Single-Trait and Multi-Trait Genome-Wide Association Analyses Identify Novel Loci for Blood Pressure in African-Ancestry Populations

Hypertension is a leading cause of global disease, mortality, and disability. While individuals of African descent suffer a disproportionate burden of hypertension and its complications, they have been underrepresented in genetic studies. To identify novel susceptibility loci for blood pressure and hypertension in people of African ancestry, we performed both single and multiple-trait genome-wide association analyses. We analyzed 21 genome-wide association studies comprised of 31,968 individuals of African ancestry, and validated our results with additional 54,395 individuals from multi-ethnic studies. These analyses identified nine loci with eleven independent variants which reached genome-wide significance (P \u3c 1.25×10−8) for either systolic and diastolic blood pressure, hypertension, or for combined traits. Single-trait analyses identified two loci (TARID/TCF21 and LLPH/TMBIM4) and multiple-trait analyses identified one novel locus (FRMD3) for blood pressure. At these three loci, as well as at GRP20/CDH17, associated variants had alleles common only in African-ancestry populations. Functional annotation showed enrichment for genes expressed in immune and kidney cells, as well as in heart and vascular cells/tissues. Experiments driven by these findings and using angiotensin-II induced hypertension in mice showed altered kidney mRNA expression of six genes, suggesting their potential role in hypertension. Our study provides new evidence for genes related to hypertension susceptibility, and the need to study African-ancestry populations in order to identify biologic factors contributing to hypertension

Abstract 121: Gstm1 Deletion Exaggerates Hypertension, Oxidative Stress, and Kidney Pathology in Angiotensin II Hypertension Independent of Nox2 and Nox4

Gstm1 gene encodes an enzyme that belongs to a superfamily of Glutathione-S-transferases that metabolize a broad range of reactive oxygen species. In the African American Study of Kidney Disease Trial (AASK), we reported that participants with decreased expression of GSTM1 as carriers of the GSTM1 null allele have accelerated kidney disease progression. To establish cause and effect, we generated Gstm1-/- (KO) mice and determined their response in angiotensin II (AngII)-induced hypertension (at 1000 ng/kg/min for 4 weeks). By radiotelemetry, Gstm1 KO mice (n=6) displayed a modest but significantly higher baseline systolic blood pressure (SBP, mmHg) compared to their wild type (WT) littermates (n=6): KO 135.8 ± 1.4; WT: 129.9 ± 1.2; P = 0.01. AngII augmented the difference in SBP between WT and KO mice (KO 175.4 ± 6.2; WT 158.0 ± 2.6, p = 0.04). There were no differences in albumin/creatinine ratio (μg/mg) either at baseline (KO 29.1± 4.1; WT 27.3 ± 2.9) or after 4 weeks of Ang II (KO 4657 ± 631.6; WT 4697.7 ± 533.8). By quantitative real-time PCR, the renal expressions of Nox2 and Nox4 genes were almost identical and not statistically different between WT and KO mice. However, by luminescence, Gstm1 KO mice had significantly increased levels of superoxide radicals: [counts/min/1 mg of dry tissue] KO 128 ± 12 vs. WT 42 ± 8.5; P < 0.0001. Furthermore, there was significantly worse kidney pathology in Gstm1 KO mice (Figure 1), including focal segmental glomerulosclerosis (FSGS), fibrosis, chronic inflammation and epithelial cell reactivity. In conclusion, loss of GSTM1 enzyme increases Ang II induced hypertension, oxidative stress and kidney injury independent of Nox2 or Nox4

Tissue-specific Deletion of Collectrin in the Proximal Tubular Epithelium Increases Arterial Pressure and Augments Salt-sensitivity

