlation between positive expression percent of DcR2, p16 and the degree of renal tissue lesion using Spearman correlation analysis. Significance standards were set at p < 0.05.Results1.Clinical data features in 36 DN patientsThere were 17 cases of the men and 19 cases of women in 36 DN patients. Compared with the control group, systolic blood pressure and 24-hour urine protein, urine NAG, urine protein to creatinine ratio, glycosylated hemoglobin, serum creatinine, cystatin C, urea nitrogen, triglyceride significantly higher, e GFR in DN group were significantly lower(P < 0.05). DN group compared with the control group, the age, gender, BMI, diastolic blood pressure, uric acid, C-reactive protein, total cholesterol, high density lipoprotein, low density lipoprotein difference had no statistical significance(P > 0.05).2.The correlation DcR2, p16 between clinical data and renal tissue lesion scroe in DNDcR2 only expressed in RTECs in DN. p16 can express in glomerular cells, RTECs and renal interstitial cells. DcR2 and p16 expression level was low in control group. The DcR2 and p16 positive percent in RTECs was significantly higher than the control group, and progressive DN is significantly higher than early DN.There was a positive correlation between DcR2 or p16 positive percent in RTECs and systolic blood pressure, 24-hour urine protein, urine NAG, urine protein to creatinine ratio, triglycerides, urea nitrogen, serum creatinine, cystatin C, negative correlation and e GFR in DN. There was no correlation between DcR2 or p16 positive percent in RTECs and age, diastolic blood pressure, BMI, albumin, glycosylated hemoglobin, cholesterol, uric acid, low density lipoprotein, high-density lipoprotein, c-reactive protein in DN.The DcR2 or p16 positive percent in RTECs were positively correlated with kidney mesangial proliferation, glomerular sclerosis, renal interstitial inflammation, renal tubular atrophy and interstitial fibrosis in DN patients.3.The co-expression DcR2 and p16 in DNConfocal microscope detected DcR2 and p16 were co-expressed in RTECs in DN, and the DcR2 less positive percent than p16. There were some p16 or DcR2 single positive RTECs, and p16 single positive percent was higher than DcR2. In addition, the p16 single positive percent in RTECs in DN was significantly higher than control, and the DcR2 single positive percent was no significant difference in each group.4.The co-expression DcR2 and apoptosis related indicators in DNConfocal microscope detected DcR2 and anti-apoptotic molecules FLIP were coexpressed in RTECs in DN. There were some FLIP single positive RTECs, and the percent was higher than DcR2. We also found FLIP single positive percent was no significant difference in each groups. In addition, DcR2 positive cells did not express or lowly express of caspase-3. There was no significant difference compared caspase-3 single positive percent in early DN with control. And caspase-3 single positive percent in progressive DN was significantly higher than control and early DN.ConclusionsDcR2 is closely related to senescent renal tubular epithelial cells and their apoptosis resistance in DN. They play important roles in renal tissue damage of DN. |
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