| Part one: Establishment of an optimal rat model of delayed ischemic preconditioning in the kidneyBackground and objective: Ischemia-reperfusion (I/R) injury is the major cause of acute renal failure (ARF) in the native as well as in the transplanted kidney. At present, there is no specific treatment for this devastating clinical syndrome with high morbidity and mortality. Brief and sublethal prior ischemia renders an organ more tolerant to subsequent prolonged I/R injury. This phenomenon is called "ischemic preconditioning" (IPC). There are two temporally and mechanistically distinct types of protection afforded by IPC, acute and delayed IPC. Appearing about 12-24 h after the preconditioning ischemia, the beneficial effects of delayed IPC require new protein synthesis and sustain for days to weeks. Because of its promising and sustained effects against I/R injury, delayed IPC has been the topic of intense research interest in other organs. Conversely, there is a limited amount of information in the literature regarding renal delayed ischemic preconditioning, and its endogenous protective mechanisms have not been adequately evaluated in the kidney. Firstly, in this part we aimed at establishing an effective rat model of delayed IPC in the kidney.Materials and methods: Male Sprague-Dawley rats were randomly divided into seven groups, including sham-operation + sham-operation (Sham/Sham), sham-operation + 40 min of bilateral ischemia (Sham/I), 10 min, 15 min, 20 min, 25 min, or 30 min of bilateral ischemia + 40 min of bilateral ischemia (I-10/I, I-15/I, I-20/I, I-25/I, I-30/I). There was a different interval (1 d, 4 d, 8 d, 16 d, 28 d) between two surgeries. Blood sample and kidneys were collected at indicated times (n=6 in each group at each time point). Serum creatinine (Scr) and blood urea nitrogen (BUN) levels, mortality, and histological change were observed throughout the study. Renal protein expression of profibrotic factors, such as a-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) and phospho-Smad2, was assessed by immunohistochemistry or western blot.Results: 1) Compared with sham-operated rats (Scr 25.52±3.01μmol/L, BUN 5.29±0.88 mmol/L), non-preconditioned rats with 40 min of bilateral ischemia and 48 h of reperfusion demonstrated a drastic increase at both Scr and BUN levels (Scr 573.85±29.64μmol/L, BUN 69.36±3.45 mmol/L, P＜0.001). With a 8-day interval between two ischemic events, rats pretreated with 10-min ischemia presented no functional protection (Scr 553.34±76.86μmol/L, BUN 67.33±13.00 mmol/L); rats pretreated with 15-min ischemia presented partial functional protection (Scr 155.93±81.83μmol/L, BUN 34.31±9.75 mmol/L); rats pretreated with 20-min, 25-min, or 30-min ischemia presented promising functional protection with similar significant decrease of Scr and BUN (I-20/I Scr 41.02±5.22μmol/L, BUN 9.95±0.65 mmol/L) (I-25/I Scr 37.86±8.95μmol/L, BUN 8.41±3.53 mmol/L) (I-30/I Scr 40.98±11.41μmol/L, BUN 6.63±1.90 mmol/L). 2) Rats pretreated with 20-min or 25-min demonstrated drastically protective effects on renal function at the interval of 4-day simlar to the interval of 8-day. Rats pretreated with 30-min demonstrated drastically protective effects on renal function at the interval of 16-day simlar to the interval of 8-day. There was a postischemic increase of Scr and BUN levels in the above rats at other intervals, but this increase is significantly less than the increase in Scr and BUN seen in the non-pretreated rats (P＜0.001). 3) Histological evaluation revealed severe tubulointerstitial damage from Sham/I group (3.53±0.22), while only the I-20/I and I-25/I group showed nearly normal renal histology (I-20/I 0.21±0.08; I-25/I 0.22±0.15). The I-10/I group showed tubulointerstitial damage similar to that of Sham/I group (3.42±0.29). The I-15/I group showed partial histological protection (1.55±0.21). The I-30/I group presented obvious renal tissue injury associated with histological change prior to the second ischemia, including tubular dilation, tubulointerstitial edema, and expansion of the interstitial compartment. 