Background: Collectrin (Tmem27) is a key regulator of blood pressure (BP) and modulator of the bioavailability of nitric oxide (NO) and superoxide. It is highly expressed in the kidney in the proximal tubule (PT), collecting duct, and throughout the vascular endothelium. We reported that collectrin plays a critical role as a chaperone for the reabsorption of all amino acids (AAs) in the PT, and for the uptake of the cationic AA L-arginine (L-Arg) in endothelial cells. Global collectrin knockout (Tmem27Y/-) mice display baseline hypertension (HTN), augmented salt-sensitive hypertension (SSH), and decreased renal blood flow. Objective and Methods: To determine the PT-specific effect of collectrin on BP homeostasis and salt sensitivity, we used the Cre-loxP approach and PEPCK-Cre to generate a mouse line lacking collectrin specifically in the PT-- PEPCK-Cre+Tmem27Y/Flox mice. PEPCK-Cre-Tmem27Y/Flox mice were used as control. Radiotelemetry was used to measure BP for 2 weeks at baseline and 2 weeks on high salt diet (HSD). Renal blood flow at baseline and on HSD was measured using contrast enhanced ultrasound in the same mice. Results: Successful deletion of collectrin in the PT was confirmed by assessing mRNA levels using real-time RT-PCR, immunohistochemistry staining of renal tissues using anti-collectrin antibody, and quantitation of protein from kidney cortex by Western analysis. Compared to control PEPCK-Cre-Tmem27Y/Flox mice (n=6), PEPCK-Cre+Tmem27Y/Flox mice (n=6) displayed significantly higher systolic BP (SBP) at baseline (120.0 ± 2.5 vs 131.6 ± 2.9 mm Hg; p = 0.014) and after HSD (135.3 ± 2.6 vs 151.5 ± 5.2 mm Hg; p = 0.019). Renal blood flow was not different between groups, at baseline nor after HSD. Conclusion: Collectrin in the PT plays an important role in blood pressure homeostasis and response to sodium intake, independent of renal blood flow. Increasing proximal tubular collectrin activity may be a novel therapeutic strategy for the treatment of hypertension and salt-sensitivity

MYH9 E1841K Mutation Augments Proteinuria and Podocyte Injury and Migration

Intronic variants of the gene that encodes the nonmuscle myosin heavy chain IIA are associated with diabetic nephropathy in European Americans and with sickle cell disease-associated nephropathy. However, the causal functional variants of have remained elusive. Rare missense mutations in cause macrothrombocytopenia and are occasionally associated with development of nephropathy. The E1841K mutation is among the common missense mutations and has been associated with nephropathy in some carriers. To determine the contribution of the E1841K mutation in kidney disease, we studied the effects of the E1841K mutation in mice subjected to high salt or angiotensin II (Ang II) as models of hypertension and in mice subjected to renal mass reduction as a model of CKD. Despite similar levels of BP among wild-type ( ) mice and mice heterozygous ( ) and homozygous ( ) for the mutation in each model, mice exhibited mildly increased albuminuria in response to high salt; severe albuminuria, nephrinuria, FSGS, and podocyte foot effacement in Ang II-induced hypertension; and early mortality in the renal mass reduction model. Treatment with candesartan during Ang II-induced hypertension attenuated kidney disease development in mice. , isolated primary podocytes from mice exhibited increased lamellipodia formation and reorganization of F-actin stress fibers. Wound healing assays revealed that podocytes had the lowest migration rate, followed by then podocytes. In conclusion, the E1841K variant alters podocyte cytoskeletal structure and renders podocytes more susceptible to injury after a damaging stimulus

Renal Collectrin Protects against Salt-Sensitive Hypertension and Is Downregulated by Angiotensin II

Collectrin, encoded by the Tmem27 gene, is a transmembrane glycoprotein with approximately 50% homology with angiotensin converting enzyme 2, but without a catalytic domain. Collectrin is most abundantly expressed in the kidney proximal tubule and collecting duct epithelia, where it has an important role in amino acid transport. Collectrin is also expressed in endothelial cells throughout the vasculature, where it regulates L-arginine uptake. We previously reported that global deletion of collectrin leads to endothelial dysfunction, augmented salt sensitivity, and hypertension. Here, we performed kidney crosstransplants between wild-type (WT) and collectrin knockout (Tmem27Y/-) mice to delineate the specific contribution of renal versus extrarenal collectrin on BP regulation and salt sensitivity. On a high-salt diet, WT mice with Tmem27Y/- kidneys had the highest systolic BP and were the only group to exhibit glomerular mesangial hypercellularity. Additional studies showed that, on a high-salt diet, Tmem27Y/- mice had lower renal blood flow, higher abundance of renal sodium-hydrogen antiporter 3, and lower lithium clearance than WT mice. In WT mice, administration of angiotensin II for 2 weeks downregulated collectrin expression in a type 1 angiotensin II receptor–dependent manner. This downregulation coincided with the onset of hypertension, such that WT and Tmem27Y/- mice had similar levels of hypertension after 2 weeks of angiotensin II administration. Altogether, these data suggest that salt sensitivity is determined by intrarenal collectrin, and increasing the abundance or activity of collectrin may have therapeutic benefits in the treatment of hypertension and salt sensitivity