Mechanism Of Na~+/H~+ Exchanger Regulatory Factors 1 Regulating Phosphate Homeostasis In The Pathogenesis Of Proximal Tubule Injury

BackgroundThe proximal renal tubule actively reabsorbs a variety of substances or metabolic products that have been filtered through the glomerulus,and its transport function is highly dependent on mitochondrial energy production.Any obstruction in the energy metabolism of mitochondria can lead to dysfunction of the proximal renal tubule.Proximal renal tubule damage can manifest as various disorders of substance reabsorption,including uric acid,phosphate,glucose,amino acids,and small molecular proteins,clinically presenting as Fanconi syndrome(FS).Secondary FS is one of the important causes of acute and chronic renal dysfunction,and there is currently a lack of systematic mechanism research.Drug use and autoimmune diseases are the two major causes of secondary FS.In our previous study of the human immunodeficiency virus(HIV)-infected patients treated with tenofovir disoproxil fumarate(TDF),we observed that 6.7%of patients had proximal renal tubule damage,with the most common clinical presentations being proteinuria(20.3%)and hypophosphatemia(12.3%),suggesting that sodium-phosphate cotransporter 2a(Npt2a)may be the transport protein that is damaged earliest in the brush border.Npt2a is located on the brush border of proximal tubular cells(PTC),responsible for more than 70%of renal phosphate reabsorption,and a single mutant encoding Npt2a can lead to complete FS or even PTC necrosis.The exact mechanism is not yet clear,but it is sufficient to illustrate the importance of intracellular phosphate homeostasis,especially the importance of brush border phosphate entering cells through Npt2a in the pathogenesis of FS.Can non-genetic factors that cause Npt2a damage also lead to FS or even PTC necrosis?Do Npt2a and its related intracellular phosphate homeostasis abnormalities play a crucial role in drug-induced(tenofovir)proximal tubule damage?The above questions and related mechanism research are worthy of exploration.On the other hand,besides the downregulation of Npt2a expression,we confirmed in vitro that various transporters,including urate transporter 1(URAT1)and sodium glucose cotransporter-2(SGLT-2),were also downregulated in human kidney-2(HK-2)proximal tubule epithelial cells treated with tenofovir,suggesting the presence of a common upstream mechanism of transporter injury.Na+/H+exchanger regulatory factor 1(NHERF1)is an important scaffolding protein that affects the localization and function of membrane proteins.NHERF1 gene knockout mice have been shown to be more sensitive to injury in cisplatin-induced acute kidney injury(AKI),and NHERF1 has been found to play an important regulatory role in the expression and function of Npt2a.Therefore,it can be speculated that the scaffolding protein may play a role in drug-related proximal tubular injury by affecting the expression or localization of proximal tubule transporters such as Npt2a.Furthermore,in our previous retrospective observation of the clinical and pathological characteristics of primary Sj?gren’s Syndrome(pSS)patients with FS(pSS-FS),we found that the most prominent clinical manifestation was hypophosphatemia,and the proportion of Npt2a involvement was about 58%-88%,higher than renal glycosuria and aminoaciduria.The target of renal tubular injury in pSS-FS patients is still unclear,and whether the scaffolding proteins NHERF1 and Npt2a related to phosphate reabsorption,as well as mitochondrial injury,also play a role in pSS-FS,requires further study.Aims1.To investigate the changes of both membrane-associated protein NHERF1 and proximal tubular transporters in the TDF-induced nephrotoxic model and prove that NHERF1 protects the TDF-induced FS by Npt2a,intracellular phosphorus,and mitochondria dysfunction pathway.2.To observe the damage of proximal renal tubular scaffold protein NHERF1 and transporters in the pSS-FS mouse model,and demonstrate that the resulting phosphorous homeostasis and mitochondrial disruption may be the common core link of TDF and pSS-mediated FS.3.To explore the mechanism of the relationship between lncRNA and proximal tubular injury in the mouse model of TDF-induced FS,and verify the above transporters and mitochondrial damage in the maleic acid-induced FS mouse model.MethodsPart Ⅰ:Proximal tubular membrane-associated protein NHERF1 and transporters dysfunctions in TDF-induced nephrotoxicity1.C57BL/6 mice(n=18)were gavaged daily with 10 mg/kg/d,50 mg/kg/d of TDF,or corn oil as the control for 8 weeks.Serum was retained for testing serum creatinine(Scr)and blood urea nitrogen(BUN),and 24-h urinary protein,phosphorus,and potassium excretion was detected.Kidney tissue was retained for Hematoxylin and Eosin(HE)staining.Immunohistochemistry(IHC),immunofluorescence(IF),and western blotting were used to detect the localization and expression of membrane-associated proteins and transporters.2.Kidney cortex of the control