Study Of The Effect Of Human Umbilical Mesenchymal Stem Cells On Mice With FSGS Based On Serum Metabolomic

BackgroundFocal segmental glomerulosclerosis(FSGS)is the primary glomerular disease(PGN)most likely to lead to end-stage kidney disease(ESKD).Prevalence of FSGS is rising with the years,while only 40-60%patients have achieved a complete or partial response by the immunosuppressive therapy,and the current therapies are also faced with difficulties and challenges,constrained by the dose and toxicity.Finding a new therapy to prevent FSGS progression is a top priority in health care.Cord mesenchymal stem cells(MSCs)with anti-inflammatory,paracrine,regenerative and other characteristics contribute to the repair of kidney injury,and have shown their potential for the treatment of acute kidney injury(AKI),kidney transplant,diabetic nephropathy,lupus nephritis and other areas,thus becoming a hotspot of research in kidney diseases.Therefore,MSCs is expected to be a new strategy for the treatment of refractory FSGS.Umbilical cord-derived mesenchymal stem cells(HUMSCs)are abundant in source,easily available,and have the better anti-inflammatory ability and other characteristics.Thus,HUMSCs is an ideal mesenchymal stem cell with therapeutic potential.Metabonomics can reflect the real-time events actually occurring in an organism,and also capture the external influence factors such as diet and intestinal flora,showing a unique advantage in the research of the markers and mechanism of kidney diseases and providing a new and broad perspective on how various kidney diseases affect the metabolites.Metabonomics has been applied to different kidney diseases,but the serum metabolic profile of FSGS has not been extensively studied,and no studies have been found on the changes of serum metabonomics after the intervention of HUMSCs on ADR-induced FSGS mouse models.ObjectiveIn this study,we used an ADR-induced FSGS mouse model to observe the efficacy of HUMSCs on FSGS mice and explored the mechanism of kidney repair.Using the UPLC-MS/MS-based metabonomics,we also analyzed the changes of serum metabolites after the intervention of HUMSCs on ADR-induced FSGS mouse models,explored the possible metabolic mechanism involved in the onset of FSGS,and analyzed the role of HUMSCs in protecting FSGS from the metabonomics perspective.MethodsIn the first part of this research,30 Balb/c male mice were divided into the normal control group(N=12)and the FSGS model group(N=18).The FSGS model group was slowly injected with ADR(2 mg/mL)at a dose of 10.5 mg/kg via caudal vein,and the normal control group was injected with an equal dose of 0.9%normal saline via caudal vein at the same time point.After the FSGS mouse models were established,six mice in the normal group and six mice in the model group were sacrificed,and then the serum creatinine and urea nitrogen at day 28 and the body weight and urine protein at day 0,8,15,21 and 28 were tested and compared of two groups.Kidney tissues were stained with HE,PAS and Masson,and the results were observed under a light microscope.The remaining 18 mice were divided into three groups:the normal control group(N=6),and the 12 mice in the FSGS model group were randomly divided into the FSGS+DiR-HUMSCs group(N=6)and the FSGS+Siane group(N=6).The normal control group and the FSGS+DiR-HUMSCs group were slowly injected with DiR-HUMSCs(2×106cell/mL)via caudal vein at day 29,30 and 31,respectively.The FSGS+Siane group was injected with an equal dose of 0.9%normal saline at the same time point;after 6 h,all rats were sacrificed,and the hearts,lungs,kidneys,spleens and livers were removed and observed under an animal imaging system for the bio-distribution of DiR-HUMSCs in mice.Kidney tissues were IHC stained with HE,PAS and Masson,and the results were observed under a light microscope.Frozen sections of kidney tissues were stained with 4',6-diamidino-2-phenylindole,dihydrochloride(DAPI)fluorescence.Paraffin section of kidney tissues were IHC stained with human and mouse mesenchymal stem cell specific anti-CD90 and anti-CD44 antibodies,and the position of HUMSCs in the kidney tissue was observed.In the second part of this research,The 18 Balb/c male mice were divided into three groups:Normal+Saline(N=6),FSGS+Saline(N=6),and FSGS+HUMSCs(N=6).FSGS+HUMSCs group:after successful modeling,FSGS mice were slowly injected with HUMSCs(2×106 cells/mL)via caudal vein at day 29,32 and 35,respectively;FSGS+Saline group:after successful modeling,FSGS mice were slowly injected with an equal dose of 0.9%normal saline via caudal vein at day 29,32 and 35,respectively;Normal+Saline group:the mice were injected with an equal dose of 0.9%normal saline via caudal vein at day 29,32 and 35,and were sacrificed at day 43.The serum of all of mice was stored in a refrigerator at-80? for metabolomics study.Kidney tissues were stained with PAS and Masson.Levels of urine protein,serum creatinine,urea nitrogen and TNF-? in the three groups were detected by ELISA;BMP-7 expression in the kidney tissue was detected by RT-PCR;TGF-?1 and FN expression in the kidney tissue was detected by IHC and semi-quantitatively scored,and the results were observed under a light microscope.In the third part of this research,the serum of 17 mice in Normal+Saline(N=6),FSGS+Saline(N=5)and FSGS+HUMSCs(N=6)is used