Clinical Characteristics And Risk Factors Of Secondary Renal Proximal Tubule Injury

BackgroundDrug nephrotoxicity is an important cause of acute and chronic renal insufficiency.The renal proximal tubule actively secretes or reabsorbs the drug(and its metabolites),which is the main site of kidney injury.The proximal tubule injury can be manifested as dysfunction,that is,reabsorption disorder for electrolytes and small molecule substances,leading to incomplete and complete Fanconi syndrome in clinical practice.In worse cases,even apoptosis or necrosis can occur,which is the most important reason of acute kidney injury(AKI).Tubule injury and repair can reproduce the development process,and it can also coordinate and interact with the glomerulus(tubuloglomerular feedback)and the tubules of different segments,which determines the outcome of the kidney.Investigation of the clinical characteristics and mechanism of proximal tubule injury related with drugs can not only help understand the common mechanisms of tubule injury and repairment,but also open an important window for exploring the interaction between tubules and their interaction with glomerulus.However,there are few studies on drug-related Fanconi syndrome all around the world,because of the limited sample size and clinical research methods.For example,tenofovir disoproxil fumarate(TDF),as the most common antiviral drug,can causes proximal tubule damage.The relationship between renal tubular dysfunction caused by TDF and decline of glomerular filtration rate(GFR)is not clear due to lack of clinical data from large cohorts,especially in the Chinese population.Long-term oral prescription of thiazide diuretics,which can block the sodium chloride co-transporter(NCC)of the distal tubule,which has been recognized to affect the function of the proximal tubule,but the underlying mechanism is largely unknown.In this study,we first observed the clinical characteristics of proximal tubule injury in the cohort of human immunodeficiency virus(HIV)infected patients treated with TDF,and explored the correlation between proximal tubule and GFR.Then we explored the relationship between transporter damage and endoplasmic reticulum(ER)stress in cell lines.Finally,we summarized the clinical characteristics of proximal tubule function in the cohort of patients with Gitelman syndrome(GS)with congenital NCC dysfunction,the same site of thiazide diuretic targeting,evaluated the glomerular renal functional reserve in GS patients,and analyzed the potential pathophysiological mechanisms.Aims1.To observe the characteristics of proximal tubule and GFR injury in HIV-infected patients receiving TDF and analyze the relationship between tubule dysfunction and GFR decline.2.To culture human proximal tubule epithelial cells in vitro,and observe the relationship between transporter injury and ER stress induced by tenofovir(TFV).3.To evaluate the function of proximal tubule and the glomerular renal functional reserve in the GS cohort,and explore the possible mechanisms of proximal tubule dysfunction and renal function secondary to distal tubule dysfunction.MethodsPart ?:Clinical characteristics of proximal tubule injury caused by TDF and potential mechanism1.Characteristics of kidney injury of HIV-infected patients treated with TDFHIV-infected patients treated with TDF who were regularly followed up in Peking Union Medical College from Sep 1,2001 to August 31,2019 were enrolled.Baseline and follow-up data were collected.Proximal tubule dysfunction was defined as meeting two or more of following criteria:hypophosphatemia,hypouricemia,low carbon dioxide binding capacity,positive urine glucose,and positive urine protein.Rapid deterioration of renal function was defined as the annual decline rate of estimated glomerular filtration rate(eGFR)exceeding 5ml/min/1.73m2.The percentage and clinical characteristics of proximal tubule dysfunction and renal function impairment were analyzed,as well as the relationship between them.2.The relationship between proximal tubule injury caused by TFV and ER stressHuman renal proximal tubular epithelial cell line(HK2)was cultured.The morphology was observed by inverted phase contrast microscope,and immunofluorescence was used for detection of specific markers(CK18 and megalin)and transporters(SGLT2,NaPi-?a,and URATI).Cell proliferation and cytotoxicity assays was conducted to explore the effect of TFV to the survival rate of HK2 cells.Appropriate concentration