Effect And Activation Of HIF-α Isoforms In The Progression Of Rat Remnant Kidney Model

PART 1 The dynamic change of H1F and its target genes in the remnant kidney modelBACKGROUNDChronic kidney disease(CKD) is a worldwide health problem,and the number of patients with CKD is increasing rapidly.Many clinical observations suggest chronic hypoxia as a final common pathway in the progression of chronic kidney disease leading to eventual kidney failure.Besides,accumulating evidence also suggests a crucial role for hypoxia in the tubulointerstitium prior to structural microvasculature damage in the corresponding region,emphasizing the pathogenic role of this condition from an early stage of kidney disease,but the situation of hypoxia at the end stage of CKD was unavailable.One of the most important factors in the cellular response to hypoxia is hypoxia-inducible factor(HIF).The steady and high expression of HIF is an important reaction and adaptation to low PO₂.Prolyl 4-hydroxylases domain(PHD) is the key regulator on the degradation of HIF-αisoforms.Two family members of PHD,PHD2 and PHD3,have also been reported to be HIF targeted genes,and they might have a feedback down-regulation of HIF-αwhich then levels off at a novel set point under chronic persistent hypoxia condition. We set this research in order to study the hypoxia condition in the end stage of RK,a well-characterized model of CKD,and test whether there is a novel set point of PHD-HIF-oxygen-sensing system in the progression of CKD.METHODSMale Sprague-Dawley rats weighing 180-200g were used in this study.The chronic kidney disease model was established with 5/6 subtotal nephrectomy(5/6 NX) operation.Rats met the enrolled criteria were randomly divided into 1wk,2wk,4wk, 6wk,8wk and 12wk groups at the first weekend after removing the right kidney.At the end of 1,2,4,6,8 and 12 wk after operation,the corresponding group rats were sacrificed and blood and kidney samples were taken.Besides,their blood pressure and 24 hour urine were also taken before sacrifice.We then compared their blood pressure, renal function,24h urine microalbumin and renal pathological injure,as well as mRNA and protein levels of HIF-1α,HIF-2αand their target genes like VEGF,HO-1, Glut-1,EPO and PHD2,PHD3 in the remnant kidney cortex among the above groups.RESULTS1.A short period of acute renal dysfunction was followed by a more stable period of chronic renal failure with values of serum creatinine between 66.0 to 117.4μmol/L.RK animals also developed proteinuria,which was significantly greater than control after week 1.The tail systolic blood pressure of RK rats rose soon after renal mass reduction,peeked at week 8,and fell a little after then,yet was still higher than control animals.2.RK cortex had marked mesangial expansion and sclerosis in the glomerulus after week 4.Tubules acquired a hypertrophied appearance starting at week 1,and foci of tubular atrophy,dilatation,and interstitial fibrosis developed at week 4,and gradually aggravated thereafter.3.Tubulointerstitial hypoxia started as early as week 1,becoming more pronounced at week 4 and 6,and still presented in the advanced stage of RK when the interstitial fibrosis and tubular atrophy were severe.4.Peritubular capillaries were narrowed and/or distorted as early as week 1, dramatically reduced in week 8 and 12,and these changes were particularly prominent in areas of interstitial expansion and tubular atrophy where pimonidazole could not be absorbed anymore.5.In RK cortex,HIF-1αwas expressed only in the atrophic and dilated tubular cells. Staining for HIF-2αhad no overlap with HIF-1α:most of the positive cells were endothelial cells,including glomeruli and peritubular endothelial cells;some of them were peritubular fibroblasts;a few of them were vascular smooth muscle cells of interlobular artery.6.The semiquantitative result of imunohistochemistry and western blot revealed that HIF-1αand HIF-2αwere both gradually up-regulated during the early stage of RK, peaked at 4 and 6wk,and then gradually down-regulated,but their mRNA levels were not significantly altered during the RK course.7.VEGF mRNA was lower in RK than in CTL kidney except for week 4 group.The expression of HO-1 was increased soon after renal mass reduction,returned to normal at week 2 and again increased at week 4 and 6.EPO expression was stronger in RK than in CTL kidney especially in week 4 and 6 while Glut-1 was stronger in RK than in CTL kidney before week 6.The imunohistochemistry results for VEGF,HO-1 and Glut-1 were identical with the mRNA results.8.PHD2 and PHD3 mRNA levels were detectable as early as week 1 after renal mass reduction,peeked at week 4 and 6,and remained elevated over the entire course of RK,which was the same as the protein levels of PHD2 and PHD3.CONCLUSIONS1.The indexes of CKD such as blood pressure,proteinuria,BUN and Scr are detcriorous with the progression of disease in 12 weeks' observation time alter renal mass reduction,especially in week 12,when tubular atrophy and interstitial fibrosis are extremely serious.Thus 12 weeks' follow-up-time can well observe the progression of CKD.2.Hypoxia exists through the whole stage of CKD,especially in foci of proliferative and hypertrophic tubules in week 4 and week 6,and even in the end stage of CKD, hypoxia still persists.3.During the progression of CKD,there might bca dynamic change of HIF-αisoforms resulting from the persistent hypoxia environment and PHD feedback adaptive mechanism.4.The physiological significance of the new set HIF-PHD oxygen-sensing circuit in the progression of CKD deserves further investigation. PART 2 The effect and mechanism of L-mimosine on remnant kidney