Bupi Yishen Formula Intervene AKI To CKD Progression By Inhibiting Renal Tubular Ferroptotic GPX4/ACSL4 Signaling

ObjectiveAKI to CKD progression has received extensive attention in recent years.Regrettably,effective clinical treatment is still lacking.The core mechanism of AKI leading to CKD is considered to be renal tubular maladaptive repair.Our previous research has confirmed the efficacy of Bupi Yishen formula(BYF)in delaying the progress of CKD.Further study has demonstrated that BYF can improve the renal tubular injury in adenine-induced CKD rat models.However,the therapeutic effect and mechanism of BYF in AKI to CKD progression are still largely unknown.Recent research suggested that ferroptosis lead to renal tubular injury and maladaptive repair directly or indirectly in AKI and renal fibrosis.As a core component of BYF,quercetin alleviates AKI by inhibiting ferroptotic GPX4/ACSL4 signaling.However,it remains unclear whether the renal tubular protective effect of BYF is attribute to ferroptosis inhibition.This study aims to explore the role of ferroptotic GPX4/ACSL4 signaling in the AKI to CKD progression and the effect of BYF on that signaling,providing an experimental evident for clinical application of BYF in the progression of AKI to CKD.MethodsNephroseq-a platform for integrative data mining of comprehensive renal disease gene expression datasets-as a replication resource.We analyzed the expression of ACSL4 in the renal tissues of normal population and patients with CKD and DN.Then the correlation between the expression level of ACSL4 and glomerular filtration(GFR)were analyzed.In order to explore the mechanism of BYF in AKI to CKD progression,the AKI non-progressive models and the AKI to CKD progressive models were established by clamping the renal artery and vein for 20 or 30 minutes respectively.Both models were sacrificed and collected serum samples and renal tissue samples at 3,7 and 14 days respectively.Serum samples were used to detect serum creatinine to evaluate renal function.The renal tissue samples were performed for histopathological staining(including HE and Masson)and detecting the protein expression level of ferroptosis indicators GPX4 and ACSL4 by WB.Then AKI to CKD progressive models were treated with BYF for 14 days.The experiment was divided into 3 groups:sham-operation group(Sham group),AKI to CKD progressive group(bIRI 30min group)and BYF treatment group(BYF group).The mice in each group were sacrificed and collected serum samples and kidney tissue samples on 14 days after AKI.Serum samples were used to detect serum creatinine to evaluate renal function.The renal tissue samples were performed for PAS staining,detecting the mRNA expression level of inflammatory indicators Ccl2,Tnfa,1133 and fibrotic indicators Colla1,Col3α1,Fn1 by qPCR and expression level of GPX4 and ACSL4 by WB.The location of ACSL4 in the human kidney tissues were analyzed through Nephroseq database.Afterwards,RSL3 induced HK-2 cell ferroptosis models were validated in vitro cellular experiments.The HK-2 cells were divided into normal control group(CTL group),RSL3 induced ferroptosis group(RSL3 group),BYF treatment group(RSL3+BYF group),ferroptosis inhibitor Fer-1 treatment group(RSL3+Fer-1 group),single BYF group(BYF group),and single Fer-1 group(Fer-1 group).The morphology of each group was observed by microscope;the survival rate of cells in each group were examined by CCK-8;the MDA level of cells in each group was detected by MDA kit.Results1.The heatmap analysis indicated that the expression of ACSL4 were significantly upregulated in CKD and DN patients compared with the healthy controls.Additionally,the expression of ACSL4 were negatively correlated with baseline GFR in CKD and DN cohort.2.Serum creatinine levels of mice were significantly elevated at 3 days but returned to the baseline and kidney morphology of mice were restored at 14 days after 20-minute IRI.However,serum creatinine levels of mice were significantly elevated at 3 days and remained higher than the baseline even at 14 days after 30-minute IRI.Renal histopathological staining experiments showed that necrotic tubular cells,tubule dilation and excessive extracellular matrix deposition at 14 days after 30-minute IRI,Consistent with that kidney function and morphology data,renal GPX4 protein abundance was dramatically decreased at 3 days after IRI,then restored in AKI non-progressive models but exhibited a sustained depression of GPX4 in AKI to CKD progressive models.Similarly,renal ACSL4 protein were significantly induced at 3 days after IRI,then restored in AKI non-progressive models and exhibited an exaggerated induction in AKI to CKD progressive models.3.In vivo study,compared with the sham group,AKI to CKD progressive group exhibited higher serum creatinine level;higher renal tubular damage score;elevated mRNA expression level of inflammatory indicators Ccl2,Tnfa,Il33 and fibrotic indicators Col1α1,Col3α1,Fn1;decreased expression levels of GPX4 protein and increased expression levels of ACSL4 protein.BYF treatment lowered serum creatinine level;reduced renal tubular damage score and extracellular matrix deposition;reduced mRNA expression level of inflammatory indicators Ccl2,Tnfa,1133 and fibrotic indicators Col1α1,Col3α1,Fn1;restored under-expression levels of GPX4 protein and inhibited over-expression levels of ACSL4 protein.4.The heatmap analysis indicated that the ACSL4 were detected in the renal tubulointerstitial samples rather than glomeruli of human kidneys.Therefore,we used RSL3 to induce ferroptosis in HK-2 cells.The morphology of HK-2 cells was observed using microscope:a large number of cells floated and died in RSL3 group compared with CTL group,while cell death was ameliorated in BYF treatment group and ferroptosis inhibitor Fer-1 treatment group.Single BYF and Fer-1 addition did not induce cell morphological change and cell death.Accordingly,CCK-8 showed that the survival rate of HK-2 cells in RSL3 group was declined significantly compared with CTL group,while BYF treatment and Fer-1 treatment elevated the cell survival rate significantly.MDA results showed that the MDA level of HK-2 cells in RSL3 group was increased significantly compared with CTL group,while BYF treatment and Fer-1 treatment decreased the cellular MDA levels.Single BYF and Fer-1 addition did not affect the cellular survival rate and MDA levels.ConclusionOur results indicate that BYF may intervene AKI to CKD progression by inhibiting sustained activation of ferroptotic GPX4/ACSL4 signaling.