for metabolomics research.After serum proteins were precipitated by methanol,serum samples used for metabonomics study were prepared.The serum metabolites of the mouse in three groups are detected based on the metabolic analysis of the widely targeted metabolome technology.We used a combination of UPLC-MS/MS detection platform and multivariate statistical analysis to study the serum metabolome differences among the three groups.ResultsResults in the first part of this research(1)At day 28,the urine protein increased,body weight decreased,and serum creatinine and urea nitrogen increased in the FSGS model group mice,compared with those of the normal group mice,and there was a statistical difference(P<0.05).HE,PAS and Masson staining results of kidney tissues in FSGS mice showed severe hyperplasia of mesangial cell and matrix,and adhesion of some balloons with segmental sclerosis.Brush border of epithelial cells of kidney tubules was exfoliated,and lumen was dilated accompanied by the formation of many protein casts.Those results proved that the FSGS mice model was successfully established.(2)The kidney of mice in the FSGS+DiR-HUMSCs group under the animal imaging system showed a strong red fluorescence distribution,which suggested DiR-HUMSCs could migrate to the kidney of the FSGS mice.(3)Human mesenchymal stem cell specific antibodies CD90+and CD44+cells could be observed in the kidney tissue of the FSGS+DiR-HUMSCs group mice,but these were not observed in the FSGS+Siane and Normal+DiR-HUMSCs group.Compared with the FSGS+Siane group mice,the mouse mesenchymal stem cell specific antibodies CD90+cells in renal tissues of the FSGS+DiR-HUMSCs group mice were slightly increased.Results in the second part of this research(1)Two weeks after HUMSCs transplant in FSGS mice,compared with the FSGS model group,the levels of the urine protein,serum creatinitine and urea nitrogen were significantly decreased,and there was a statistical difference(all P-values<0.01).(2)Compared with the FSGS+Saline group,the pathological injury of kidney tissues in the FSGS+HUMSCs group mice was significantly improved.(3)The BMP-7mRNA expression level of kidney tissue significantly was decreased,the TGF-?1 and FN expression levels significantly were increased,and the serum TNF-? level was significantly increased in the FSGS+Saline group,and there was a statistical difference between the FSGS+Saline group and the Normal+Saline group(all?-values<0.01).(4)Two weeks after HUMSCs transplant in FSGS mice,the BMP-7 level was significantly increased,the TGF-?1 and FN expression levels were decreased,and the serum TNF-? level was significantly decreased in the FSGS+HUMSCs mice,and there was a statistical difference between this group and the FSGS+Saline group(all P-values<0.01).Results in the third part of this research(1)The trend of serum samples separation in Normal+Saline,FSGS+Saline and FSGS+HUMSCs group was obvious.(2)Compared with the Normal+Saline group,among the serum metabolites of ADR-induced FSGS mice,the levels of Trimethylamino oxide,3'-Sialyllactose,N-Acetyl-valine,3-Chloro-L-tyrosine and Bile acid metabolites were significantly up-regulated,and the levels of serum palmitoleic acid and L-thyroxine were significantly down-regulated.(3)After the transplant of HUMSCs in FSGS mice,compared with the FSGS+Saline group,the levels of L-thyroxine and palmitoleic acid were significantly up-regulated,and the level of 3'-Sialyllactose was significantly down-regulated.Those abnormal circulating metabolites by the regulation of HUMSCs were near the normal level.(4)Branched chain amino acid mechanism,synthesis of unsaturated fatty acids,thyroid hormone metabolism and bile metabolic pathways were involved in the occurrence and development of ADR-induced FSGS and the protection of HUMSCs on FSGS.(5)The serum Trimethylamino oxide and 3'-Sialyllactose were positively correlated with the level of inflammatory factor TNF-? in FSGS+Saline group(all P-values<0.05).(6)There was a significant positive correlation between serum 3'-Sialyllactose and the urinary protein and urea nitrogen levels in FSGS+Saline and FSGS+HUMSCs groups mice(all P-values<0.05).Conclusion(1)HUMSCs can be homing to the injured kidney tissue of mice with ADR induced FSGS.(2)HUMSCs can regulate the expressions of pro-inflammatory TNF-? and pro-fibrotic factors TGF-?1 and anti-fibrosis factor BMP-7,thereby inhibiting the immuno-inflammatory response and extracellular matrix of kidney tissue,which could improve the function and structure of kidney of FSGS mice and protect the FSGS.(3)In the UPLC-MS/MS-based metabonomics study,we identified a group of serum differential metabolites(Trimethylamino oxide,3'-Sialyllactose,N-Acetyl-valine,3-Chloro-L-tyrosine,Bile acid metabolites,Palmitoleic acid and L-thyroxine)involved in the occurrence and development of ADR-induced FSGS.Branched-chain amino acid metabolism,bile acid metabolism and synthesis,and thyroxine metabolic pathways are involved in the occurrence and development of FSGS,HUMSCs may reverse the abnormal metabolic pathways by regulating the levels of L-thyroxine,Palmitoleic acid and 3'-sialyllactose in the circulation,which could inhibit the immunity of the kidney tissue Inflammation.Our findings may provide a new metabolic mechanism and preclinical data of HUMSCs therapy for FSGS.