4) Evaluation of tubulointerstitial fibrosis was studied by Masson's trichrome staining at 5 wk postischemia. Mild to moderate tubulointerstitial injury was found in kidneys of rats subjected to 40-min ischemia alone or pretreated with 10-min ischemia and further worsened in kidneys pretreated with 30-min ischemia (tubulointerstitial fibrosis area %, 18.92±2.63 vs. 17.50±3.16 vs. 25.30±3.78, respectively), the degree of tubulointerstitial fibrosis even observed for 10 weeks was not attenuated in the above three groups. Worsened tubulointerstitial fibrosis in the I-30/I group correlated with renal hypertrophy and increased levels of Scr and BUN. In contrast, tubulointerstitial fibrosis area was minimal in kidneys with 20-min ischemia pretreatment (1.12±0.49%). 5) the expression ofα-SMA and TGF-β1 were significantly increased at 5 wk postischemia in kidneys of Sham/I and I-10/I groups and further increased in that of I-30/I group, when compared to sham-operated group. However, the increased expression of these profibrotic factors was significantly attenuated in rats pretreated with 20-min ischemia.Conclusions: 1) At a certain extent, the renoprotective effect is positively correlated with the duration of preconditioning ischemia. Synergic effects of prolonged preconditioning ischemia and injurious ischemia lead to worsened tubulointerstitial fibrosis. 2) Ischemic renal fibrosis is associated with increased numbers of myofibroblasts (α-SMA positive) and activation of TGF-β/Smad signaling pathway. 3) Preconditioning with 20-min ischemia has short- and long-term protective effects on the kidney. 4) To optimize renoprotective effects, the interval between two ischemic events must be suitable. In conclusion, 20-min preconditioning ischemia-the interval of 4 to 8 d-40-min of bilateral ischemia is the optimal schedule of renal delayed IPC. Part two: Evaluation of endogenous protective mechanisms involved in renal delayed ischemic preconditioningBackground and objective: Ischemic ARF is characterized by massive tubular epithelial cell necrosis. Vascular dysfunction and inflammatory response play an important role in the pathogenesis of ARF. The beneficial effects of delayed IPC on renal morphology and function have been evaluated by our previous studies. However, its endogenous protective mechanisms have not been adequately evaluated in the kidney. We therefore tested the hypothesis that renoprotective effects of delayed IPC may be associated with changing tubule cell fate, based at least in part on its ability to ameliorate inflammatory response and enhance blood flow.Materials and methods: Male Sprague-Dawley rats were randomly divided into three groups, including sham-operation + sham-operation (Sham), sham-operation + 40 min of bilateral ischemia (I/R), and 20 min of bilateral ischemia + 40 min of bilateral ischemia (IPC). The interval between two surgeries was 4-day. Blood sample and kidneys were collected at indicated reperfusion time, including 4 h, 24 h, 48 h, and 72 h (n = 6 in each group at each time point). Serum creatinine (Scr) and blood urea nitrogen (BUN) levels, mortality, and histological change were observed throughout the study. Transmission electron microscopy (TEM) was used to observe cell ultra-structure of tubule and endothelium. Apoptosis was confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay. Protein expression and distribution in the kidney were determined by western blot and immunohistochemical analysis, and mRNA expression was quantified by realtime RT-PCR. Concentrations of MIP-2, endothelin-1, and 6-keto-PGF1αwere measured by ELISA kit or radioactive immunoreaction kit. The DNA binding activity of NF-kappa B in rat kidney was measured by eletrophoretic mobility shift assay (EMSA).Results: 1) At 4 h postischemia, levels of serum Scr, AST and urinary NAG of IPC group were significantly decreased when compared with that of I/R group (Scr 63. 