The Effect Of MUTYH And Dahuang Fuzi Decoction (DFD) On Renal Fibrosis:Role Of Mitochondrial Dysfunction

Chronic kidney disease(CKD)now has been a serious public problem worldwide at present.Epidemiological investigation showed that the prevalence of CKD has been as high as 10.8%in China.In clinic,CKD mainly present proteinuria and renal function decrease which was aggravated by glomerulosclerosis and tubular interstitial fibrosis.Therefore,relieve the interstitial fibrosis means a lot to slow down the progress of CKD.In recent years,role of mitochondrial dysfunction(MtD)in the progress of CKD hasbeen investigated a lot.Researches showed that MtD increased the production of reactive oxygen species(ROS),promoted epithelial-mesenchymal transition(EMT)and stimulated transforming growth factor-β(TGF-β)releases which in turn aggravated renal interstitial fibrosis.Then,it is important for CKD to protect mitochondrial function.Human MUTYH gene is located in the no.1 autosome.It contains 16 exons and the size of it is about 11.2Kb.Two different proteins are produced by the human MUTYH gene:type 1 protein is located on mitochondria because it has a mitochondrial targeting signal at the amino terminus while type 2 protein is located on nucleus.MUTYH is a specific DNA glycosylase which initiates BER system to repair oxidative DNA damage by recognizing 8-oxoG and removing the mispaired A with 8-oxoG.Dysfunction of MUTYH lead to a variety of tumors and nervous systerm diseases,however,the role of MUTYH on renal fibrosis and mitochondrial function has not been studied.Mitochondria is similar to "qi" in Chinese medicine.Both of them can provide energy for human activities and maintain body temperature.Dysfunction of them can cause multiple systerm diseases.Some researches showed that Chinese medicine like cynomorium and turmeric which had the function of "warming qi" could protect mitochondrial function.As a typical traditional formula that could "warming qi",the effect of Dahuang Fuzi Decotion(DFD)on renal fibrosis and mitochondrial function has not been investigated.We assume that MUTYH can decrease DNA mutations caused by oxidative stress,protect mitochondrial function and delay the progress of CKD.DFD can protect kidney function by relieving renal interstitial fibrosis and ameliorating mitochondrial dysfunction.Part 1 MUTYH deletion aggravates renal fibrosis and mitochondrial dysfunction in UUO modelObjective:To find out the influence of MUTYH deletion on renal fibrosis and mitochondrial function in UUO model.Methods:Wild type(WT)and MUTYH deletion mice were used to build up unilateral ureteral obstruction(UUO)model.Each type of them was randomly assigned to the following three groups:the UUO group for 3 days,the UUO group for 10 days and the control group.We observed the degree of renal interstitial fibrosis in UUO mice by Masson staining.The regulation of MUTYH in WT UUO mice was detected by qRT-PCR.The expression differences of α-SMA,TGF-β,collagen Ⅰ and collagen Ⅲ were determined by Western blotting and qRT-PCR.The inflammatory factors such as interleukin-1β(IL-1β),intercellular adhesion molecule 1(ICAM-1),tumor necrosis factor-α(TNF-α)and monocyte chemoattractant protein 1(MCP-1)were detected by qRT-PCR.Mitochondrial function markers like ROS production,mitochondrial DNA(mtDNA)copy numbers and mitochondrial membrane potential(MMP)were also observed.Results:(1)MUTYH was up-regulated in the WT UUO model.(2)Masson staining showed significant deposition of extracellular matrix in kidneys of WT mice with UUO.Deletion of MUTYH significantly increased tubulointerstitial fibrosis compared with WT UUO mice.Levels of the components of extracellular matrix including α-sma,collagen 1,collagen 3 and TGF-β were obviously increased in WT UUO mice,which was more severe in UUO mice with MUTYH deletion.The inflammatory factors were released much more in MUTYH deletion UUO mice than in WT UUO mice.(3)mtDNA copy numbers and MMP decreased while ROS production increased in WT UUO mice compared to normal WT mice,which was more severe in MUTYH deletion UUO mice.Conclusions:MUTYH deletion aggravates renal fibrosis and mitochondrial dysfunction in UUO model.Part 2 Overproduction of human MUTYH protects against fibrosis and inhibits mitochondrial dysfunction in TGF-β1-treated HK-2 cellsObjective:To explore the effect of human MUTYH overproduction on fibrosis and mitochondrial function in TGF-β1-treated HK-2 cells.Methods:Immortalized human kidney(HK-2)proximal tubular epithelial cells were cultured.5ng/mL of recombinant TGF-β1 was used to stimulate the cells while control group were treated with vehicle medium.For over-expression of human MUTYH,pcDNA3.1 expression human MUTYH were transfected into HK-2 cells using Lipofectamine 2000.Levels of α-SMA,collagen Ⅰ,collagen Ⅲ and MUTYH were detected by Western blotting and qRT-PCR.Elisa was used to determine the level of 8-oxoG.Meanwhile,mtDNA copy numbers,MMP and ROS were also observed.Results:(1)The qRT-PCR results showed that expression of MUTYH was increased significantly at 48h after TGF-β1 stimulated.Using western blotting,we detected two bands of MUTYH:the mitochondrial MUTYH(～57kDa)and the nuclear MUTYH(～53kDa).After TGF-β1 stimulating,expression of mitochondrial MUTYH was decreased and nuclear MUTYH expression increased at 24h.This phenomenon was more obvious at 48h.(2)Level of 8-oxoG and expressions of α-sma,collagen 1 and collagen 3 were all increased significantly after 24h stimulation.With overproduction of human MUTYH,fibrosis of HK-2 cells was improved obviously and level of 8-oxoG was decreased.(3)In TGF-β1 stimulated HK-2 cells,mtDNA copy numbers,MMP were decreased and ROS production was increased significantly.After overproduction of human MUTYH,mitochondrial dysfunction was improved obviously.Conclusions:Overproduction of human MUTYH protects against fibrosis and inhibits mitochondrial dysfunction in TGF-β1-treated HK-2 cells.Part 3 Dahuang Fuzi Decoction attenuates renal fibrosis and ameliorates mitochondrial dysfunction in chronic aristolochic acid nephropathyObjective:To study the effect of Dahuang Fuzi Decotion(DFD)on renal fibrosis and mitochondrial function in chronic aristolochic acid nephropathy(AAN).Methods:Mice were divided into the following six groups randomly:the control group,the AAN model group(AAN),the saline-treated group(AAN+Vehicle),the normal-dose DFD-treated group(AAN+NDFD),the high-dose DFD-treated group(AAN+HDFD)and the rosiglitazone-treated group(AAN+Rosi).After treating for 8 weeks,24h urine and blood samples were collected and the mice sacrificed to study the biochemical parameters associated with renal function.The samples were analyzed for renal fibrosis and mitochondrial dysfunction(MtD)markers.To achieve that,collagen Ⅲ,collagen Ⅰ,mtDNA copy numbers,MMP,ATP content and ROS production were detected.Results:(1)Proteinuria,kidney function and the renal pathological characteristics were improved by DFD and rosiglitazone.(2)The expression of collagen Ⅲ and Ⅰdecreased after treating with either DFD or rosiglitazone.(3)Mitochondrial dysfunction represented as decrease in mitochondrial DNA copy numbers,reduction of MMP and ATP content,increase in ROS production,was improved by DFD and rosiglitazone.Conclusions:DFD could protect against renal fibrosis and ameliorate mitochondrial dysfunction in chronic AAN mice.