1. Identification of new biomarkers for disease progression in autosomal dominant polycystic kidney disease
- Author
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Magayr, Tajdida and Ong, Albert
- Subjects
610 - Abstract
Introduction: ADPKD is the most common renal genetic disease and the fourth most common cause of end-stage renal disease (ESRD) world-wide. Although PKD1 and PKD2 patients have different phenotypes, there is also significant intra-familial variability in disease progression suggesting that other genetic or environmental factors have major influences on disease progression. With the availability of new therapies, there is an urgent need to identify novel biomarkers which can identify ADPKD patients at risk of rapid disease progression at an early stage. Methods: Spot urine specimens were collected from consecutive patients with ADPKD (n = 130) attending a PKD clinic at the Sheffield Kidney Institute and healthy controls (n = 33). Urinary exosomes were isolated from urine samples by ultracentrifugation and confirmed by electron microscopy and western blotting. The expression of exosomes associated proteins, namely polycystin-1, polycystin-2 and ErbB4, cell free microRNAs, exosomes associated microRNAs and angiogenesis related proteins were examined in spot urine samples from healthy volunteers, ADPKD patients with early (eGFR > 60 ml/min) or late (eGFR < 60 ml/min) disease using western blotting, quantitative PCR, next generation sequencing, angiogenesis proteomics array and ELISA. Results: Electron microscopy confirmed the presence of multiple (< 100 nm) vesicles in the pelleted fraction and western blotting confirmed that the exosome specific protein TSG-101 was only detectable in the pellet. A significant decrease in exosome associated PC1 and PC2, normalised for TSG-101, was clearly detected in ADPKD patients with both early and late disease compared to healthy volunteers. I also detected an ErbB4 positive band at 80 kDa, corresponding to the known C-terminal intracellular domain (ICD) generated by ?-secretase mediated cleavage. In contrast to PC1 and PC2, I found that expression of the 80 kDa band was significantly increased in ADPKD patients with late disease compared to healthy controls as defined by baseline eGFR. These changes in ErbB4 confirm our recent findings in human cystic cells and murine Pkd1 tissue. Cell free urine contains very low levels of microRNAs compared to exosomes. A specific set of microRNAs was found downregulated in urine samples from patients with ADPKD when compared to the controls, including miR-30 family, miR-192-5p, miR193b-3p and miR-194-5p. The differentially expressed microRNAs were found negatively correlated with eGFR and positively correlated with mean kidney length, and were significantly better predictors of the rate of disease progression in ADPKD compared to mean kidney length and together had a combinatory effect. In addition, a specific signature for angiogenesis related proteins was found in urine samples from patients with ADPKD compared to healthy controls. Relative expression of MCP-1 in urine is significantly correlated with eGFR and mean kidney length, and it is a significantly better predictor of ADPKD progression compared to mean kidney length. Conclusions: This study has shown that exosomes purified from ADPKD patients are a promising source of urinary biomarkers. Progressive ADPKD is associated with alteration in urine profiling of exosomes associated proteins, microRNAs as well as angiogenesis related factors. ErbB4, miR-30 family, miR-192-5p and miR-193b-3p were the best markers for disease progression (eGFR slope) outperforming mean kidney length. The discovery of distinct biomarker profiles will allow detection of those at high risk of disease progression so as to identify those who might benefit from earlier and more intensive intervention.
- Published
- 2017