69±12.19μmol/L vs. 109.89±15.73μmol/L, P＜0.001; AST 329.75±22.13 IU/L vs. 834.884±225.50 IU/L, P＜0.001; NAG 34.42±9.63 IU/L vs. 51.344±7.49 IU/L, P＜0.01, respectively). The decrease in Scr levels peaked at 48 h postischemia (36.26±6.89μmol/L vs. 575.93±36.26μmol/L, P＜0.001), companied by improved survival rate in IPC group (100 % vs. 68 %). 2) Extensive proximal tubular necrosis was found at 48 h after renal I/R injury, accompanied by inflammatory cell infiltration. Ischemic preconditioning significantly attenuated acute tubule necrosis (ATN) (4.03±1.62 % vs. 69.174±8.93 %, P＜0.001). Significant positive correlation was observed between the level of Scr and the degree of ATN (r = 0.99, P＜0.001). There was a small number of apoptotic cells in kidneys of I/R group and IPC group, when compared with that of Sham group (20.2±3.5/HPF; 14.8±4.3/HPF; 4.03±1.62/HPF, respectively). Compared to kidneys subjected to I/R only, pretreated kidneys with 20-min-ischemia displayed a marked increase in proliferating proximal tubule cells (35.5±5.4/HPF vs. 20.1±4.0/HPF, P＜0.001). 3) In rats subjected to I/R injury, ED-1~+ cells markedly accumulated into the tubulointerstitium, and increased to 25.3±3.5 cells/HPF at 24 h post-ischemia. Ischemic pretreatments significantly (P＜0.01) decreased the number of macrophages to 6.2±1.2 cells/HPF, accompanied by decreased concentrations of macrophage inflammatory protein-2 (MIP-2) in the preconditioned kidney. 4) Furthermore, our results showed that both protein and mRNA expression of ICAM-1 and TNF-αwere significantly reduced by IPC (P＜0.05). 5) I/R injury led to increased degradation of IκB-αprotein, in correlation with increased p65 positive nucleus. This process was almost reversed by IPC. I/R-induced NF-κB binding to target oligonucleotides was almost completely blocked by administration of IPC (159.3±11.8% versus 110.2±8.8%, P＜0.05). 6) Endothelial abnormity with denaturation and swell was obvious at 24 h after I/R injury, while the integrity of vascular endothelium surrounding tubules was preserved in preconditioned kidneys. 7) Levels of endothelin-1 (ET-1) in plasma and kidney tissue were significantly decreased in IPC group in comparison with I/R group (93.64±20.66 pg/ml vs. 116.76±15.55 pg/ml, P＜0.05; 108.204±15.48 pg/mg protein vs. 136.73±4.33 pg/mg protein, P＜0.05, respectively). At the same time, increased gene expression of ET-1 in I/R group was also suppressed by ischemic preconditioning. Renal protein expression of endothelial nitric-oxide synthase (eNOS) and serum concentrations of 6-keto-PGF1αwere significantly elevated in preconditioned rats compared to that in nonpreconditioned rats (P＜0.05). 8) Hypoxia-inducible factor-1α(HIF-1α) positive cells were rare in the medulla of sham-operated rats. HIF-1αpositive tubule cells were increased in kidneys of I/R group, distributing mainly in the inner medulla or along damaged area in the outer medulla. Delayed IPC further increased the expression and distribution of HIF-1αespecially in proximal tubules of the outer medulla. The distribution of heme oxygenase-1 (HO-1) in kidneys of experimental groups was similar to that of HIF-1α.Conclusions: 1) Delayed IPC has the ability to ameliorate tubule cell death and enhance tubule cell proliferation and recovery. 2) Ischemic pretreatment can suppress NF-κB-associated inflammation caused by renal I/R injury. 3) IPC can attenuate vascular dysfunction, through preserving the integrity of endothelial cell and regulating the release of endothelium-derived active factors. 4) Enhanced ischemia tolerance by delayed IPC may be attributed to higher levels of HIF-1αin the outer medulla. Taken together, our results indicate that delayed ischemic preconditioning offers both functional and histological protection on the kidney, which may be related to its ability to tilt the balance of tubule cell fate toward survival, suppress inflammatory response, and improve renal microvascular function.
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