group and high-dose TDF group mice were obtained.RNA was extracted for mRNA and lncRNA transcriptome sequencing,and differential genes were screened for GO and KEGG analysis to search for the possible molecular pathway of TDF-induced FS.3.Transmission electron microscopy was used to observe mitochondrial morphology changes in TDF-induced FS mice’s kidney cortex.Western blot analysis was performed to determine the expression levels of mitochondrial DNA replication helicase Twinkle,mitochondrial respiratory chain-associated proteins(Pgclα and cytochrome b),and mitochondrial fusion and fission-related proteins(mitochondrial fusion protein Opal and mitochondrial fission protein Drpl)in kidney tissues.Part Ⅱ:Scaffold protein NHERF1 protects Tenofovir-induced nephrotoxicity by regulating Npt2a and intracellular phosphorus balance1.The human renal tubular epithelial cell line(HK-2)was grown and received 72 h exposure to 16 μM Tenofovir or vehicle.Cell proliferation and cytotoxicity assay was conducted to explore the effect of Tenofovir on the survival rate of HK-2 cells.ATP levels were assessed using the ADP/ATP Ratio Bioluminescence Assay Kit.Quantification of phosphate was measured using the Phosphate Assay Kit.2.HK-2 cell line overexpressed with NHERF1 was constructed and treated with 16 μM Tenofovir or 0.1%DMSO.Protein expression levels and phosphorus concentration in cells and mitochondria were detected by the above methods.Part Ⅲ:NHERF1,Npt2a,and mitochondrial dysfunctions in pSS-FS mouse model 1.Proteins extracted from the salivary glands(SG)of normal mice were emulsified in an equal volume of Freund’s complete adjuvant(FCA)at a concentration of 2 mg/mL(the SG group),while renal tubular proteins at a concentration of 1 mg/mL(the RT group).For disease induction,each 8-week-old female C57BL/6 mouse received subcutaneous multiinjections on the back with 0.1 mL of the above emulsion on days 0 and 7,respectively.On day 14,a booster injection was carried out with a half dose of the proteins emulsified in Freund’s incomplete adjuvant(FIA).Mice injected with phosphate buffered saline(PBS)or adjuvant served as the control groups.At 6/8/10/12 weeks postimmunisation,Scr,BUN,24-hour water intake,urine volume,and urine electrolyte excretion were determined.Serum levels of anti-SSA and anti-SSB autoantibodies were determined by enzyme linked immunosorbent assay(ELISA).2.The kidney,SG,and liver tissues were sent for pathological examination,and the ultrastructure of renal tubular epithelial cells,especially mitochondrial structure,was observed by transmission electron microscopy.3.IHC and IF were used to detect the expression of membrane-related proteins(NHERF1),and proximal renal tubular transport proteins(Npt2a and Megalin)in kidney tissue,and to observe the formation of CD21+ectopic germinal centers in the renal interstitium.Part Ⅳ:Association analysis of proximal tubular transporter damage and AKI1.We systematically searched four databases to identify the lncRNA expression studies of AKI in animal models and patients.The lncRNA expression data were extracted from 38 included studies,and the lncRNA vote-counting strategy was applied to identify significant lncRNA biomarkers.The predicted targets of lncRNA biomarkers were obtained by searching Co-LncRNA,RBPmap,and LncBase v.3.Further,GO enrichment analysis and KEGG pathway analysis were performed.2.The expression results of differential lncRNAs obtained by systematic review and transcriptome sequencing were interlaced,and then the obtained differential lncRNAs were verified in the kidney cortex samples of TDF-induced FS mice by real-time quantitative PCR(qPCR).3.Single-cell RNA sequencing was used to verify that transporters damage also exists in maleic acid-induced proximal renal tubule injury.ResultsPart Ⅰ:Proximal tubular membrane-associated protein NHERF1 and transporters dysfunctions in TDF-induced nephrotoxicity1.Chronic high-dose TDF administration resulted in significantly in creased Scr(42.63 ±7.284 vs.13.65 ± 0.87 μmol/L,P<0.05)and BUN levels(11.30 ± 1.22 vs.7.64 ±0.27mmol/L,P<0.05)compared to that in non-AKI control mice.In addition,albuminuria,urinary phosphorus excretion,and uric acid(UA)excretion were all increased by high-dose TDF treatment compared with that in the control mice.HE staining of kidney tissue showed swollen and exfoliated tubular epithelial cells,lodging of brush border cilia,cellular degradation,and overall fewer cells,along with significantly higher renal tubular injury scores in TDF-treated mice.Transmission electron microscopy of these tissue samples verified the disorder and cellular damage in brush border cilia,which was more severe in the high-dose TDF group.Western blot analysis indicated that NHERF1 and PTC transporters proteins were lower in TDF-treated mice than in the control group.2.A variety of mRNA and lncRNA were differentially expressed in the renal cortex of mice in the high-dose TDF group and the control group.These mRNAs or lncRNAs are located