• GFR
• dog
• cTnI
• CRP
• CKD
• cat
• Ca x P product
• alpha-tochopherol
• ionized calcium

SUMMARY

Chronic kidney disease (CKD) is a clinical syndrome with a high prevalence both in human and veterinary medicine. Being able to formulate an early diagnosis of CKD can allow veterinarians to introduce a dietary and medical therapy, which can dramatically reduce progression towards end stage renal disease (ESRD)1. At the same time, strong evidences of a deep relationship between heart and kidney in the progression of CKD in humans, have led to a greater attention towards potential markers of prognosis and negative outcome of the disease, even in small animal population. The present research project has been organized in two sections2,3.

FIRST SECTION
The aim of the first part of the study has been to validate a glomerular filtration rate (GFR) method, at a low number of plasma samples, through the plasma clearance of iohexol in both CKD and clinically healthy cats.
MATERIALS AND METHODS - After the owners’ informed consent, 53 clinically healthy and 14 CKD cats have been submitted to a first blood sample (0) and to an eight-hour clearance study. Iohexol (Omipaque® 300 mgI/ml) has been intravenous injected at the dose of 64.7 mg/kg body weight. Heparinised blood samples have been taken at 5 and 30 minutes and 1, 2, 4, 6 and 8 hours from the completion of iohexol injection. Plasma has been obtained and each sample has been stored at -20 C° till extraction process and HPLC analysis. Pharmacokinetic analysis has been performed through the software Easy Fit® for Macintosh (Istituto Mario Negri, Milano, Italia) and, for each subject of HC and CKD group, plasma concentration of iohexol/time curves have been analyzed through a non-compartimental kinetic model. Then, a pharmacokinetic analysis has been carried on after the application of simplified models (Model A, Model B, Model C and Model D) with a lower number of blood samples. Statistical analysis has been performed by using the software GraphPad Prism 4 for Macintosh, USA.
RESULTS – t-test analysis (p<0.05) between GFR of CH and CKD patients has shown a significant difference between the two groups of subjects, not only for reference method (p=0.003), but also for Model A (p=0.0005), Model B (p=0.01), Model C (p=0.001) and Model D (p=0.004). Pearson correlation analysis (p<0.05) between each simplified model and reference method has shown a positive linear correlation with vey high values of Pearson r and R2.
CONCLUSIONS - The present study has validated a safe, simple and accurate three-sample HPLC method (5’ – 30’ – 1 hour) for the determination of GFR through the plasma clearance of iohexol in feline patients. This model represents an attractive and cheap alternative to cumbersome plasma clearance methods, with a dramatic applicatory potential in different clinical settings.

SECOND SECTION
The aim of the second part of the study has been to assess serum ionized calcium, total calcium, calcium corrected for albumin (cCaAlb), calcium corrected for total proteins (cCaPt), Ca x P product (Ca x P), cardiac troponin I (cTnI), C-reactive proteine (CRP) and α-tochopherol in CH and CKD canine patients at different stages of the disease.
MATERIALS AND METHODS – serum ionized calcium, total calcium, cCaAlb and cCaPt have been determined in 301 CKD and 125 CH patients, while Ca x P, cTnI, CRP and α-tochopherol have been assessed in 13 IRIS 1, 7 IRIS 2, 13 IRIS 3 and 11 IRIS 4 subjects. Ionized calcium has been determined through a selective ion method (STAT PROFILE® pHOx Plus, GEPA, Milano, Italy), cTnI through an immunometric method (IMMUNOLITE 2000® Immunoassay System), CRP through an immunometric method (RANDOX immunoturbidimetric kit for CRP, Vet Med Lab, IDEXX, Germany) and α-tochopherol through HPLC (Chromosystems-Diagnostic Kit HPLC & LC/MS, Munchen, Germany). Statistical analysis has been performed by using the software GraphPad Prism 4 for Macintosh, USA.
RESULTS – One-way ANOVA has reported a significant difference (p<0.0001) in ionized calcium concentration among CH, IRIS 1, IRIS 2, IRIS 3 and IRIS 4 and χ2 analysis has shown a significant difference (p<0.0001) in the number of patients with hyper, hypo and normocalcemia according to the progression of the disease. One-way ANOVA among CH subjects and IRIS 1, IRIS 2, IRIS 3 and IRIS 4 patients has reported a significant difference in the mean value of Ca x P (p<0.0001). No significant correlation has been found between Ca x P and plasma creatinine in any of IRIS classes. The number of patients with Ca x P above 70 mg/dl has been reported to increase significantly (p<0.0001) with the severity of CKD, as well as the number of dead patients (p<0.0008). Finally, Kaplan-Meier survival curve has shown a significantly higher percentage of survival (p<0.0002) of CKD patients with Ca x P below 70, compared to patients with Ca x P above 70. One-way ANOVA among CH subjects and IRIS 1, IRIS 2, IRIS 3 and IRIS 4 patients has reported a significant difference in the mean value of cTnI (p<0.02). No significant correlation has been found between cTnI and plasma creatinine in any of IRIS classes. The number of patients with cTnI above 0.20 ng/ml has been reported to increase significantly (p<0.0001) with the severity of CKD, as well as the number of dead patients (p<0.02). Finally, Kaplan-Meier survival curve has shown a significantly higher percentage of survival (p<0.0002) of CKD patients with cTnI below 0.20 ng/ml, compared to patients with cTnI above 0.20 ng/ml. One-way ANOVA among CH subjects and IRIS 1, IRIS 2, IRIS 3 and IRIS 4 patients has reported a significant difference in the mean value of CRP (p<0.0001). No significant correlation has been found between CRP and plasma creatinine in any of IRIS classes. The number of patients with CRP above 9.7 mg/l has been reported to increase significantly (p<0.0001) with the severity of CKD, as well as the number of dead patients (p<0.0009). Finally, Kaplan-Meier survival curve has shown a significantly higher percentage of survival (p<0.001) of CKD patients with CRP below 9.7 mg/l, compared to patients with CRP above 9.7 mg/l. One-way ANOVA among CH subjects and IRIS 1, IRIS 2, IRIS 3 and IRIS 4 patients has reported a significant difference in the mean value of α-tochopherol (p<0.0002). A significant correlation (p=0.00) has been found between α-tochopherol and plasma creatinine in IRIS 2. The number of patients with α-tochopherol below 21.6 ppm has been reported to increase significantly (p<0.0001) with the severity of CKD. No significant difference in the number of survived and dead patients has been found between subjects with α-tochopherol below and above 21.6 ppm. Finally, Kaplan-Meier survival curve has shown no significant difference.
CONCLUSIONS – The present study has demonstrated a significant increase in Ca x P, cTnI and CRP serum concentration according to the progression of CKD. Ca x P, cTnI and CRP have shown a prognostic, not IRIS stage-dependent, value and a significant correlation towards mortality. In CKD dogs, as well as in humans, alterations of calcium-phosphate metabolism, cardiovascular injury and inflammation seemed to play a significant role in the progression and negative outcome of CKD. No correlation has been reported between mortality and α-tochopherol, although a significant serum reduction with the progression of CKD has been shown.