rat modelBACKGROUNDOne of the most important factors in the cellular response to hypoxia is hypoxia-inducible factor(HIF).The high expression of HIF is an important reaction and adaptation to low PO₂,and so might retard the progression of CKD.Cobalt is the traditional drug in studying the biological mechanism of HIF.It was reported that rat pretreated with cobalt had good prognosis after I/R injure.Besides,cobalt also had different extent of morphological protection on CKD.Unfortunately,the possible benefits of cobalt given in clinical practice to CKD patients are sharply limited by its toxicity.PHDs inhibitor can effectively stabilize and activate HIF without oxygen dependent.L-mimosine is just a PHDs inhibitor,which has little side effect and has been confirmed to reduce the kidney I/R injure by increasing the expression of HIF-αin kidney.We thus chose L-Mim to study the effect of PHDs inhibitor on CKD.In our previous study we found that HIF-αisoforms presented a low-high-low dynamic change and then reached a new set point of HIF-PHD-oxygen systerm at last during the persistent hypoxia condition in RK model,which suggested that different administration period of PHDs inhibitor might have different effect on retarding the progression of CKD.Meanwhile,recent studies suggest that HIF activation promotes epithelial to mesenchymal transition(EMT) and renal fibrogenesis.In the present study,we together investigated whether long-term HIF activation had side effect on CKD.METHODSMale Sprague-Dawley rats weighing 180-200g were used in this study.The chronic kidney disease model was established with 5/6 subtotal nephrectomy(5/6 NX) operation.Rats met the enrolled criteria were randomly divided into early treatment group(treatment phase:1wk-12wk),progression treatment group(treatment phase: 5wk-12wk) and advanced treatment group(treatment phase:9wk-12wk) at the first weekend after removing the right kidney.At the end of week 12 after operation,the corresponding group rats were sacrificed and their blood and kidney samples were taken.Besides,their blood pressure and 24 hour urine were also taken before sacrifice. We then compare their blood pressure(Bp),renal function(BUN and Scr), hemoglobin(Hb),24h urine microalbumin(U_alb),renal pathological injure and hypoxia condition,as well as protein levels of HIF-1α,HIF-2αand their target genes like VEGF,HO-1,Glut-1,PHD2,PHD3 and fibrosis markers collagenâ…¢,α-SMA, TGF-β1 and macrophage cells infiltration in the remnant kidney cortex among the above groups.RESULTS1.Early treatment group had no difference with control group in BUN,Hb,U_alb and Bp,but had a higher Scr and a more serious condition in tubular atrophy, dilatation and interstitial fibrosis.Progression treatment group had lower BUN, Scr and Ualb,higher Hb and less server tubular interstitial injure than control. There was no significant difference in the above parameters between advanced treatment group and control group.2.In early L-Min treated rat,the atrophic and dilated tubular had more HIF-1αexpression than control ones,especially in interstitial fibrosis area;the early treatment group also had a higher expression of HIF-2αin the same place,and the main expression cells were interstitial cells.The progression treatment group also had stronger nuclear staining of HIF-1αand HIF-2αthan control group in the same place and the same cells as the early group,but the expression was a little less.There was no expression difference of the above two isoforms between advanced treatment group and control group.Western blot analysis confirmed the above result.3.Both the early and progression treatment groups had higher expression of PHD2 and PHD3 than control group,especially the former,while no expression difference between the advanced group and control group.4.The immunostaining of HIF targeted genes,VEGF,HO-1 and Glut-1,were stronger than in control ones after progression L-Min treatment.Early L-Min treated rats only had increased expression of Glut-1.These targeted genes had no difference between the advanced group and control group.5.The expressions of collagenâ…¢,α-SMA and TGF-β1 were higher in Early L-Min treated group and lower in progression treated group than control ones,but the difference was not significant between the advanced group and control group.6.The peritubular capillary network evaluated using CRI was restored by progression L-Min treatment and no difference after early and advanced L-Min treatment.7.The pimonidazole positive staining area was larger in early treatment group than in control group.Although the positive area was also larger in progression treatment group than in control group,it had no difference with the one in early treated group.Also there was no difference between the advanced group and control group in the positive area.8.Compare with control group,the number of ED-1 positive cells was larger in early treatment group,smaller in progression treatment group and no difference in advanced treatment group.CONCLUSIONS1.Early L-Min treatment(1wk-12wk) may deteriorate the renal function of CKD and aggravate tubular interstitial fibrosis due to the selective expression of fibrosis related genes after long-tern high expression of HIF-αisoforms.2.Progression treatment(5wk-12wk) can improve renal function and anemia, decrease albuminuria and tubular interstitial injure of CKD,which is resulted from the secondary expression of protective HIF target genes,VEGF and HO-1,etc, after L-Min treatment at a proper time.3.The end stage renal injure is irreversible,thus advanced L-Min treatment can not retard the progression of CKD and also has no effect on HIF-αisoforms and their targeted genes.