Mechanism Of Klotho/Vasopressin Regulating Ectopic Renal Lipid Deposition Through Brain-kidney Axis In Type 2 Diabetic Nephropathy

BackgroundChina’s"sugar kidney era"has arrived,according to statistics,China has nearly 500 million people in pre-diabetes,every 10 people have diabetes,nearly 40%of the patients can develop diabetic kidney disease.Data from the kidney disease dialysis registry show that diabetes has surpassed glomerulonephritis to become the first cause of new-set dialysis.The social and economic burden caused by diabetes has become a major global public health problem worthy of further study.In recent years,with the deepening of the research,and constantly emerge a batch of oral glucose-lowering drugs with heart and renal protection effect,such as sodium-glucose transporter protein 2 inhibitor（SGLT2i）,although achieved very good hypoglycemic effect,can delay kidney development to a certain extent,but still can cause kidney gluconeogenesis increased,osmotic diuresis and other side effects.This suggests that we need to further explore the pathogenesis of diabetic nephropathy and find new therapeutic targets.The current view is that diabetes has a"metabolic memory"and even if blood glucose levels are well controlled,there is always a risk of kidney progression.New drugs,such as glucagon-like peptide-1 receptor agonists（GLP-1Raliraglutide,etc.）and metabolic surgery for weight loss,mainly play a role in glucose control based on weight loss and regulation of energy metabolism.Therefore,people gradually change the concept of treatment mode,from"glucose centered treatment"to"lipid centered treatment".It is suggested that the neuroendocrine mechanism of hypothalamus energy metabolism mediates the development of DM by regulating the disorder of protein-energy-lipid metabolism in peripheral organs.The disorder of protein-energy-lipid metabolism is not only manifested as obesity with elevated lipid profile,but also as hypercatabolism,leading to protein energy wasting（PEW）,and even progressing to cachexia.Both obesity and PEW are the results of hypothalamus energy wasting disorder.The kidney is not only a excretory organ,but also an important organ of energy metabolism.However,besides renal sympathetic nerve activation,whether the hypothalamus regulates renal energy metabolism and its neural circuit remains unclear.The increase of plasma osmotic pressure in the body leads to the secretion of Vasopressin in the paraventricular nucleus and supraspinal nucleus of the hypothalamus,which further targets the collecting tube of the kidney to participate in the regulation of water metabolism balance.The new study found that Vasopressin is involved in the process of liver steatosis in type 2 diabetes mellitus,and the anti-aging protein Klohto plays an important role in glucose and lipid metabolism and energy balance.Our research group,previous animal model studies found that at the early stage of progression from DM to DKD,there was obvious lipid deposition in kidney tissue,suggesting that lipid toxicity was involved in the progression of DKD kidney.Therefore,the purpose of this project was to explore the disorder of protein-energy-lipid metabolism in patients with type 2 diabetic nephropathy.The relationship between plasma osmotic pressure and renal progression,and from the new perspective of neuro-endocrine regulation of renal metabolism,actively explore the possible mechanism of action and central intervention target of hypothalamic Klotho/Vasopressin mediated DKD renal ectopic fat deposition,so as to provide a strong macro for preventing T2DM from developing into DKD and preventing it from developing into ESRD Theoretical basis,has become an innovative project in the field of kidney disease research.Methods1.Correlation between protein-energy-lipid metabolism disorder and renal progression in patients with type 2 diabetic nephropathy1.1 A total of 1219 non-dialysis diabetic nephropathy patients were selected to collect general information,blood routine,renal function,blood lipid profile,electrolytes,urinary microalbumin and urinary creatinine data,and calculate the urinary albumin creatinine ratio（UACR）,remnant cholesterol（RC）and plasma osmotic pressure（Os P）.The relationship between UACR,e GFR,RC and Os P was analyzed by linear regression and logistic regression.1.2 The RMR and body composition（body cell mass and fat mass）of 774 patients at the end of dialysis were assessed by bioimpedance.Anthropometric data were collected using standard measurement methods,and the muscle circumference of the upper arm（AMC）was calculated.Daily protein and energy intake were calculated using a 3-day food diary.Biochemical,nutritional and dialysis parameters were obtained.Blood samples from 204 patients were randomly collected for ELISA detection of Klotho/FGFs levels,and the relationship between RMR,Klotho/FGFs and diabetes mellitus and protein energy consumption was analyzed by linear regression and logistic regression.2.Correlation analysis of renal ectopic fat deposition and Hypothalamus Klotho/Vasopressin in T2DN ratsSTZ-induced T2DN rats were used as research subjects for 4w,8w,12w and 16w,and the expression trend of adipose deposition molecules AQP7,ADRP and CD36 in kidney and Klotho/FGF23 in hypothalamus were detected by immunohistochemistry.According to the peak of molecular expression trend,it was confirmed that the study on renal lipid metabolism was most obvious at 12w of the disease course.The data of food intake,water intake and