Single-cell RNA Sequencing To Explore The Renal Protective Effects Of SGLT2i And ARB On Diabetic Mice And The Molecular Mechanisms

Background: Diabetic kidney disease(DKD)is one of the main microvascular complications of diabetes and the main cause of end-stage renal disease(ESRD).It was reported that the prevalence of DKD in diabetic patients ranges from 20% to 40% in foreign countries.The prevalence of DKD in China is also increasing year-by-year,ranging from30% to 50% in community and around 40% in hospitalized patients.The current treatment methods of DKD are limited,angiotensin converting enzyme inhibitor(ACEI)/angiotensin receptor blocker(ARB)as the standard therapy can prevent the progression of proteinuria and reduce the risk of cardiovascular events.In addition,numerous clinical studies have confirmed that sodium-glucose cotransporter 2 inhibitor(SGLT2i)not only reduce blood glucose,but also significantly reduce the occurrence of renal endpoint events.Therefore,ACEI/ARB and SGLT2 i have been recommended by guidelines as standard therapy for DKD patients.However,how these two drugs exert additional renal protection remains unclear,how SGLT2 i synergizes with ARB is also completely unknown.Objective: In order to clarify whether SGLT2 i and ARB have synergizes effects and explore the renoprotective mechanisms of ARB and SGLT2 i by single cell RNA-sequencing(sc RNA-seq).Methods: The male db/db mice was used as type 2 diabetic kidney disease model,the male db/m mice were used as their normal controls.The mice were received saline,dapagliflozin,Irbesartan or dapagliflozin combined with irbesartan for 8 weeks.Body weight,blood glucose and blood pressure levels were monitored weekly.After 8 weeks,random urine was collected from mice in each group to detect urine albumin/creatinine ratio.24-hour urine was collected before sacrifice to detect the 24-hour urinary albumin excretion(UAE).Serum were collected to detect biochemical index such as serum creatinine after anesthesia.The mice were deep anesthetized and sacrificed to prepare kidney single-cell suspensions for 10×Genomics sc RNA-seq,and analyzed Differentially expressed gene(DEG)and gene notology(GO)et al.The kidney of remaining samples were removed and embedded for further staining such as periodic acid-schiff stain(PAS)and immunofluorescence,fixed in the electron microscope preservation buffer for the ultrastructure observation,and the RNA of the kidney was extracted for RT-PCR to verify the results of the analysis.Results: The blood glucose of db/db mice was significantly higher than db/m mice.The blood glucose of SGLT2 i and SGLT2i+ARB group was significantly lower than that of db/db mice with ARB intervention.The UACR in the db/db group was significantly higher than db/m mice,and decreased significantly after intervention in each group,as well as UAE.Kidney sc RNA-sequence identified 10 different kidney cell types,of which proximal tubule(PT)cells were the major cell type.The analysis found that the genes specifically regulated by ARB were mainly related to inflammation and fibrosis,while the genes specifically regulated by SGLT2 i were mainly related to the function of mitochondrial.Moreover,our further analysis of PT cells identified a novel PT cell subcluster that was increased in DKD and decreased after all treatment.Immunofluorescence showed that the specific cell marker(APOC3,GC and KCNK1)of this new cluster co-localized well in PT cells,and their expression were increased in db/db mice and DKD patients,but decreased after treatment.Conclusion: ARB and SGLT2 i can significantly protect renal function in DKD mice,and ARB and SGLT2 i may exert an additive effect rather than a synergistic effect in renoprotection.ARB had more anti-inflammatory and anti-fibrotic effects,while SGLT2 i affected more mitochondrial function in PT.We also identified and validate a new PT subcluster that may be involved in the pathogenesis of DKD,providing new insights into the pathogenesis of DKD.