The Inhibitory Role On Fibroblast Of Proliferative HKC At G1/S Phase Arrested By Hypoxia

Renal failure, one of the most seriously clinical severe cases, which is leadby kinds of acute or chronic renal damage, is the most important problem onkidney research. And the main factor leading renal failure is renal tubule atrophyand interstitial fibrosis, which are closely correlated with non-adaptive renaltubule rebuilt. So if we make clear with the mechanism about regeneration andreparation of RTEs, then we can find a way to treat with renal tubule atrophy andinterstitial fibrosis, in order to prevent our kidney from failure.The growth of kidney is controlled by cell cycle choose. There are cellspopulation which are proliferative, hyperplasive, apoptosis or differentiated innormal renal tubular. The renal tubule grows when there are more proliferativecell population. G1/S phase is the most important key point in cell progress. Thereis little cell proceeding in mature kidney but more when they are in pathologystate. The cell progress is happened as in embryonic times. For example, in the early state when ischemia or anoxemia happened, RTEs can be synchronized toenter into cell cycle and arrested at G1/S phase to choose to proliferate orapoptosis. The"healthy cells"can go through the G1/S point and enter into cellcycle and synthesis DNA. So we conclude that the proliferating RTEs arrested atG1/S phase by hypoxia can lead all tubular cells to proliferate and develop. At thesame time, they can inhibit the growth of the fibroblast in kidney. So our researchare planed to co-culture the proliferating RTEs arrested at G1/S phase by hypoxiaand the fibroblast, and then research the role of inhibition by RTEs to prove ourdeduce. It is the basis to research and prevent the renal failure at final stage byrenal tubule atrophy and interstitial fibrosis.【AimAims】Our research are planed to co-culture the proliferating RTEs arrested at G1/Sphase by hypoxia and the fibroblast, and then research the role of inhibition byRTEs to prove that the proliferating RTEs arrested at G1/S phase by hypoxia caninhibit the growth of the fibroblast in kidney, and lead all tubular cells toproliferate and develop.【Methods】1.Research for synchronization at G0/G1 phase by serum starvation to human renal tubular epithelialcells.Find out the suitable concentration and time for synchronization of HKC cellat G0/G1 state by the methods of serum starvation. The results were verified byimmunocytochemistry and FCM.2. Research for the arrest of RTEs at G1/S phase by hypoxia.Hypoxia arrested the G0/G1 HKC cells at G1/S phase by culture for 12~16 h.The results were verified by FCM, immunocytochemistry, Western blot and electron microscope.3. Research for the inhibition roles to fibroblast by proliferating RTEs arrestedat G1/S phase by hypoxia.Co-culture fibroblast with the proliferating RTEs arrested at G1/S phase byhypoxia through Transwell co-culture system. Check the change of expression ofPCNA and Cyclin D1 in WI-38 by Western blot.【Results】1. Synchronization by DMEM with 0.1% FBS for 48 h is the best condition.We synchronized the HKC for 24 h or 48 h or 72 h with the concentrations ofFBS, 0% or 0.1%. By FCM, we found that after culture with 48 h at 0% or 0.1%concentration, the proportion of G0/G1 phase is(90.9±0.9)% or(93.6±2.0)%,which has markedly differences with control group. Results from MTT told usthat synchronized HKC by DMEM with 0.1% FBS for 48 h could less effect thegrowth of HKC than with 0% FBS. The results were further verified byimmunocytochemistry of Brdu.2. HKC were arrested at G1/S phase by hypoxia after culturing for 12~16h.By FCM, we found that majority of HKC, after synchronized at G0/G1 phaseby serum starvation, enter into G1/S after cultured in hypoxia for 12~16 h. Theresults were verified by the expression of cdk2 and Cyclin E. The proliferation ofthe HKC was verified by immunocytochemistry of PCNA. Electron microscopeshow that there aren't obviously apoptosis happened.3. Fibroblast can be inhibited by the proliferating RTEs arrested at G1/S phase by hypoxia. The results of Western blot indicate that the expression of PCNA and CyclinD1 are lower in fibroblast (WI-38) which are co-culture with HKC arresterd byhypoxia after synchronizated. But the expression of PCNA and Cyclin D1 havelittle charge in fibroblast (WI-38) when were co-cultured with the undisposedHKC. The results indicated that the growth of fibroblast is inhibited after coculturingfor 2 h or 4 h with the proliferating RTEs which were arrested at G1/Sphase by hypoxia.【Conclusion】After synchronized at G0/G1 phase by serum starvation, the proliferatinghuman renal tubular epithelial cells which was arrested at G1/S phase by hypoxiacan inhibit the the growth of fibroblast.