Genetic background influences cardiac phenotype in murine chronic kidney disease

Nephrol Dial Transplant (2018) 33: 1129–1137 doi: 10.1093/ndt/gfx332 Advance Access publication 22 December 2017 Samantha Neuburg1, Corey Dussold1, Claire Gerber1, Xueyan Wang1, Connor Francis1, Lixin Qi1, Valentin David1, Myles Wolf2 and Aline Martin1 1 Division of Nephrology and Hypertension, Department of Medicine, Center for Translational Metabolism and Health, Institute for Public Health and Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA and 2Division of Nephrology, Department of Medicine, Duke Clinical Research Institute, Duke University, Durham, NC, USA Correspondence and offprint requests to: Aline Martin; E-mail: ABSTRACT Keywords: chronic kidney disease, Col4a3 null mouse, FGF23, Background. Levels of fibroblast growth factor 23 (FGF23) increase early in chronic kidney disease (CKD) and are independently associated with left ventricular hypertrophy (LVH), heart failure and death. Experimental models of CKD with elevated FGF23 and LVH are needed. We hypothesized that slow rates of CKD progression in the Col4a3 knockout (Col4a3KO) mouse model of CKD would promote development of LVH by prolonging exposure to elevated FGF23. Methods. We studied congenic Col4a3KO and wild-type (WT) mice with either 75% 129X1/SvJ (129Sv) or 94% C57Bl6/J (B6) genomes. Results. B6-Col4a3KO lived longer than 129Sv-Col4a3KO mice (21.4 6 0.6 versus 11.4 6 0.4 weeks; P < 0.05). 10-week-old 129Sv-Col4a3KO mice showed impaired renal function (blood urea nitrogen 191 6 39 versus 34 6 4 mg/dL), hyperphosphatemia (14.1 6 1.4 versus 6.8 6 0.3 mg/dL) and 33-fold higher serum FGF23 levels (P < 0.05 versus WT for each). Consistent with their slower CKD progression, 10 week-old B6-Col4a3KO mice showed milder impairment of renal function than 129SvCol4a3KO mice and modest FGF23 elevation without other alterations of mineral metabolism. At 20 weeks, further declines in renal function in B6-Col4a3KO mice was accompanied by hyperphosphatemia and 8-fold higher FGF23 levels (P < 0.05 versus WT for each). Only the 20-week-old B6-Col4a3KO mice developed LVH (LV mass 125 6 3 versus 98 6 6 mg; P < 0.05 versus WT) in association with significantly increased cardiac expression of FGF receptor 4 (FGFR4) messenger RNA and protein and markers of LVH (Atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta-myosin heavy chain (b-MHC); P < 0.05 versus WT for each). Conclusions. In conclusion, B6-Col4a3KO mice manifest slower CKD progression and longer survival than 129Sv-Col4a3KO mice and can serve as a novel model of cardiorenal disease. genetic background, left ventricular hypertrophy INTRODUCTION Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that regulates phosphate homeostasis. In chronic kidney disease (CKD), disordered bone and mineral metabolism is a common complication that begins early and worsens progressively as kidney function declines. In CKD, an increased level of FGF23 is currently the earliest detectable sign of disordered mineral metabolism and a powerful risk factor for left ventricular hypertrophy (LVH), heart failure and death [1–4]. FGF23 induces LVH through direct activation of FGF receptor 4 (FGFR4) and phospholipase C gamma signaling in cardiac myocytes [5, 6] independent of its coreceptor aKlotho, which exerts cardioprotective effects [7]. However, the exposure time and concentration thresholds required for FGF23 to induce LVH in vivo during CKD progression are unknown. Indeed, investigation of cardiac effects of FGF23 in CKD has been limited by a lack of experimental models that recapitulate the early elevations of FGF23 levels and development of LVH that characterize human CKD. The most frequently used CKD models involve surgical reductions of kidney function in rats and mice (i.e. 5/6 nephrectomy, unilateral ureteral obstruction, ischemia reperfusion) or administration of nephrotoxic diets (high phosphate, adenine). Surgical models are limited by substantial risk of mortality, heterogeneous effects on kidney function and the difficulty to dissociate the specific effects of kidney injury from the general effects of invasive surgery on the downstream phenotype. Although 5/6 nephrectomy can induce alterations of mineral metabolism and LVH, producing the full phenotype often necessitates dual administration of high-phosphate diets, and the amplitude of the C The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. V 1129 ORIGINAL ARTICLE Genetic background influences cardiac phenotype in murine chronic kidney disease MATERIALS AND METHODS Animals Serum and urine biochemistry We collected overnight urine samples from animals housed in metabolic cages and serum samples by intracardiac exsanguination. We used a murine intact FGF23 (iFGF23) enzymelinked immunosorbent assay (ELISA) to measure the active iFGF23 protein and a C-terminal FGF23 (cFGF23) ELISA that recognizes the full-length protein and its C-terminal cleavage fragments:measure total FGF23 (both from Immutopics, Carlsbad, CA, USA). We calculated the intact: to total FGF23 ratio as a surrogate marker of FGF23 cleavage [22, 24]. We measured parathyroid hormone (PTH) using a mouse intact ELISA (Immutopics), 1,25-dihydroxyvitamin D [1,25(OH)2 D] by immunoassay (Immunodiagnostic Systems, Gaithersburg, MD, USA) and calcium, phosphate, blood urea nitrogen (BUN), albumin and creatinine using colorimetric assays (Pointe Scientific, Canton, MI, USA). Echocardiography and blood pressure We performed echocardiography under isoflurane anesthesia 1 week prior to sacrifice (at 9 and 19 weeks of age) using a Vevo 770 High-Resolution Imaging System (VisualSonics, Toronto, ON, Canada). We used the parasternal short- and long-axis views to obtain 2-dimensional and M-mode images. We acquired at least 10 independent cardiac cycles per each experiment. We measured cardiac output, ejection fraction (EF) and stroke volume across the long axis and calculated the cardiac index using the following equation: cardiac index ¼ cardiac output/body weight. We measured blood pressure (BP) in sentient mice using a computerized mouse tail-cuff system (CODA, Kent Scientific, Torrington, CT, USA). We performed acquisitions for 20 cycles, once a day during three consecutive days, to familiarize each mouse with the system and to reduce environmental stress. We analyzed the third-day data from habituated mice. Heart and kidney histology tm1Dec mutant mice We purchased 129X1/SvJ-Col4a3 KO ) from Jackson Laboratory (Bar Harbor, ME, USA). (Col4a3 We first outcrossed 129X1/SvJ heterozygotes with C57BL/6J wildtype (WT) mice. We further backcrossed the F1 heterozygotes with either strain to generate incipient congenic strains: first, for one generation with 129X1/SvJ WT mice to obtain a mixed background strain (N2) that contained 75% 129X1/SvJ genome (129Sv); second, for three generations to C57BL/6J WTs to obtain B6.129-Col4a3tm1Dec (N4) that contained 94% (...truncated)