New methods of detecting kidney disease biomarkers in biofluids

Early diagnosis is crucial for facilitating treatment and limiting progression of chronic kidney disease (CKD). Current diagnostic approaches mainly focus on blood tests. However, blood analysis is invasive and requires trained personnel and specialised equipment. Hence, this thesis sought to use ATR-FTIR spectroscopy to detect components in non-invasive biofluids (urine and saliva) as early diagnostic tools. APRT-deficiency is characterised by the presence of typical crystalline 2,8 dihydroxyadenine (DHA) in the kidney and/or in urine which is currently detected by phase-contrast microscopy or more complex blood analysis of APRT enzyme activity. In this study, an optimised ATR-FTIR spectroscopy protocol for the detection of DHA was developed and used to analyse urine samples from patients with APRT-deficiency. The spectra showed typical crystalline DHA can be identified in urine and distinguished from other urinary crystals. The concentration of typical crystalline DHA was also quantitated using a calibration curve. Further, this study identified a different form of DHA in some of patients’ urine. Imaging of different forms of DHA showed differences in appearance and structure between the two forms. A method for purification of endogenous typical crystalline DHA from patient urine was developed. In vitro treatment of human proximal tubular epithelial cells (HKC-8) with the different forms of DHA (0-200 µM) showed all forms of DHA induced a dose-dependent increase in expression of inflammatory genes (TNF-a, MCP-1, Kim-1). ATR-FTIR spectroscopy was also used to quantitate salivary urea in CKD patients to determine whether salivary urea can provide an indicator of CKD stage. The data showed an increase in salivary urea in CKD3-5 compared to healthy controls. A positive correlation between blood urea and salivary urea suggested that salivary urea could be an additional parameter for CKD diagnosis. Renal cells are shed into urine and the populations of cells are likely to vary with disease. The third part of the study explored the potential utility of urinary cells as biomarkers of kidney disease. Normal urine (N=18) contains low numbers of viable cells (1000-4000 cells/100 mL). Normal urinary cells exhibited limited in vitro growth with inadequate numbers for downstream analyses of cell type-specific markers. Analysis of freshly isolated cells showed wide variation in RNA yield and in expression of renal cell type-specific markers confounding comparisons of normal and diseased urines. Further investigations are required to determine the wider utility of this approach. Studies in this thesis showed that ATR-FTIR spectroscopy provides a simple, cost-effective method for detecting urinary DHA and salivary urea for diagnosis of APRT deficiency and CKD, respectively, with wider application to analysis of other urinary components. Both the method and instrumentation can be adapted for clinical use as a point-of-care screening tool