Adaptive Regulation Of EPO In Adult Yak Renal Interstitial Fibroblasts-like Cells Under Hypoxia

Polycythemia is a kind of representative mountain sickness with hypoxic stimulation for long period being one of the main causes.It is not only the most common chronic mountain disease in people living on the plateau,but also one of the representative mammalian diseases that is maladaptive to high altitude environments.The yak(Bos grunniens)lives between 3000-5000 meters above sea level and adapts well to the high-altitude and hypoxic environment.There is a an increase in the number red blood cells with increasing altitude,but this is only a kind of adaptive physiological change and has not caused pathological results such as erythrocytosis.Studies in human and mouse have found that the erythropoietin(EPO),which is the core factor during the erythropoiesis is regulated by the hypoxia induced factor-2α(HIF-2α).However,studies about the EPO producing cell in the kidney and its related regulation in yak has not been reported.Because of the tissues protective effect of EPO in addition of its hematopoietic function,and the heart and lung are the most important organs in yak hypoxia adaptation,therefore,this work aims to explore the expression of EPO,HIF-1α and HIF-2α in heart,liver,lung and kidney firstly,and then to study the EPO producing cell in the yak kidney and its regulation.To achieve the aims stated above,three-part experiment was conducted and the results are as following:The expression of EPO,HIF-1α and HIF-2α in different tissues(heart,liver,lung and kidney)of adult yak were detected by qRT-PCR and immunohistochemistry.The results showed that the EPO,HIF-1α and HIF-2α could be dectected in the above tissues,but the expession of mRNA in some tissues are not consistent with its expression intensity of protein.The expression of EPO mRNA in the kidney was relatively higher than others.Also,results from immunohistochemistry analysis showed that the expression of EPO could be detected in renal collecting tubule epithelial cells,renal tubular epithelial cells,and peritubular interstitial cells.The expression of HIF-1α and HIF-2α had some overlapping positions with EPO,and these results are consistent with findings in humans and mice,which implies that the regulation of EPO in yak kidney may be related to HIF-1α and HIF-2α too.In addition to the kidney,the high expression of EPO,HIF-1α and HIF-2α could also be dectected in the lung and heart,it is not means the tissues protective effect of EPO only,but also indicates that the two organs are likely to be the most critical organs for the adaptation of the yak to hypoxia.Different methods were used to separate and culture the renal tubule epithelial cells(RTECs)and renal interstitial fibroblast-like cells(RIFs).The result shows that,digestion with combined enzyme(1 mg / mL collagenase I and 1 mg / mL collagenase II)could obtain cells in the shortest time and with best proliferation capacity.The identification results showed that the purified RTECs had a classic cobblestone appearance,the expression of cytokeratin-18 is postive,and negtive for vimentin and CD31.These characteristics are consistent with those found in humans and mice.While the purified RIFs had an irregular triangle shape or spindle-shaped appearance,it was positive for vimentin and negtive for cytokeratin-18 and α-SMA.These results indicate that the morphological and biochemical characteristics of RTECs and RIFs are consistent with the typical renal tubular epithelial cells and renal interstitial fibroblastlike cells.To further investigate the cell that produces EPO in yak kidney and its regulation mechanism,the RTECs and RIFs were treated with dimethyloxalylglycine(DMOG),an inhibitor of prolyl-4-hydroxylase domain(PHD),which could inhibit the degradation of HIF and thereby simulate hypoxia.As expected,increasing the concentration of DMOG caused a corresponding increase in the expression of HIF-1α and HIF-2α in RIFs and RTECs.The expression of EPO was correspondingly increased in RIFs,but no expression of EPO could be detected in RTECs after treating with any of the concentrations of DMOG.This means that the RIFs were the EPO-producing cells in adult yak kidney,and the PHD has a negative effect during the EPO production regulation.HIF-1α siRNA and HIF-2α siRNA were used separately to disturb the expression of HIF-1α and HIF-2α in RIFs,which aims to investigate the regulatory effect of HIF-1α and HIF-2α to EPO.In the cells that were treated with HIF-1α siRNA,the expression of HIF-1α mRNA was decreased significantly,this means the expression of HIF-1α in RIFs is disturbed steady.After that,both undisturbed(name to DMOG)and disturbed(name to DMOG+HIF-1α siRNA)RIFs were treated with 200 μmol/L DMOG,and the results of WB showed that the expression of HIF-1α in group DMOG+HIF-1α siRNA was significantly lower than it in the DMOG group,while the expression of HIF-2α,EPO and EPO mRNA were basically the same in the two groups,these results indicate that the expression of EPO in RIFs was not regulated by HIF-1α.While the expression of HIF-2α mRNA was decreased significantly in the cells in which the expression of HIF-2α was disturbed,the expression of HIF-1α in the group of DMOG and DMOG+HIF-2α siRNA were basically the same,but the expression of HIF-2α,EPO and EPO mRNA in group DMOG+HIF-2α siRNA were significantly lower than that in the DMOG group.The above results indicate that the upstream regulator of EPO in RIFs was HIF-2α,and not HIF-1α,and the HIF-2α has a positive effect during the EPO production regulation..Geldanamycin(GA),which was a kind of inhibitor of heat shock protein 90(HSP90),was used later to explore the effect of HSP90 to EPO regulation in RIFs.The results show that the expression of HSP90,HIF-1α,HIF-2α,and EPO decreased in synchrony as the concentration of GA increased suggesting that EPO production in RIFs is also regulated by HSP90.In conclusion,(1)This work systematically studied the expression of HIF-1α,HIF-2α,and EPO in adult yak heart,liver,lung and kidney for the first time,the positive expression of EPO,HIF-1α and HIF-2α in renal tubular epithelial cells and peritubular interstitial cells was consistent with what found in humans and mice.The significantly expression of EPO,HIF-1α and HIF-2α in the heart and lung indicated that the two organs are likely to be the most critical organs for the adaptation of the yak to hypoxia.(2)The morphology and characteristics of isolated and purified RTECs and RIFs were basically the same as those of humans’.Hypoxic environment was simulated by DMOG addition,it was revealed that the cell with the ability to produce EPO was the RIF but not RTEC.(3)The PHD had a negative effect on the EPO regulation via HIF-2α,and the expression of HSP90 had a positive effect on the regulation of EPO.