Identification of Biomarkers for PKD1 Using Urinary Exosomes

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals inherit a defective copy of either PKD1 or PKD2, which encode polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 and PC2 are secreted on urinary exosome-like vesicles (ELVs) (100-nm diameter vesicles), in which PC1 is present in a cleaved form and may be complexed with PC2. Here, label-free quantitative proteomic studies of urine ELVs in an initial discovery cohort (13 individuals with PKD1 mutations and 18 normal controls) revealed that of 2008 ELV proteins, 9 (0.32%) were expressed at significantly different levels in samples from individuals with PKD1 mutations compared to controls (P<0.03). In samples from individuals with PKD1 mutations, levels of PC1 and PC2 were reduced to 54% (P<0.02) and 53% (P<0.001), respectively. Transmembrane protein 2 (TMEM2), a protein with homology to fibrocystin, was 2.1-fold higher in individuals with PKD1 mutations (P<0.03). The PC1/TMEM2 ratio correlated inversely with height-adjusted total kidney volume in the discovery cohort, and the ratio of PC1/TMEM2 or PC2/TMEM2 could be used to distinguish individuals with PKD1 mutations from controls in a confirmation cohort. In summary, results of this study suggest that a test measuring the urine exosomal PC1/TMEM2 or PC2/TMEM2 ratio may have utility in diagnosis and monitoring of polycystic kidney disease. Future studies will focus on increasing sample size and confirming these studies. The data were deposited in the ProteomeXchange (identifier PXD001075).

Keywords: ADPKD; genetic renal disease; polycystic kidney disease.

Figure 1.

Flow diagrams for discovery and confirmatory studies. (A) Discovery: The cohort comprises individuals with PKD, aged <40 years and controlled with a maximum of two antihypertensive drugs, who were nonsmokers, were all of European ancestry, and had an eGFR>60 ml/min per m². The sample is then prepared and mutation analysis is completed. Only individuals with PKD1 mutations are further analyzed by one-dimensional SDS-PAGE, slice recovery, and MS/MS label-free proteomics. Peptide intensities are measured and then Loess normalized and the average intensity per protein is derived. Proteins are analyzed in the band in which they are most highly represented and very low intensity proteins are removed. Each gel slice across the 13 PKD1 and 18 normal individuals is treated as a separate experiment and analyzed using a Welch t test and Bonferroni correction and a q test. (B) The only criteria for entry into the confirmatory cohort is MRI or ultrasonography-positive polycysts in both kidneys. Crude exosomes (exosomes and THP prepared by simple one-step ultracentrifugation) are prepared. Western blotting is performed for TMEM2 and three other proteins with low P and q values and good antibodies (PC1, PC2, and SAHH2 [IRBIT]). Analysis is by quantitative Western blotting with a CCD camera and is quantified using Alphaview software. One-way ANOVA and Tukey’s HSD are used to calculate P values.

Figure 2.

Purification of ELVs. (A and B) ELVs from normal individuals (A) and individuals with PKD1 (B) with 4%–12% SDS-PAGE stained with Coomassie brilliant blue (to note, these samples are run in a randomized manner order in the original gels; however, for this figure, individual lanes are cut and pasted into a montage with the normal and PKD individuals in separate groups). The gel slices are labeled A–J, with A being at the highest molecular mass (250–500 kDa) and J being the lowest (10–15 kDa). (C) ELVs observed at ×40,000 and ×80,000 magnification using transmission electron microscopy (TEM) (note the “punched-out soccer ball” appearance of classic exosomes). DP, definitely pathogenic; F, female; HLP, highly likely pathogenic; LP, likely pathogenic; M, male.

Figure 3.

Volcano plot. Proteins found at significantly different levels in normal and PKD1 individuals, and color maps to the gel slice in which they were found (see key). The x axis shows the log₂ fold change between normal and PKD1 individuals, whereas the y axis shows the −log₁₀ of the P value (A) and q value (B). The diameter of the symbols varies as the square root of the mean ion intensity in the normal cohort.

Figure 4.

Discovery MS/MS data show that PC1/TMEM2 and PC2/TMEM2 ratios are decreased in individuals with PKD1 mutations. (A) The ratios of PC1/TMEM2 and PC2/TMEM2 are decreased in individuals with PKD1 mutations, with P values of 3.3×10⁻¹⁰ and 7.2×10⁻⁷, respectively. Data are not obtained from gel slice B from one normal individual so n=17 for normal individuals. (B) The PC1/TMEM2 ratio correlates well with HtTKV (adjusted R²=0.63; P=0.001 for a linear model; Pearson correlation coefficient r=−0.81), whereas the PC2/TMEM2 ratio had an R²=0.12 (r=−0.45).

Figure 5.

Quantitative Western blotting for PC1, PC2, and TMEM2. (A) Representative Western blots for crude exosomes from four PKD1 individuals (P) and five normal individuals (N) show that PC1 and PC2 tend to be at lower levels in PKD1 individuals than normal individuals, whereas TMEM2 appears to behave in the opposite manner. (B) Tukey boxplots of PC1/TMEM2 ratios (left) and PC2/TMEM2 ratios (right), displaying means and interquartiles, as assessed by quantitative ratiometric Western blot analysis. Statistics: one-way ANOVA with genotype as levels, post hoc Tukey’s HSD. Normal versus PKD1 P<0.001 for PC1/TMEM2; normal versus PKD1 P<0.001 for PC2/TMEM2, and normal versus PKD2 NS for both ratios.

Figure 6.

“Float up” fractionation of human urinary exosomes. Pooled exosomes from three normal individuals are prepared on an initial 5%–30% D₂O gradient, with the sample layered on the top of the gradient. The entire gradient is harvested without subfractionation and the THP pellet is discarded. The recovered total exosomes minus the THP, are pelleted and resuspended in 60% sucrose D₂O (final 50% sucrose) and are underlaid on a second 5%–30% sucrose D₂O gradient and centrifuged for 18 hours at 200,000g. (A) Resultant gradient with position of 14 fractions with their refractive indexes (η), (F#, fraction number). (B) Western blots of the fractions. TMEM2 copurifies with fibrocystin, PC1, PC2, and the MVB/exosome marker ALIX.