of TFV was used to stimulate HK2 cells,and the impact on transporters and ER stress was evaluated.Part ?:Clinical characteristics of renal proximal tubule function in GS patientsPatients who were diagnosed with GS(confirmed by gene sequencing)in Peking Union Medical College Hospital from August 1,2005 to December 31,2019 were enrolled,as well as the selected gender-and age-matched healthy controls.For patients whose SLC12A3 gene was sequenced as a single heterozygous mutation,further whole exome sequencing was performed to clarify the gene mutation.Clinical data including demographic information,symptoms,and hematuria tests were collected.The hydrochlorothiazide test was performed to assess the function of NCC.Characteristics of calcium and phosphate metabolism and uric acid of patients were analyzed.The glomerular renal functional reserve in GS patients were evaluated by acute protein load test.ResultsPart ?:Clinical characteristics of proximal tubule injury caused by TDF and potential mechanism1.A total of 375 HIV-infected patients receiving TDF were enrolled,mainly males(90.1%),with an average follow-up duration of 37.9±26.1 months,up to 116 months.The proportion of proximal tubule injury was 6.7%,and the most common clinical manifestations were proteinuria(20.3%)and hypophosphatemia(12.3%).Proximal tubule dysfunction was significantly associated with low body weight,but was not associated with age,TDF course,baseline viral load,baseline CD4+T lymphocyte count.2.A total of 373 patients had complete creatinine result during the follow-up.The eGFR at the end of the follow-up was significantly lower than the baseline(104.6±15.2 vs.110.6±14.2 ml/min/1.73m2,P<0.001).The average annual decline rate of eGFR was 5.0±22.7 ml/min/1.73m2,of which 23.6%of patients had an annual decline rate of eGFR exceeding 5 ml/min/1.73m2.Female was significantly related to the high annual decline rate of eGFR.3.Cell experiments in vitro:HK2 cells were elliptical or polygonal,and looked like paving stone when fused together.The immunofluorescence detection was positive for CK18,megalin,SGLT2,NaPi-?a,and URAT1.Cell proliferation and cytotoxicity assays showed that the survival rate of HK2 cells was more than 95%under different concentrations of TFV for 24h,while below 95%under 2.5?mol/L for 48h.When given TFV with a concentration of 1 ?mol/L for 48h,the expression level of transporters involving SGLT2,URAT1,and NaPi-IIa,were declined,while the expression level for GRP78/BiP showed no difference.Part ?:Clinical characteristics of renal proximal tubule function in GS patients1.A total of 125 patients with genetic diagnosed GS were enrolled,and 33 patients(26.4%)were with only a single heterozygous mutation.Twenty-five patients completed whole exome sequencing,and 9 patients(36%)were finally diagnosed with compound heterozygous mutations.Seven mutation sites were newly detected by whole exome sequencing,including 3 missense mutations,1 splice-site point mutation and 3 intron mutations.2.Compared with healthy controls,GS patients had higher blood calcium level.However,urinary calcium,serum magnesium,parathyroid hormone,and 25-hydroxyvitamin D were lower in GS patients.Blood phosphate was not significantly different between the two groups.Lower blood magnesium level was associated with lower parathyroid hormone level.Low levels of ionized calcium are associated with metabolic alkalosis.3.Serum uric acid in GS patients was significantly higher than that in healthy controls.The proportion of men and women with hyperuricemia were 19.4%and 34.5%,respectively.Urinary fraction excretion of uric acid in the hyperuricemia group was significantly lower than that of patients with normal serum uric acid.4.In 4 GS patients,the mean glomerular renal functional reserve was 66.7±21.5 ml/min/1.73m2 by creatinine clearance,and 12.1±4.4 ml/min/1.73m2 by cystatin C-based eGFR,both of which showed no differences with health controls.ConclusionUnder the research conditions of this study:1.Among the HIV-infected patients treated with TDF,6.7%had proximal tubule dysfunction and 23.6%showed annual decline rate of eGFR exceeding 5ml/min/1.73m2.Proximal tubule dysfunction wasn't related to annual decline rate of eGFR.2.The protein levels of proximal tubular transporters(SGLT2,URAT1,NaPi-IIa)were downregulated by TFV.3.In GS patients,low urinary calcium,high serum calcium and high serum uric acid were related to the compensation of proximal tubular function.