A 2DE map of the urine proteome in the cat: effect of Chronic Kidney Disease

Introduction Urine is considered an ideal source of clinical biomarkers as it can be obtained noninvasively, repeatedly and in adequate amounts. In veterinary medicine, the application of proteomics techniques is still very limited. The aim of our work was to produce a preliminary map of the urine proteome of the healthy cats (Felis catus) and to compare it with the proteome of cats affected by chronic kidney disease (CKD). For that we have used an approach based on two-dimensional electrophoresis and protein identification using mass spectrometry (MS). Materials and Methods Urine samples were collected by cystocentesis from 4 healthy and 4 cats affected by CKD and analyzed by 2DE. The first dimension was performed by isoelectric focusing on 17 cm long IPG strips (pH 3-10); the second dimension was performed on 10% SDS-PAGE and stained with colloidal Coomassie. Spots were excised from the gel, reduced, alkylated and digested with trypsin and identified using ESI-Q-TOF MS (Campos et al., 2013). Results and Discussions 2DE allowed the separation of 66 spots in the urine proteome of healthy and CKD cats. Eighteen spots were overrepresented in CKD and nine spots were underrepresented. The 27 differentially expressed spots and the nine most abundant common spots were excised from the gels for MS identification (Figure 1; Table 1). Preliminary 2D map. 21 spots yielded significant results by MS, producing a preliminary feline urine map, including 13 proteins that may be functionally classified as transport (38%), immune and cellular response (38%), and cellular communication and growth (15%). The most abundant protein was cauxin, a serine esterase produced by healthy tubular cells, specifically excreted in urine of cats and probably involved in the synthesis of felinine pheromone (Miyazaki et al., 2007). The transport proteins, albumin, transferrin, haemopexin and haptoglobin all derive from plasma and have been identified as common components of urine also from healthy humans (Candiano et al., 2010). Among the proteins involved in immune and cellular defence response, we identified IgK light chain, protein AMBP and uromodulin. Differently from dogs (Brandt et al., 2014) and humans (Lhotta, 2010), uromodulin is not the most abundant urine-specific protein in cats. The remaining proteins, perlecan and fetuin-A, are involved in cell communication and growth. In particular, perlecan, a negatively charged proteoglycan of the glomerular filtration barrier, has also been identified in dog urine (Nabity et al., 2011). Effect of CKD. Regarding the effect of CKD on the urine proteome, seven differentially represented proteins have been identified These proteins can be indicative of tubular dysfunction when not reabsorbed (e.g. RBP) or not secreted (e.g. uromodulin and cauxin) and could be studied as putative biomarkers of nephropathy. Among the overrepresented proteins, retinol binding protein (RBP) is a 22 kDa protein freely filtered by the glomerulus and reabsorbed by the tubules. The appearance of RBP in urine is a marker of impaired tubular function in humans (Pallet et al., 2014) and it has also been reported in dogs (Nabity et al., 2011) and cats (van Hoek et al., 2008). Interesting underrepresented proteins were uromodulin and cauxin. Uromodulin is a glycoprotein produced by healthy tubular cells and its disappearance has been already proved in humans affected by CKD (Lhotta, 2010) and could be applied also in cats. Regarding cauxin, according to Miyazaki et al., (2007) this protein could be a promising biomarker for the determination of tubular damage in CKD cats. Conclusions 2DE was essential in fractionation of the complex urine proteome in cats, producing a preliminary map that included 13 proteins. In particular, uromodulin, cauxin and perlecan, specifically secreted in urine, could help in the evaluation of renal function. Seven proteins were differentially represented in CKD cats suggesting their use as a putative biomarker of nephropathy in cats and possibly in other veterinary species