The Potential Role And Pathogenesis Of IL-34 In Diabetic Kidney Disease And Renal Interstitial Fibrosis

BackgroundDiabetic Kidney disease(DKD)is a threatening microvascular complication of diabetes mellitus,which is a pivotal cause of end-stage renal disease(ESRD).The pathogenesis of DKD is complex,including genetic susceptibility,epigenetic regulation,hyperglycemic-induced renal hemodynamic changes,local metabolic abnormality,and oxidative stress.Inflammatory mediators can directly inhibit insulin signaling,participating in tissue insulin resistance(IR),by promoting renal fibrosis through various transcription and posttranscriptional mechanisms.Macrophagemediated inflammation also contributes to insulin resistance and target organ damage.IL-34 is a cytokine that promotes the differentiation and viability of monocytes and macrophages through binding with the colony-stimulating factor-1 receptor(CSF1-R or cFMS).Until now,the potential role and the pathogenesis of IL-34 in DKD and its effect on the quantification and polarization of macrophages have not been elucidated.Objective1)To investigate the potential role of IL-34 in DKD and its role in macrophage.2)The role of IL-34 in the process of renal interstitial fibrosis and the influence of macrophages.3)To elucidate the regulation pathogenesis of IL-34 in HK-2 cell lines under the high glucose condition in vitro.Materials and methods1)Based on the Nephroseq database,we searched the expression of IL-34 mRNA in DKD patients.Through collecting the DKD patients who completed renal biopsies in our hospital from January 2018 to December 2018,we investigated the expression levels of IL-34 by immunohistochemical staining.2)To elucidate the role of IL-34 in DKD,the mouse model of progressive DKD(unilateral nephrectomy +60%high-fat diet+STZ)was established based on IL-34 KO mice and WT respectively,the group was divided as follows:①WT-Sham②WTDKD③IL-34 KO-Sham④IL-34 KO-DKD.The pathophysiological indexes,renal pathological changes,protein and mRNA levels of IL-34 and its homologous factor CSF-1 were evaluated by RT-PCR,and Western Blot detection.We also evaluated the expression levels of IL-34 and its related receptors cFMS,PTPζandα-SMA protein,quantification and polarization of renal macrophage among these four groups.3)Meanwhile,we also established the UUO(unilateral ureteral obstruction,UUO)mice model based on WT mice and IL-34 KO mice to investigate its specific role in renal fibrosis.The group was divided as follows:① WT-Sham②WT-UUO③IL-34 KO-Sham ④ IL-34 KO-UUO.The pathophysiological indexes,renal pathological changes,the protein and mRNA levels of IL-34 and the quantification of macrophage in renal were performed same as the DKD model.4)In vitro,HK-2 cells were treated with hyperglycemia(HG).Setting group as follows:① Normal glucose,D-glucose=5.5mmol/L(NG),②High glucose,D-glucose=30 mmol/L(HG),and③Mannitol,D-glucose=5.5mmol/L+Mannitol=24.5mmol/L(HM).The phenotype of IL-34 in HK-2 under different conditions were evaluated by RT-PCR and Western Blot respectively.The potential transcription relationship between FOXO1 and IL-34 were investigated by luciferase reporter gene assay.The pcDNA3.1-FOXO1 plasmid was transfected into HK-2 cells and the expression of FOXO1 was evaluated by RT-PCR and Western Blot under NG or HG conditions.Finally,HK-2 cells were treated with LY294002(PI3K inhibitors)or DMSO under NG or HG respectively to investigate whether the changes of IL-34 stimulated by HG were regulated by PI3K/AKT/FOXO1 signaling pathway.5)Statistical:Correlations between IL-34 and clinical parameters were determined by Pearson or Spearman analysis.Data were presented with mean±SD or median and interquartile range,as appropriate.For multiple comparisons,data were analyzed by one-or two-way ANOVA as appropriate,with post hoc comparison using Tukey’s test.P<0.05 indicates statistically significant differences.Results1)IL-34 was up-regulated in renal of DKD patients and the expression of IL-34 in renal tubule was positively correlated with proteinuria,but not with eGFR,sCR,Alb.2)In wild C57BL/6J mice prescribed with unilateral nephrectomy,fed with a 60%high-fat diet and