| PART 1: The Role of Hypoxia in Renal Injury of UUO Rat ModelBackground and objective: Progressive renal diseases (PRD) are a group of diseases with diverse etiologies defined by the fact that there is a progressive decline in renal function despite the elimination or amelioration of the initiating stimulus. Although many progresses have been achieved in recent years, the mechanisms underlying PRD are not very clear. At the end of last century, Fine and his colleagues proposed the "chronic hypoxia hypothesis" which states the chronic oxygen deprivation to the tubulointerstitial compartment is the underlying reason for the scarring process and that the basis for this is compromise of the postglomerular capillary circulation. Many evidences have shown that tubulointerstitial injury and fibrosis are associated with capillary loss and hypoxia is indeed exists in different stages of several PRD models like the remnant kidney. We propose that hypoxia also exists in the obstructed kidney of unilateral ureteral obstruction (UUO) rat model and in early stage it could induce the initial renal injury and in late stage it could induce the renal scarring.Methods: Male Sprague-Dawley rats weighting about 180g were randomly assigned to eight groups with four animals in each group, including sham, 12h, 24h, 48h, 3d, 5d, 7d and 10d after UUO. With anesthesia, the left ureter was ligated to establish UUO rat model. At each time point, the blood and urine samples were collected to test the biochemical parameters and the ligated kidneys were collected to examine the inflammatory cell infiltration, interstitial fibrosis, hypoxia, transforming growth factor beta-1 (TGF- β1), vascular endothelial growth factor (VEGF) and its receptors Flk-1(VEGF-R2), Flt-1(VEGF-R1) with histology, immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and western blot techniques.Results: There were no significant differences in body weight of different group rats. It was found that serum creatinine (Scr) (values of sham, 3d, 5d, 7d and 10d were 40.6 ±2.4, 65.6+12.3, 55.5±5.9, 56.7 + 2.0, 72.5±7.0 u mol/L ) significantly increased from 3d after UUO (p<0.01), while blood urea nitrogen (BUN)( value of sham, 3d, 5d, 7d and lOd were 4.33 + 0.57, 5.90 ±1.48, 5.58 ±1.48, 7.50 + 0.42, 8.80 ± 1.31mmol/L ) significantly increased from 7d (p<0.01), same as the total serum cholesterol (value of sham, 3d, 5d, 7d and lOd were 2.04 + 0.23, 2.09 + 0.24, 1.97± 0.14, 2.60 ±0.20, 2.66 + 0.29mmol/L) (p<0.05). However, there were no difference in24h urine protein excretion (value of sham, 3d, 7d and lOd were 6.33 + 2.28, 8.06± 2.45, 5.37 + 2.06, 9.05±3.10mg/24h) and blood pressure at each time point. ED-1 positive cell infiltration of the cortex and medulla interstitium was found to increase from 12h after ligation and there were significant increase of ED-1 positive cells' number in every high power field at 3d and 7d (p<0.01). The mRNA level of III type collagen was also up-regulation and kept in high level from 3d after UUO which was about 6-7 times higher than sham group. It was shown in Masson stain that fibrosis in interstitium of obstructed kidney became severe with time going. Hypoxia detected by Hypoxyprobe was found to significantly expand from 12h after ligation, reach maximum at 3d and persist until 1 Od. Hypoxia mainly focused on tubular epithelium cells. The mRNA level and protein expression of TGF- 3 1 began to increase from 48h after ligation and became higher with time. The increased expression of TGF- P 1 also distributed in tubular epithelium cells. There were basic expression of the mRNA and protein of VEGF, Flk-1 (VEGF-R2), and Flt-1 (VEGF-R,) in sham kidney. The mRNA level and protein expression of VEGF increased from 12h after ligation and reached maximum at 3d, then both of them began to go down gradually. The mRNA level and protein expression of Flk-1 (VEGF-R2) had the same trend as VEGF. The mRNA level and protein expression of Flt-1 (VEGF-R 1) increased from 24h after ligation, reached maximum at 3d and kept in high level until lOd. The expression changes of VEGF, Flk-1 (VEGF-R2) and Flt-1 (VEGF-Ri) also mainly focused on tubular epithelium cells. In 7 days after ligation, in the obstructed kidney, hypoxia demonstrated a positive correlation with the infiltrated ED-1 positive cell number (r=0.755, PO.05), with the score of interstitial fibrosis (r=0.821, PO.05), with protein expression of TGF-pi (r=0.834, PO.05), with protein expression of VEGF (r=0.850, PO.05), with protein expression of Flk-1 (VEGF-R2) (r=0.966, PO.01), with protein expression of Flt-1 (VEGF-Ri) (r=0.838, PO.05).Conclusion: Our data shows that the renal function of UUO rat model has a slight decrease in 10 days, but there is little influence on 24h urine protein excretion and blood pressure. Inflammatory cell infiltration and interstitial fibrosis are marked characters of UUO rat model. Hypoxia indeed exists in the obstructed kidneys and demonstrates a strong correlation with the infiltrated ED-1 positive cell number, with the score of interstitial fibrosis, with protein expression of TGF-(31, VEGF, Flk-1 (VEGF-R2) and Flt-1 (VEGF-Ri). Hypoxia maybe involves initiating the renal injury of UUO and promoting the injury process.PART 2: The Renoprotective Role of Losartan, Hirudin or Rapamycin in Renal Injury of UUO rat model------The Influence on HypoxiaBackground and objective: In the first part, it has been shown that hypoxia indeed exists in the obstructed kidneys and demonstrates a strong correlation with the number of infiltrated ED-1 positive cell, with the score of interstitial fibrosis, with protein expression of TGF-pl, VEGF, Flk-1 (VEGF-R2) and Flt-1 (VEGF-R|). So, hypoxia maybe involves initiating the renal injury of UUO and promoting the injury process. In this part, we will continue to find more evidence to confirm the role of hypoxia in the renal injury of UUO rat model. We choose several drugs used widely in clinical practice and we presume that they maybe improve hypoxia of ligated kidney in UUO rat model. These drugs include angiotensin II receptor 1 antagonist - losartan, specific and selective inhibitor of thrombin -hirudin and immunosuppressive agent -rapamycin. We will investigate their influence on hypoxia, infiltrated ED-1 positive cell, interstitial fibrosis, TGF-pl, VEGF, Flk-1 (VEGF-R2) and Flt-1 (VEGF-R,) in the obstructed kidneys of UUO rat model.Methods: Male Sprague-Dawley rats weighting about 180g were randomly assigned to eight groups with four animals in each group, including 3d after ligation without treating, 3d treated with losartan (SOmg.kg^.d"1), 3d treated with hirudin (1.5mg.kg''.d''), 3d treated with rapamycin (0.2mg.kg''.d"'), 7d after ligation without treating, 7d treated with losartan (50mg.kg'1.d"1), 7d treated with hirudin (1.5mg.kg"'.d'1) and 7d treated with rapamycin (0.2mg.kg"'.d"'). With anesthesia, the left ureter was ligated to establish UUO rat model. At each time point, the blood and urine samples were collected to test the biochemical parameters and the ligated kidneys were collected to examine hypoxia, the inflammatory cell infiltration, interstitial fibrosis, transforming growth factor beta-1 (TGF- 3 1), vascular endothelial growth factor (VEGF) and its receptors Flk-1 (VEGF-R2), Flt-1 (VEGF-R,) with histology, immunohistochemistry, reverse transcription polymerase chain reaction(RT-PCR) and western blot techniques.Results: There were no significant differences in body weight of different group rats. Comparing with untreated group, in losartan-treat group, there were not significant changes in the value of Scr, BUN and 24h urine protein excretion, while there were significant decrease in the blood pressure at 3d (116 + 4.8 vs. 101 ±6.3 mmHg, p<0.01) and 7d (114±4.8 vs.96±4.8 mmHg, p<0.01) after ligation. In the obstructed kidneys of losartan-treat group, there were significant improvement of hypoxia at 3d (p<0.05) and 7d (p<0.01), significant reduce of the infiltrated number of ED-1 positive cell at 7d (p<0.01), significant down-regulation in the mRNA level of type III collagen at 3d (pO.Ol) and 7d (p<0.01), significant decrease in the score of interstitial fibrosis at 7d (p<0.05), significant decrease in the mRNA level and protein expression of TGF- 3 1 at 3d (p<0.05, pO.Ol) and 7d (pO.Ol, p<0.05), significant decrease in the mRNA level and protein expression of VEGF at 3d (both pO.Ol), while slight increase in the mRNA level (pO.Ol) and protein expression (p>0.05) of VEGF at 7d, significant increase in the mRNA level and protein expression of Flk-1 (VEGF-R2) at 3d (p<0.01, pO.Ol) and 7d (pO.Ol, p<0.01), significant decrease in the mRNA level and protein expression of Flt-1 (VEGF-Ri) at 3d (pO.Ol, pO.Ol) and 7d (pO.Ol, p<0.05). Comparing with untreated group, in hirudin-treate group, there were significant decrease in the value of Scr (56.7+2.0 vs.51.8±3.4 u mol/L, p<0.05), BUN (7.50 ± 0.42 vs.5.75±1.22 mmol/L, p<0.05) and total serum cholesterol (2.60±0.20 vs.2.07 + 0.24 mmol/L, p<0.05) at 7d after ligation. In the obstructed kidneys of hirudin-treat group, there were significant improvement of hypoxia at 3d (pO.Ol) and 7d (p<0.05), significant reduce in the infiltrated number of ED-1 positive cell at 3d (p<0.05) and 7d (pO.Ol), significant down-regulation in the mRNA level of type III collagen at 3d (pO.Ol) and 7d (pO.Ol), significant decrease in the score of interstitial fibrosis at 7d (p<0.01), significant decrease in the mRNA level and protein expression of TGF- P 1 at 3d (pO.Ol, pO.Ol) and 7d (p<0.01, p<0.01), significant decrease in the mRNA level and protein expression of VEGF at 3d (both p<0.01), while significant increase in the mRNA level and protein expression of VEGF at 7d (both p<0.01), significant increase in the mRNA level and protein expression of Flk-1 (VEGF-R2) at 3d (p<0.01, p<0.01) and 7d (pO.Ol, p<0.01), significant decrease in the mRNA level and protein expression of Flt-1 (VEGF-R,) at 3d (pO.Ol, pO.Ol) and 7d (p<0.01, pO.Ol). Comparing with untreated group, in rapamycin-treat group, there were not significant changes in the value of Scr, BUN and 24h urine protein excretion. In the obstructed...
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