The Mechanism Of Diabetes Aggravating Renal Ischemia Reperfusion Injury

Object:To investigate the effect of diabetes on renal ischemia/reperfusion injury in rats and the underlying relationship with mitochondrial function and mitophagy.Methods:1.Cell culture:human kidney proximal tubular epithelial cells(HK-2)were used for the experiment and randomly assigned to the following four groups,normal control(NC);normal+H/R group(NH/R),high glucose control(HG)and high glucose+H/R(HG+H/R).The NC group cells were cultured in an incubator with DMEM+10%fetal bovine serum(FBS)under the condition of 37°C 5%CO2 and 21%O2.Cells in the NH/R group were placed in the hypoxic environment,filled with the mixture of 94%N2+5%CO2 and 1%O2 and subjected to hypoxia for 12h under 37°C,then reoxygenated for 3h by exposing the cells to a cell incubator.Cells in HG group were cultured in the normal glucose medium+10%FBS to the density of about 50%,then cultured in the high glucose(30m M)medium+10%FBS for 36h.In the group HG+H/R,cells exposed to high glucose,then switched to culture in the normal glucose and serum free medium before inducing hypoxia.After 12h of hypoxia,high glucose medium+10%FBS was used for 3h during reoxygenation.2.Animal model construction:SD rats were randomly divided into four groups:normal+sham group(NS);normal+I/R group(NI/R);DM+sham group(DS);DM+I/R group(DI/R).Type 1 diabetic was induced in male rats by a single intraperitoneal(i.p.)injection of 65 mg/kg STZ as previously described.Rats were considered to be a successful diabetic rat model when serum glucose levels≥16.7 mmol/L in three consecutive measurements(three consecutive days).Nondiabetic rats were injected with an equal volume of sodium citrate buffer.All rats’right kidneys were excised via midabdominal incision.Then,nephrectomy was performed by ligating the right kidney vascular pedicle with silk thread.The left kidney vascular pedicle was subjected to ischemia for 45 minutes with a microvascular clamp.Reperfusion was allowed for 24h.The sham-operated group underwent the same surgical procedure without left kidney vascular pedicle occlusion.After reperfusion,the rats were sacrificed to collect blood and tissue samples for subsequent experiments.3.CCK-8 assay kit was used to detect cell damage.We use commercial DCFH-DA molecular probes to measure Intracellular ROS levels.Mitochondrial membrane potential(MMP)was qualitatively assessed using MMP assay kit with JC-1.ATP content was measured using ATP assay kit.Western blot was used to detect the level of mitophagy related proteins.Apoptosis were determined by flow cytometry and Western blot.Lentiviral transduction was used to construct cell lines with stable overexpression of PINK1 gene.4.Blood samples were collected through the inferior vena cava,serum was separated by centrifugation,and serum creatinine(Cr)and urea nitrogen(BUN)were detected by the biochemical kit to evaluate renal function.One part of the left kidney tissue was stored in 10%formaldehyde,the other part was immediately stored in liquid nitrogen,and then about 1mm~3 tissue was retained and fixed in 2.5%glutaraldehyde.HE staining and electron microscopy were used to observe the renal tissue and mitochondrial damage in rats.TUNEL assay was used to detect the apoptosis of renal tubular cells.Results:1.HG aggravates HK-2 cells injury and mitochondrial dysfunction induced by H/R.ATP content,mitochondrial ROS and MMP levels,which are considered markers of mitochondrial function,were measured in cell experiment.Compared with the NC group,ATP content in the H/R group decreased,mitochondrial ROS level increased and MMP decreased,while high glucose aggravated this phenomenon.2.Pink1-Parkin mediated mitophagy was induced after H/R.By inhibiting the PINK1-Parkin pathway,HG reduced the mitophagy level in HK-2 cells which induced by H/R.This suggests that tubular cells have an increased ability to clear damaged mitochondria.Compared with the NC group,the expression of PINK1 and Parkin in HK-2 cells increased after H/R.However,H/R-induced mitophagy is reversed when exposed to HG.3.We transfected the cells with a PINK1 overexpression vector(p LVX-Neo-PINK1)or p LVX-Neo empty vector as the negative control group.The protein expression of PINK1 was significantly increased following transfection with the overexpression plasmid,as compared with that in the Empty vector group.With the overexpression of PINK1,the expression of LC3B-II and Parkin were also increased,which was accompanied by decreases in TOMM20 and TIMM23,indicating induction of mitophagy.We also found that cell damage,mitochondrial dysfunction,and apoptosis can be ameliorated by reversing pink1-related mitochondrial autophagy.4.Based on the results of cell experiments,we constructed the model of type 1diabetes and renal ischemia reperfusion injury in SD rats.Diabetic rats were more sensitive to renal I/R.and we also detected mitochondrial damage in renal I/R posterior renal tubules.Rats with I/R injury had a higher percentage of broken and swollen mitochondria in their renal tubule cells than the sham control group.In addition,the ultrastructural changes of mitochondria in diabetic rats after I/R injury were more serious,manifesting as mitochondrial swelling,cristae deletion and matrix vacuolization.