in the respiratory chain complex,mitochondrial inner membrane,and brush border,and are closely related to oxidative phosphorylation and energy metabolism.3.The mitochondrial markers,Pgc1α,cytochrome b,Drp1,and PiC were markedly decreased in the TDF-treated FS model mice in a dose-dependent manner.Electron microscopy imaging further revealed disordered membrane boundaries,damaged membrane structures,fewer cristae,and fragmented or abnormally large mitochondria in the PTC of mice treated with TDF.Part Ⅱ:Scaffold protein NHERF1 protects Tenofovir-induced nephrotoxicity by regulating Npt2a and intracellular phosphorus balance1.Although cell viability was unaffected by 0-16 μM Tenofovir,intracellular ATP concentrations decreased in HK-2 cells by 24 h after treatment with 2 μM or higher Tenofovir,and 0.5 μM or higher Tenofovir by 48 h.In addition,16 μM Tenofovir led to significantly lower intracellular Pi concentrations within 24 h,while mitochondrial phosphorus concentrations decreased after 72 h exposure.We also noted that ATP and intracellular Pi levels decreased in a time and dose-dependent manner.Western blots showed that the protein levels of the transport-related scaffold protein NHERF1,and the associated transporters,Npt2a,URAT1,and SGLT-2,were all decreased significantly in cells treated with 16 μM Tenofovir compared to the control group.Similarly,significant decreases in the mitochondrial functional markers were also detected.2.Overexpression of NHERF1 in HK-2 cells can improve the downregulation of Npt2a and other transporters caused by Tenofovir treatment,and the downstream mitochondrial damage is also rescued.Part Ⅲ:NHERF1,Npt2a,and mitochondrial dysfunctions in pSS-FS mouse model 1.Both the SG group and the RT group mice developed a typical disease profile of pSS(6 weeks postimmunization),including increased water intake,reduced saliva secretion,positive serum anti-SSA and anti-SSB autoantibodies and pathologically lymphocytic infiltrations in SG tissues.2.For the kidney,despite similar Scr and BUN levels,both the SG group and the RT group presented with renal tubular dysfunctions including decreased urine osmolality,increased urine output,proteinuria,decreased serum potassium levels,and increased urinary potassium and phosphorus excretion compared to the control group.Kidney pathology showed swollen and exfoliated tubular epithelial cells,brush border cilia lodging,and dissolving with focal lymphocytic infiltrations in both two groups.2.Compared with controls,the expression of NHERF1,megalin,and Npt2a on the pSS mice proximal tubule was decreased.The lymphocytic infiltration of the renal interstitium in all pSS mice at 10 weeks postimmunization showed several CD21+focuses.In addition,as shown in semiquantitative analyses,the positive area staining ratio of megalin(0.103±0.008 vs.0.133 ± 0.005,P=0.016)and Npt2a(0.048 ± 0.006 vs.0.070 ± 0.005,P=0.029)in the RT group mice was significantly lower than that in the SG group.The electron microscopy showed the disorderly dissolution of brush border cilia,a decreased or even absent mitochondrial ridge,and increased mitochondrial membrane density in the two pSS groups(10 weeks postimmunization).Part Ⅳ:Association analysis of proximal tubular transporter damage and AKI1.The meta-analysis identified 31 significant dysregulated lncRNAs from 38 studies.TapSAKI,XIST,MALAT1,CASC2,and HOXA-AS2 were considered as the potential predictive biomarkers and therapeutic targets of AKI.2.lncRNA PVT1 was significantly increased in the renal cortex of high-dose TDF-treated mice,which was consistent with transcriptome sequencing and literature reports.Single-cell RNA sequencing analysis showed that the maleic acid-induced FS mouse model also had downregulated proximal renal tubule transporters.ConclusionUnder the research conditions of this study:1.TDF treatment leads to several nephrotoxicity-related pathologies,such as damage or injury to proximal tubules,mitochondrial dysfunction,and ultimately renal failure.TDF-related reductions in NHERF1 and Npt2a were associated with concomitant deterioration of renal function,while URAT1 and SGLT2 levels were also diminished.2.NHERF1 protects the Tenofovir-induced proximal tubular injury by Npt2a,intracellular phosphorus,and mitochondria dysfunction pathway.3.A novel mouse model of pSS-FS with NHERF1 and transporters disorder was generated by immunizing mice with renal tubular proteins.It will contribute to the characterization of multiple facets of pSS with kidney injuries and further study of innovative therapeutic strategies.The damage of NHERF1 and the resulting phosphorous homeostasis and mitochondrial disruption may be the common core link of TDF and pSS-mediated FS.4.The maleic acid-induced proximal renal tubule injury mouse model also verified the transporters injury,indicating the universality of the mechanism that the damage of Npt2a leads to the disturbance of phosphorus homeostasis and mitochondrial damage in proximal renal tubule injury.