Effect of renal failure, sex, and BMP2 modulation on gene expression in mouse aorta

Renal failure and male sex strongly predict cardiovascular disease. Understanding the impact of sex and kidney function on the biology of cardiovascular disease will clarify the differential presentation, progression, and response to treatments observed in each sex. Elucidating the molecular differences also may reveal new and effective therapeutic strategies. Deficiency of KLOTHO promotes ectopic calcification of soft tissues such as aorta, aortic valves and kidneys. The rapid, synchronous onset of calcification particularly in the 129S1/SvlmJ (129) mouse strain facilitates molecular studies. (Kuro-o et al. Mutation of the mouse Klotho gene leads to a syndrome resembling ageing. Nature 390: 45-51, 1997; Salloum JS, Garsetti DE, Rogers MB. Genetic Background Influences the Impact of KLOTHO Deficiency. Physiological Genomics. 52, 512-516 (2020) We previously assessed microRNA gene expression differences associated with aorta calcification in healthy or Klotho deficient mice. (Tang Y, Shah TA, Yurkow EJ, Rogers MB. MicroRNA Profiles in Calcified and Healthy Aorta Differ: Therapeutic Impact of miR-145 and miR-378. Physiological Genomics. 52: 517–529, 2020) Here we used RNA seq to profile messenger RNAs in aortas from control (ctrl) healthy and Klotho homozygous deficient mice of both sexes with renal disease that causes aortic calcification. In addition, we profiled messenger RNAs in aortas from male mice bearing a Bone Morphogenetic Protein (BMP)2 regulatory mutation. BMP2 is a potent osteogenic factor involved in pathological vascular and valve calcification. The Bmp2 mutation (Bmp2?UCS) is a 300 nucleotide deletion of an ultra-conserved sequence (UCS) in the 3' untranslated region (UTR) of the Bmp2 gene. This sequence mediates a post-transcriptional block that prevents BMP2 synthesis in specific cell types, including the aortic valves, coronary vasculature, and aorta. (Shah, T.A., Y. Zhu, N.N. Shaikh, M.A. Harris, S.E. Harris, and M.B. Rogers, Characterization of new bone morphogenetic protein (Bmp)-2 regulatory alleles. Genesis, 2017. 55(7)). All experimental parameters (sex, kidney health, and BMP2 mutation) modulated mRNA profiles. Overall design: Both the Bmp2 and Klotho mutations were made fully congenic on the 129S1/SvlmJ (129) by backcrossing more than 10 times. RNA was extracted from male and female mice between 44 and 53 days of age for RNA-seq profiling. Control mice with healthy kidneys were either homozygous wild type (++) or heterozygous for the recessive Klotho mutation (Kl+). Affected mice with renal failure were homozygous for the recessive Klotho mutation (KlKl). Additional groups of control or Klotho deficient male mice were homozygous for the BMP2 mutation (?UCS/?UCS).