Associations Between Urinary Protein Biomarkers And Disease Progression In Patients With Autosomal Dominant Polycystic Kidney Disease

Aim of the study: In the current study,we measured the spectrum of urine protein from patients with autosomal dominant polycystic kidney disease(ADPKD),which reflects the injuries in each nephron segment,the renal inflammation and the activity of intra-renal complement system.In order to screen out the useful urinary biomarkers for ADPKD patients,we studied the correlation of the spectrum of urine protein with renal functional parameters,kidney volume,and PKD gene mutations,which may reflect ADPKD disease progression.Methods:The first part of study is a multi-center(8 hospitals)observational cross-sectional study,starting from March 2014 to August 2016.The hospitalized patients or outpatients with ADPKD were recruited according to their CKD stage and the male to female ratio is 1:1.The medical history and data from routine lab test were collected.The protein concentrations of fasting morning urine samples and fasting serum specimens were measured by enzyme-linked immunosorbent assay(ELISA),scattering turbidimetry,and transmission turbidimetry.The magnetic resonance imaging(MRI)was used to measure the total kidney volume(TKV).The normal control subjects from the medical examination center were matched to patients in CKD1 group and with the male/female ratio of 1:1.Ten urine proteins were studied and standardized with the urine creatinine concentration.Parameters for glomerular damage: the urinary albumin/creatinine ratio(ACR),urinary transferrin/creatinine(TRF/Cr),urinary immunoglobulin G/creatinine(IgG/Cr);parameters for proximal renal tubular damage: urinary kidney injury molecule-1/creatinine(KIM-1/Cr),urinary liver type fatty acid binding protein/creatinine(L-FABP/Cr),urinary neutrophil gelatinase-associated lipocalin/creatinine(NGAL/Cr),urinary retinol binding protein/creatinine(RBP/Cr),urine alpha 1-microglobulin/creatinine(?1-MG/Cr);inflammatory factors: urine monocyte chemoattractant protein 1(MCP-1/Cr);complement system: urine complement factor B(CFB/Cr).The second and the third part is a single-center,prospective,longitudinal cohort study,and is followed up for three years from March 2014 to December 2016(ADPKD patients recruited with baseline estimated glomerular filtration rate(eGFR)above 15 ml/min*1.73 m2 and the majority of patients with eGFR above 30 ml/min*1.73 m2).During the follow-up,patients were re-visited every half year or 1 years.In each visit,the medical history was collected,and the physical examination,routine laboratory test,MRI based TKV measurement,and urine protein assessment were performed.In the third part,the PKD gene mutations were detected using a high-throughput sequencing method.Results:The first part "the multicenter cross-sectional study for the relationship of urine protein spectrum and disease progression in autosomal dominant polycystic kidney disease patients” : this study recruited 200 patients with ADPKD,including 40 patients in each CKD stage(CKD1-5)with male/female ratio of 1:1,and also enrolled 40 healthy control subjects matched to CKD1 patients.Three glomerular damage parameters urinary ACR/Cr,IgG/Cr,and TRF/Cr were significantly increased in CKD1 stage compared with healthy controls(P < 0.01),and were kept steady during CKD1-2 stage,but were further increased till CKD3-5 stage(P < 0.05,TRF/Cr were further increased till CKD4-5 stage).Urine ?1-MG/Cr and RBP/Cr,two markers for the proximal tubule injury,were not changed at the early stage of CKD,but were increased till CKD stage 3(P < 0.05);urinary NGAL/Cr was significantly increased in CKD1 stage compared with healthy controls(P < 0.05),but L-FABP/Cr was not changed at the early stage of disease compared to normal controls(P > 0.05),while among patients,both were increased significantly at CKD4-5 stage.Interestingly,the serum concentration of NGAL was increased at CKD2 stage(P < 0.05);at CKD1 stage,the urinary KIM-1/Cr was significantly higher than normal controls,and with the progress of CKD,KIM-1/Cr was increased gradually,which was peaked at CKD4 stage(P < 0.05).Complement system: the CFB/Cr at CKD1 to CKD3 stage was similar to that in normal subjects(P > 0.05),but at CKD4-5 the urinary CFB/Cr was significantly increased(P < 0.001).Inflammation factor: at CKD1 stage,the urine MCP-1/Cr were significantly higher in ADPKD patients compared with normal controls(P < 0.05),and with the decline of eGFR,urinary MCP-1/Cr was increased(P < 0.001).Interestingly,the serum concentrations of MCP-1 was not changed with the disease progress(P > 0.05).All urinary parameters were negatively correlated with eGFR(P < 0.001).For patients at the early stage(eGFR ? 60 ml/min * 1.73 m2),the correlation between urinary MCP-1/Cr and HtTKV was better than other urinary markers(r = 0.396,P < 0.001).For CKD1-3 patients,a further multiple regression analysis found that urinary ACR,?1-MG/Cr,MCP-1/Cr were independently related to HtTKV.The second part “the prospective cohort study for the relationship of urine protein spectrum and disease progression in autosomal dominant polycystic kidney disease patients”: in this cohort,a total of 106 ADPKD patients were included,and the median follow-up time was 2.0 [1.7-2.2] years.Most of the patients in this study had relatively preserved renal function.During the follow-up,the average annual change TKV in ADPKD patients was 79.4 [23.8-148.4] cm3,and the average annual rate of change of TKV(? TKV % / year)was 5.33% [2.48%-10.65%].The average annual change of eGFR was-3.2±6.6 ml/min * 1.73 m2,and the average annual rate of eGFR change was-4.2 ±8.7%.Using ROC curve analysis,we found that except the urine CFB/Cr and MCP-1/Cr,all other urine protein had diagnostic value for the baseline eGFR < 60 ml/min * 1.73 m2.Among these urine proteins,the urine of ?1-MG/Cr had the best diagnosis efficiency,and urine L-FABP/Cr had the second best diagnosis efficiency;Baseline urine MCP-1/Cr could predict ?TKV%/year>5% and ?TKV%/year?6%.Using the multi-factor Logistic regression analysis,we found that baseline urine MCP-1/Cr was an independent predictor for?TKV%/year>5% and ?TKV%/year?6%.The third part "the relationship of urine protein spectrum and gene mutations in autosomal dominant polycystic kidney disease" : in this cohort,a total of 93 patients,mostly from the second part longitudinal cohort study,were recruited,and the median follow-up time was 2.0 [1.8-2.2] years.The positive rate for detecting PKD gene mutations was 78.5%,and the pathogenic mutations were not detected in 20 patients.The proportion of PKD2 mutations,non-truncated mutations in PKD1 gene and truncated mutations in PKD1 gene was 12.9%,20.4% and 45.2% respectively.The negative detection rate for ADPKD patients with no family history was slightly higher than those with family history(36.8% vs 17.6%,P = 0.13).The type of PKD mutations did not affect the growth rate of TKV(P > 0.05).The urinary MCP-1/Cr was higher in patients with PKD1 truncated mutations than those with PKD1 non-truncated mutations(P < 0.05).Multivariate analysis found that the age of patients was negatively correlated with eGFR,and male gender and PKD1 gene mutations(especially PKD1 truncated mutations)were independent risk factors for eGFR decline.Moreover,the multivariate analysis found that urinary ACR,L-FABP/Cr,and KIM-1/Cr were independently negative correlated with eGFR;and if the baseline HtTKV was the dependent variable,only age and urine MCP-1/Cr were independently and positively correlated with HtTKV.Conclusions: Urine MCP-1 had a good correlation with renal function and HtTKV,and it was a risk factor and independent predictor for the fast growth of kidney volume in ADPKD patients.