Serum intact parathyroid hormone levels in cats with chronic kidney disease

Chronic kidney disease (CKD) is frequently observed in cats and it is characterized as a multisystemic illness, caused by several underlying metabolic changes, and secondary renal hyperparathyroidism (SRHPT) is relatively common; usually it is associated with the progression of renal disease and poor prognosis. This study aimed at determining the frequency of SRHPT, and discussing possible mechanisms that could contribute to the development of SRHPT in cats at different stages of CKD through the evaluation of calcium and phosphorus metabolism, as well as acid-base status. Forty owned cats with CKD were included and divided into three groups, according to the stages of the disease, classified according to the International Renal Interest Society (IRIS) as Stage II (n=12), Stage III (n=22) and Stage IV (n=6). Control group was composed of 21 clinically healthy cats. Increased serum intact parathyroid hormone (iPTH) concentrations were observed in most CKD cats in all stages, and mainly in Stage IV, which hyperphosphatemia and ionized hypocalcemia were detected and associated to the cause for the development of SRHPT. In Stages II and III, however, ionized hypercalcemia was noticed suggesting that the development of SRHPT might be associated with other factors, and metabolic acidosis could be involved to the increase of serum ionized calcium. Therefore, causes for the development of SRHPT seem to be multifactorial and they must be further investigated, mainly in the early stages of CKD in cats, as hyperphosphatemia and ionized hypocalcemia could not be the only factors involved

Osteokines and the vasculature: a review of the in vitro effects of osteocalcin, fibroblast growth factor-23 and lipocalin-2

Bone-derived factors that demonstrate extra-skeletal functions, also termed osteokines, are fast becoming a highly interesting and focused area of cross-disciplinary endocrine research. Osteocalcin (OCN), fibroblast growth factor-23 (FGF23) and lipocalin-2 (LCN-2), produced in bone, comprise an important endocrine system that is finely tuned with other organs to ensure homeostatic balance and health. This review aims to evaluate in vitro evidence of the direct involvement of these proteins in vascular cells and whether any causal roles in cardiovascular disease or inflammation can be supported. PubMed, Medline, Embase and Google Scholar were searched for relevant research articles investigating the exogenous addition of OCN, FGF23 or LCN-2 to vascular smooth muscle or endothelial cells. Overall, these osteokines are directly vasoactive across a range of human and animal vascular cells. Both OCN and FGF23 have anti-apoptotic properties and increase eNOS phosphorylation and nitric oxide production through Akt signalling in human endothelial cells. OCN improves intracellular insulin signalling and demonstrates protective effects against endoplasmic reticulum stress in murine and human endothelial cells. OCN may be involved in calcification but further research is warranted, while there is no evidence for a pro-calcific effect of FGF23 in vitro. FGF23 and LCN-2 increase proliferation in some cell types and increase and decrease reactive oxygen species generation, respectively. LCN-2 also has anti-apoptotic effects but may increase endoplasmic reticulum stress as well as have pro-inflammatory and pro-angiogenic properties in human vascular endothelial and smooth muscle cells. There is no strong evidence to support a pathological role of OCN or FGF23 in the vasculature based on these findings. In contrast, they may in fact support normal endothelial functioning, vascular homeostasis and vasodilation. No studies examined whether OCN or FGF23 may have a role in vascular inflammation. Limited studies with LCN-2 indicate a pro-inflammatory and possible pathological role in the vasculature but further mechanistic data is required. Overall, these osteokines pose intriguing functions which should be investigated comprehensively to assess their relevance to cardiovascular disease and health in humans

Molecular Mechanisms by which Phosphate Modulates Angiotensin II-Induced Hypertension

Hypertension or high blood pressure is a critical health burden. Adherence to certain diets lowers the risk of hypertension. Preliminary data have shown a significant decrease in BP in mice receiving high dietary phosphate. High dietary phosphate stimulates the release of FGF23 from bones, and binds to its renal FGFR, causing downregulation in the expression of sodium-phosphate-dependent cotransporters (NaPIIa/c) located at the level of proximal convoluted tubules and encoded by SLC34A1/3 respectively, thus contributing to a decrease in the reuptake of phosphate coupled with sodium ion and increased excretion in urine. Our aim is to investigate the underlying mechanisms by which phosphate modulates hypertension by influencing the activity of these transporters. Initially, hypertension was induced by infusing male mice with Ang II pumps subcutaneously and fed low: 0.15% P, Control: 0.3%, High: 1.5% P. Mice were sacrificed, and kidneys were harvested to determine mRNA expression levels of FGF23 and SLC34A1-3. Urine and plasma phosphorous were analyzed. To assess the effect of phosphate on renal damage, DHE stain for ROS and NGAL were done. Our results showed that in hypertensive mice fed high dietary phosphate there was an alteration in FGF23 and downregulation in SLC34A1-3, and changes in urine phosphate excretion with variation in plasma phosphate. In addition, ROS was found in all groups of hypertensive mice and NGAL was increased in hypertensive mice receiving a high dietary group. Dietary phosphate might be used as a non-pharmacological intervention in the prevention and control of hypertension, however, further investigations into phosphate regulatory mechanisms and its effect on kidney damage should be assessed

EARLY DIAGNOSTIC AND PROGNOSTIC MARKERS OF CHRONIC KIDNEY DISEASE (CKD) IN CANINE AND FELINE PATIENTS

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

Tumoral calcinosis: Identification of a novel recessive mutation in fibroblast growth factor-23 (FGF-23)

Tumoral calcinosis (TC) is a rare genetic disorder characterized by periarticular cystic and solid tumorals calcifications. It is characterized by hyperphospatemia and an elevated serum of calcitriol concentration in every patients. The hyperphosphatemia results form an increase in capacity of renal tubular phosphate reabsorption. The identification of phosphotonin family hormones suggest that mutations of these molecules could be involved in the pathogenesis of TC. One of these molecules is represented by FGF-23. The TC phenotype is similar to that described in the FGF-23 knockout mice. In the present study we described a new FGF-23 mutation in a subject affected by TC.A Caucasian women (years 67) was examined for a history ofectopic calcification. Biochemical exams showed and hyperphosphathemia and hyperphosphaturia with normal value ofPTH and inappropriately normal level of 1–25 (OH)2 D3. The patient presented a big shoulder calcification and also a calcification of femoral artery. We expanded the family tree through detailed family histories, which importantly revealed that parents were consanguineous. Hystologically the mass was characterized by calcium deposition and granulomatous reaction around the mass. Genomic DNA was extracted from blood collected from the patient, her daughter and her grandchild by standard procedure. DNA was not available from her parents. All three FGF-23coding exons, as well as conserved splice sites, were amplified by standard PCR procedure. Nucleotide sequences were determined by direct sequencing with a DNA kit and an automated DNA sequencer (ABI PRISM 3100 - Perkiln-Elmer Corp). We discovered a new homozygous codon 41, His/Gln (CAC-CAA) substitution in exon 1 of FGF-23 gene in the affected patient. A heterozygous substitution was present in the daughter. No mutation were found in the two children. FGF-23 gene mutation was not found in the SNP database (www.ncbi.nih.gov/snp). In summary, a recessive mutation in FGF 23 causes TC. Understanding the functional significance and molecular physiology of this novel mutation will reveal critical information regarding the role of FGF-23 in states of normal and of disorder of phosphate homeostasis