urinary protein of12w T2DN rats were collected and the correlation analysis was conducted with R language"corrr"packages.3.Effects of central administration of recombinant Klotho on renal lipidomics and metabolomics of db/db miceA 12-week-old db/db mouse was selected as the research object.After 3 days of single lateral ventricular injection of recombinant Klohto and Dapagliflozin and their combination,partial renal tissues were analyzed by chromatography-mass spectrometry.The prediction model was established by partial least square discriminant analysis（PLS-DA）and original data analysis to correct p-value（FDR）<0.25,VIP>1.Differential lipids and metabolites were screened,the volcano map was drawn with R package ggplot2,and the multi-scale metabolite co-expression network（MMCEN）was constructed with R package MEGENA and Cytoscape（3.6.0）.4.Central recombinant Klotho improves renal ectopic lipid deposition by down-regulating Vasopressin in the hypothalamus of db/db miceThe changes of daily body weight,food intake,water intake,blood glucose,urinary protein creatinine ratio and other indicators of db/db mice were collected by central recombinant Klotho,and the changes of Vasopressin,Klotho/FGFR1 and SGLT2 in hypothalamus were detected by immunohistochemistry.Kidney tissue samples were collected for paraffin section,and the expression of lipid deposition molecular protein was detected by immunohistochemical method.In vitro,the expression of Vasopressin was detected by immunofluorescence after the intervention of GT1-7hypothalamic neuroendocrine cells and FGFR receptor inhibitor PD173074,PI3K inhibitor Wortmannin and JAK/STAT inhibitor Tofacitinib.The levels of Vasopressin,Klotho and IL-6 in serum and cell supernatants were determined by ELISA.5.Study on the mechanism of ectopic lipid deposition in renal tubular epithelial cells induced by VasopressinThe proximal renal tubules were selected as the research object,and 0.2m M palmitic acid（PA）model was made.Exogenous administration of 10^-6M Vasopressin,A-769662 and Balovaptan was performed to quantify AQP7 m RNA level by q-PCR.The protein expressions of AQP7,ADRP,AMPK/INSIG2 and AVPRs were detected by Western Blot,and the changes of AQP7 membrane translocation were detected by immunofluorescence.Results1.The proportion of patients with diabetes progression to diabetic nephropathy was 48.89%,which was significantly higher than the 40%reported in previous studies;Elevated RC levels were associated with an increased risk of proteinuria in non-dialysis T2DN patients;The decrease of RC level was associated with the increase of Os P in non-dialysis T2DN patients.Reduced RMR levels were associated with an increased risk of PEW in dialysis patients;Klotho,but not FGFs,predicted the course of coexisting DM and PEW in dialysis patients.2.The expression levels of ADRP,CD36 and AQP7 in the kidney of T2DN group were significantly different from 8w to 12w（P<0.05）;The peak of lipid metabolism molecules in kidney was found between 4w and 16w,and the peak time of ADRP was at 12w,which was later than that of AQP7 and CD36.Vasopressin expression was increased in hypothalamus of T2DN rats,and was positively correlated with RC,related factors promoting lipid deposition（AQP7,ADRP,CD36,SREBP-1C）and FGF23,and negatively correlated with INSIG1 and ACC.The expression of Klotho decreased in hypothalamus and kidney of T2DN rats,and was negatively correlated with Vasopressin,RC,AQP7,ADRP,CD36,SREBP-1C and FGF23,and positively correlated with INSIG1 and ACC.Except triglycerides,FGF23 had no correlation with the factors of lipid metabolism.3.In lipidomics,Phosphatidylcholine[PC（16:1/18:3）],phosphatidylethanolamine[PE（18:1/18:3）],sphingomatidylcholine[SM（D14:3/30:1）]and digalactose diglyceride[DGDG（16:0/16:0）]were changed by central recombinant Klotho or combined with Dapagliflozin.The relationship between Klohto and sphingomyelin was first discovered.In non-target metabolomics,Klotho combined with daaglizin altered leukotriene B4,hypoxanthine,and diarylheptanone,which were enriched in arginine and proline metabolic pathways,although no changes were observed in Klotho intervention.4.α-Klotho,as a hypothalamic hormone,can improve body weight and blood glucose,and reduce food intake and water intake in diabetic nephropathy mice.Central administration of recombinant Klotho down-regulated Vasopressin expression in hypothalamus and improved renal ectopic lipid deposition in T2DN mice,but had no effect on urinary protein creatinine ratio.Recombinant Klotho may inhibit Vasopressin expression and secretion in GT1-7 cells（hypothalamus neuroendocrine cells）through FGFR1/PI3K and JAK/STAT pathways.Central administration of Dapagliflozin may promote the expression of Klotho in hypothalamus and kidney,and its mechanism remains to be further explored.5.Exogenous addition of Vasopressin can aggravate the increase of lipids and triglyceride level in MPTEC cells induced by PA.Vasopressin promotes lipid deposition in MPTEC cells mediated by binding AVPR1A,dephosphorylation of AMPK/INSIG2 pathway,up-regulation of AQP7 membrane translocation and ADRP lipodrop-forming protein.ConclusionKlotho can predict renal progression and PEW in patients with type 2 diabetic nephropathy.Central Vasopressin-targeted kidney binds to AVPR1A and intensifies renal ectopic fat deposition through AMPK/INSIG pathway;Central supplementation of Klotho down-regulates Vasopressin through FGFR1/PI3K and JAK/STAT pathways to improve renal ectopic fat deposition.