Identification of Polymorphisms in Genes Involved in Aldosterone Synthesis and Activity in Feline Hypertensive Chronic Kidney Disease
Systemic hypertension is a common disorder of aged cats. It is most often diagnosed in cats with chronic kidney disease (CKD) with around one-third of such cats being hypertensive; however, cats that are normotensive at the time of CKD diagnosis rarely develop hypertension subsequently. Hypertensive cats have significantly lower plasma potassium than normotensive cats with CKD, possibly due to hyperaldosteronism, which could in turn be due to polymorphisms in relevant encoding genes. Polymorphisms in genes involved in aldosterone synthesis (11-beta hydroxylase, aldosterone synthase) and activity (amiloride-sensitive epithelial sodium channel; ENaC) are associated with altered risk of developing essential hypertension in humans and were investigated in this study.
Cats with naturally-occurring CKD (plasma creatinine >2.0 mg/dL and with inappropriately low urine specific gravity <1.035) were selected for this study. Hypertension was diagnosed if systolic blood pressure measured by Doppler was >175 mmHg on at least two occasions or on one occasion if hypertensive ocular lesions were present. Genomic DNA was extracted by silica-gel membrane adsorption (QIAamp® DNA blood kit; QIAgen) from buffy coat-enriched packed cells collected into potassium EDTA tubes. Sequencing primers were designed from contigs built from the Felis catus whole genome sequence trace archive (NCBI). Sections of genes encoding 11-beta hydroxylase, aldosterone synthase and the three subunits of ENaC were amplified by polymerase chain reaction and sequenced in three normotensive and three hypertensive cats. Single nucleotide polymorphisms (SNPs) were identified. Selected SNPs were genotyped by single base extension (SNaPshotTM, Applied Biosystems) in a further 24 normotensive and 24 hypertensive cats to establish allele frequencies.
Genes for aldosterone synthase and 11-beta hydroxylase were largely monomorphic (minor-allele frequencies of SNPs: 0.03-0.09). Genes for the alpha, beta and gamma ENaC subunits respectively contained two SNPs with minor-allele frequencies 0.38 and 0.49, four SNPs with minor-allele frequencies 0.20-0.44, and five SNPs with minor-allele frequencies 0.11-0.49.
This study has identified numerous polymorphisms that can now be evaluated to determine if they are associated with hypertension in the cat. Work to genotype 250 more cats with CKD and known blood pressure status is currently ongoing.