Sensitivity Of Diabetic Mice And High Glucose-conditioned Renal Tubular Cells To Acute Injury Induced By Ischemia And ATP Depletion

BackgroundRecent epidemiological research has revealed an important relationship between acute kidney injury (AKI) and chronic kidney disease (CKD). On one hand, AKI may contribute to the development and progression of CKD; on the other hand, CKD patients are predisposed to AKI. And following AKI, CKD patients show a worse outcome than non-CKD patients. The mechanism underlying the AKI sensitivity of CKD patients is unknown.Many diseases may cause CKD, including diabetes mellitus (DM), hypertension, and glomerulonephritis. Among them, DM is now the leading cause of CKD. Diabetic nephropathy (DN) is a serious complication for patients with DM, which is characterized by thickening of the glomerular basement membranes (GBM) and and progressive nodular glomerulosclerosis. While the main pathological changes are in glomerular, the pathological change of AKI induced by ischemia is in kidney tubular cells, expecailly the apoptosis in the proximal tubular cells. DM patients showed higher injury, poor kidney function and severe tubular cell damage than ND patient after AKI. But how does high glucose or diabetes increase the acute kidney injury level induced by ischemia? The mechanism is not clear.Part ⅠObjective To investigate the effect of diabetes in acute kidney injury induced by ischemia in mice.Methods Akita diabetic mice (DM) and the non-diabetic mice (ND) were used for this study, the renal pedicles were clamped to induce ischemic injury,23minutes later the clamp was released for reperfusion.(1) Mice survival, serium creatinine (Scr), Blood urea nitrogen (BUN) were examined, HE staining and Tunel staining were used to determin the histological damage in mice tissues.(2) The relationship between mice glucose level and BUN level after injury was analyzed.Results (1) DM mice showed higher mortality and poorer survival after ischemia injury than ND mice. Besides, the BUN level at0,24,48hours post-injury was25.9±1.13,65.8±18.82,63.1±27.52mg/dl respectively in ND mice, but26.2±3.14,172.6±28.04,244.6±54.22mg/dl in DM mice; the Scr level in ND mice at48hours post-injury was0.58±0.1mg/dl, but was2.29±0.72mg/dl in DM mice, BUN and Scr level were significantly higher in DM mice than in ND mice (P<0.05). In addition, the histological analysis showed that kidney injury score was significantly higher in DM mice than in ND mice at48hours post-injury (P<0.05). Tunel staining results showed that the apoptosis in kidney tubular cells was38.09±11.75/mm2in ischemic ND kidney tissues, but was119.04±31.07/mm2in ischemic DM kidney tissues, the apoptosis level was significantly higher in DM mice kidney tissues than in ND mice kidney tissues (P<0.05).(2) Kaplan-Meier analysis showed that the BUN level at48hours post-injury was correlated with fasting blood glucose level (R=0.85, P<0.05)Conclusion Diabetes increased ischemic AKI in mice; the injur level was correlated with the blood glucose level, suggesting that high level of blood glucose is an important factor in sensitizing diabetic mice to ischemic AKI.Part IIObjective To investigate the role of mitochondrial apoptotic pathway in higher tubular cell injury induced by ATP depletion or ischemia in high glucose or diabetic condition.Methods (1) Immortalized rat kidney proximal tubular cell line (RPTC) was cultured in low glucose medium (5.5mM glucose), high glucose medium (30mM glucose), and mannitol control medium (5.5mM glucose±24.5mM mannitol), respectively, for2weeks, then treated with sodium azide or anoxia to induce apoptosis, the apoptosis rate and caspase3activity were then examined.(2) Bax translocation and cytochrome c release after apoptosis were examined in RPTC cells cultured in low glucose, or high glucose, or mannitol control medium.(3) Bax translocation and cytochrome c release were examined in Akita diabetic and non-diabetic mice kidney tissues after ischemia AKI.Results (1) The apoptosis rate after azie or anoxia treatment was15%,60%,15%in RPTC cells cultured in low glucose, or high glucose, or mannitol control medium, respectively, the apoptosis level in RPTC cells cultured in high glucose medium was significantly higher than in low glucose or mannitol medium (P<0.05). And the caspase activity in RPTC cells cultured in high glucose medium after Azide or anoxia treatment was79.95±5.08,75.71±3.26nmol AFC/mg/hour, but was34.18±3.35,29.83±4.09nmol AFC/mg/hour in RPTC cells cultured in low glucose medium, the caspase activity was also significantly higher in RPTC cells cultured in high glucose medium than RPTC cells cultured in low glucose or mannitol medium after ATP depletion (P<0.05).(2) Bax translocation and cytochrome c release were higher and earlier in RPTC cells cultured in high glucose medium than those in low glucose or mannitol medium after azide or anoxia treatment (P<0.05).