NOVEL BIOMARKER IN CATS AFFECTED WITH SPONTANEOUS CKD: FOCUS ON HYPERTENSION AND PROTEINURIA

Chronic kidney disease (CKD) is a common disease of elderly cats characterized by a progressive course and a high mortality rate. The development and the progression of CKD depend on the presence of complications such as proteinuria and hypertension. Hence, a correct staging of the disease based to these latter pathologic states together with serum creatinine, as suggested by the International Renal Interest Society (IRIS) guidelines, is recommended to properly treat affected cats. In this regard, hypertension is often difficult to confirm given the high frequency of white-coat hypertension in this specie. Moreover, although decisive progresses have been achieved in the last years, an early diagnosis of CKD or of hypertension and proteinuria associated with CKD is still challenging in cats. Early identification of ongoing kidney injury may allow veterinarians to prevent worsening of the disease and ultimately to improve the quality of life of affected feline patients. The first part of this thesis was focused on a preliminary evaluation of preanalytical and analytical variability of parameters used in IRIS staging of CKD: preanalytical and analytical variability of urinary protein-to-creatinine (UPC) ratio (study 1); the effect of hematuria and pyuria on UPC (study 2); and the physiologic concentration of the novel biomarker of glomerular filtration rate symmetric dimethylarginine (SDMA) in Holy Birman cats (study 3). The second main part of this thesis was aimed to assess how biomarkers such as serum big endothelin-1 (big-ET1), homocysteine (Hcy), aldosterone and urinary big-ET1, alpha-1 microglobulin (A1MG) and the presence of tubular proteins in urine (evaluated with sodium dodecyl sulfate-agarose gel electrophoresis, SDS-AGE) may allow an early diagnosis of CKD, may identify cats at risk of severe worsening of the disease, or may be a predictor of hypertension (study 4). To this aim, privately owned cats (i.e. clinically healthy cats at risk to develop CKD and cats affected with CKD) were prospectively enrolled and sampled over time. The results of study 1 demonstrated that proteinuria was stable up to to 6 hours at room temperature, 1 week at refrigeration temperature, four freeze-thaw cycles and 4 weeks at −20°C, whereas the use of different analytical methods resulted in inaccuracy and suboptimal concordance in classifying samples according to IRIS substaging, that in turn can potentially affect clinical decisions, make questionable the comparison of UPC results between different laboratories, and have significant impact in substaging cats affected by CKD. Study 2 showed that hematuria could affect proteinuria, suggesting that diagnosis and staging of renal proteinuria should be avoided or interpreted with caution in samples with high hematuria. Study 3 highlighted that SDMA may be a better marker of CKD than creatinine in Birman cats and the analysis of both creatinine and SDMA could help prevent errors in diagnosing and staging CKD in Birman cats. In study 4, different results were found with the different biomarkers. Big-ET1 did not give satisfying results in serum, whereas in urine the ELISA method yielded satisfying validation results, supporting its introduction in this specie. Urinary big-ET1 was associated with the severity of CKD and proteinuria, revealing that it could be a promising aid in nephropatic cats and could shed light on the pathogenesis of tubulo-interstitial and glomerular damage in cats with CKD. The method to measure Hcy can be considered reliable in cats according to the validation tests. Serum Hcy increased progressively with the progressive increase in severity of CKD and the detection of high Hcy in some non-azotemic patients with CKD could add this new marker to those currently available for the identification and staging of the kidney disease of the cat. Conversely, no direct relationship was found between Hcy and hypertension. Aldosterone was not associated with severity of CKD, proteinuria and SBP. Therefore, neither Hcy nor aldosterone can be considered an indicator of hypertension in cats affected with CKD. The ELISA kit used for A1MG measurement failed all the validation tests and results were considered unacceptable; further studies are therefore needed to investigate the presence of A1MG in cats with CKD and tubular damage. SDS-AGE showed the consistent presence of bands with high molecular weight in healthy cats, suggesting that this pattern is normal in this specie and has to be taken into account while evaluating proteinuria in cats; tubular bands were frequent in patients with CKD at any stage, confirming the predominant tubule-interstitial damage of this disease in cats and suggesting that SDS-AGE can be considered a valuable aid in diagnostic approach to feline CKD.