injected with STZ treatment,the blood glucose was increased.After being injected with STZ for 16 weeks,the level of sCR and uACR were elevated.The renal pathology was presented with mesangial matrix dilatation,interstitial inflammatory infiltration,and renal fibrosis.Compared with the group of WT-DKD,the phenotype of clinical and pathological in the group of IL-34 KO-DKD were alleviated.The expression of IL-34 homologous factor CSF-1 and IL-34 receptors in cFMS,PTPζ,and SDC-1 were also up-regulated in WT-DKD mice.The macrophage infiltration in renal of IL-34 KO DKD mice were less than that of WT-DKD mice,with the reduction of M1 macrophages was the most significant.3)The expression of IL-34 was also up-regulated in WT-UUO mice and was highest on the 3rd day.The quantify of macrophage and fibrosis phenotype of IL-34KO-UUO mice in kidney were alleviated compared with WT-UUO.4)In vitro,the results indicated that the expression of IL-34 was elevated in HK-2 cells after treated with HG,while the NG or HM were not.The elevated expression of IL-34 was regulated by the PI3K/AKT/FOXO1 signaling pathway.Meanwhile,FOXO1 also inhibited IL-34 expression by binding to the IL-34 promoter region.Overexpression of FOXO1 inhibited the expression of IL-34 as well as the activation of the PI3K/AKT signal pathway.Conclusion1)The expression of IL-34 was up-regulated in the renal tissues of DKD patients.2)In the group of WT-DKD,the urinary protein,renal fibrosis,and local renal macrophage infiltration and the expression of IL-34 were upregulated in the group of WT-DKD.The clinical and pathological expression were also exist in the group of IL34 KO DKD,which was alleviated compared with the group of WT-DKD.3)The expression of IL-34 was also elevated in renal tissue of WT-UUO mice,which reached its peak on third day and then declined.The clinicopathological phenotype and renal macrophage infiltration were reduced in IL-34 KO mice after UUO induction compared with WT-UUO mice.4)FOXO1 inhibits IL-34 expression by binding to IL-34 promoter regions.The upregulation of IL-34 in HK-2 cells after treated with high glucose was regulated by the PI3K/AKT/FOXO1 signaling pathway.BackgroundThe widely used Renin-angiotensin-aldosterone system inhibitor(RASI)may increase the risk of hyperkalemia and acute kidney injury(AKI).We aimed to analyze the RASI-related AKI or hyperkalemia reported in the FAERS database to optimize patients’ treatment and provide a reference for a clinically safe and rational prescription.MethodsWe obtained data in FAERS recorded from January 2004 to December 2020.Disproportionality analysis and Bayesian analysis were used in data mining to screen the suspected AKI or hyperkalemia after RASI.The time to onset,hospitalization,and prognosis of RASI-associated AKI or hyperkalemia were also investigated.ResultsWe identified 11,3 01 RASI-related adverse events(AEs)of hyperkalemia and AKI in the FAERS database;4997 were due to ACEIs,5658 were due to ARBs,and 646 were due to the combination of ACEI and ARB.AKI was more commonly reported in patients with ARB(78.42%)than ACEI users(57.27%).Hyperkalemia cases were reported more in ACEI users(28.70%)than ARB users(14.14%).The median time to onset of RAS-associated AKI was 135.0(17.0-620.0)days.RASI-associated hyperkalemia occurred relatively later in ACEI users,with a median onset time of 261.0(43.0-1097.7)days,compared with that of 200.5(52.0-636.0)days in ARB users(p<0.001).Among all AEs,72.39%of cases received hospitalization.Death occurred in 6.3%of the renal AE cases.The elderly and heart failure were potential risk factors for death in patients who developed RASI-associated renal AEs,with an increased OR compared with younger age(OR=1.32)and hypertension patients(OR=2.55).Based on the criteria of the four algorithms,the ACEI and ARB combination further increased the incidence of AKI and hyperkalemia,demonstrating the highest RORs,PRRs and EBGMs.ConclusionsPatients who indicated RASI for heart failure demonstrated a higher death risk when AEs occurred.ACEI combined with ARB can increase the incidence of hyperkalemia and AKI.Careful and individualized management is necessary.