(3) Bax translocation and cytochrome c release were significantly higher in kidney tissues in DM mice than in ND mice after ischemic AKI.Conclusion High glucose or diabetes sensitized kidney tubular cells to ATP depletion or ischemia induced apoptosis by increasing mitochondrial apoptotic pathway.PartⅢObjective To investigate the role of p53in higher tubular cell injury induced by ATP depletion or ischemia in high glucose or diabetic condition.Methods (1) P53protein level was examined in RPTC cells cultured in different medium and in kidney tissues in DM mice or ND mice.(2) RPTC cells were stablely transfected with dominant negative p53(p53-DN) plasmid or control plasmid, then cultured in low glucose, or high glucose, or mannitol control medium for2days, followed by azide or anoxia treatment to induce apoptosis, then the apoptosis level, caspase activity, Bax translocation and cytochrome c release were examined.(3) RPTC cells cultured in low glucose, or high glucose, or mannitol control medium were treated with20μM pifithrin-α for2days, followed by azide or anoxia treatment to induce apoptosis, then the apoptosis level, caspase activity, Bax translocation and cytochrome c release were examined.(4) Akita DM mice and ND mice were injected with2mg/kg pifithrin-α,30minutes later these mice were given renal pedicles clamping for23minutes to induce AKI. Then mice survival, serium creatinine (Scr), Blood urea nitrogen (BUN) were examined, HE staining and Tunel staining were used to determin the histological damage in mice tissues.Results (1) High glucose activated p53and induced p53phosphorylation in RPTC cells, and following ATP depletion, there was more p53activation in RPTC cells cultured in high glucose medium than RPTC cells cultured in low glucose medium. P53base level was a little higher in DM mice kidney tissues than in ND mice kidney tissues, at48hours post-ischemia, there was more p53activation in ischemic kidney tissues in DM mice than in ND mice.(2) Inhibition of p53with p53-DN significantly reduced Azide induced apoptosis from60%to15%in RPTC cells cultured in high glucose medium (P<0.05), caspase activity was also reduced from61.63±5.92to26.76±2.94nmol AFC/mg/hour (P<0.05). P53-DN did not affect apoptosis and caspase activity in RPTC cells cultured in low glucose medium.(3) Inhibition of p53with pharmacological inhibitor, pifithrin-a significantly reduced Azide induced apoptosis from61%to25.6%in RPTC cells cultured in high glucose medium (P<0.05), caspase activity was also reduced from67.73±5.13to44.83±8.93nmol AFC/mg/hour (P<0.05). Pifithrin-a did not affect apoptosis and caspase activity in RPTC cells cultured in low glucose medium.(4) The BUN and Scr level in DM mice injected with0.9%saline were230.89±45.48mg/dl,2.36±0.56mg/dl, respectively, but reduced to100.72±45.27mg/dl, O.83±0.10mg/dl in DM mice injected with pifithrin-a, pifithrin-a significantly protected kidney function in DM mice after ischemic AKI (P<0.05), but did not affect the BUN and Scr level in ND mice. Tunel staining showed that the apoptosis rate in DM mice injected with0.9%saline was131.21±31.07/mm2, but reduced to48.32±15.32/mm2in DM mice injected with pifithrin-a, pifithrin-a significantly reduced the apoptosis in DM mice after ischemic AKI (P<0.05), but did not affect apoptosis in ND mice. However the HE staining showed that pifithrin-α did not significantly reduce the histolocia damage, which was assessed with kidney injury score, in DM mice and ND mice after ischemic AKI.Conclusion P53level was higher in high glucose cultured RPTC cells and diabetic ischemic kidney tissues. Inhibition of p53with p53-DN or pifithrin-α significantly inhibited azide induced apoptosis through inhibition of intrinsic mitochondrial apoptotic pathway in RPTC cels cultured in high glucose medium. Pifithrin-α also significantly inhibited ischemia induced kidney tubular cell apoptosis, protected kidney function in DM mice, but did not affect injury level in ND mice.SummaryThis study has determined the sensitivity changes induced by high glucose, and the roles played by mitochondrial apoptotic pathway and p53in sensitivity changes in renal tubular cells. High glucose or diabetes sensitized kidney tubular cells or tissues to ATP depletion or ischemia induced cell death, which is accompanied by higher and earlier Bax translocation and cytochrome c release, higher caspase activity and more activation of p53. Pharmlogical inhibition of p53abrogated the sensitivity changes in cultured cells and kidney tissues. These studies suggested that mitochondrial apoptotic pathway and p53appears to contribute to the higher sensitivity in diabetic tissues. However, other signaling pathways might also participate in the sensitivity changes in diabetes.