Rationale: SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single-nucleotide polymorphism in SH2B3 (rs3184504) results in substitution of tryptophan for arginine at amino acid 262 and is a top association signal for hypertension in human genome-wide association studies. Whether this variant is causal for hypertension, and if so, the mechanism by which it impacts pathogenesis is unknown. Objectives: Test the hypothesis that the tryptophan-encoding allele of rs3184504 promotes hypertension development and end-organ damage through loss of SH2B3-mediated repression of cytokine signaling to enhance T-cell activation. Methods and Results: We used CRISPR-Cas9 technology to create mice homozygous for the major (arginine/arginine) and minor (tryptophan/tryptophan) alleles of this SH2B3 polymorphism. Tryptophan/tryptophan mice exhibited 10 mm Hg higher systolic blood pressure during chronic Ang II (angiotensin II) infusion compared with arginine/arginine controls. Renal injury and perivascular fibrosis were exacerbated in tryptophan/tryptophan mice compared with arginine/arginine controls following Ang II infusion. In addition, renal and ex vivo stimulated splenic CD8 + T cells from Ang II–infused tryptophan/tryptophan mice produced significantly more IFN (interferon)-γ compared with arginine/arginine controls. IL (Interleukin)-12-induced IFN-γ production was greater in tryptophan/tryptophan compared with arginine/arginine CD8 + T cells. In addition, IL-12 enhanced Stat4 (signal transducer and activator of transcription 4) phosphorylation to a greater degree in tryptophan/tryptophan compared with arginine/arginine CD8 + T cells, suggesting that tryptophan-encoding SH2B3 exhibits less negative regulation of IL-12 signaling to promote IFN-γ production. Finally, we demonstrated that a multi-single-nucleotide polymorphism model genetically predicting increased SH2B3 expression in lymphocytes is inversely associated with hypertension and hypertensive chronic kidney disease in humans. Conclusions: Taken together, these results suggest that the tryptophan-encoding allele of rs3184504 is causal for blood pressure elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T-cell IL-12 signaling leading to enhanced IFN-γ production.

SH2B3, also known as LNK, is an adaptor protein that negatively regulates growth factor and cytokine signaling. The minor allele of a single nucleotide polymorphism in SH2B3 (rs3184504) encodes a tryptophan (Trp) at amino acid 262 as opposed to arginine (Arg) and is strongly associated with hypertension in genome-wide association studies. Whether this variant is causal and how it impacts hypertension development and end-organ damage is unknown. We used CRISPR-Cas9 to engineer mice homozygous for the major and minor alleles of this SH2B3 polymorphism, resulting in Arg/Arg and Trp/Trp mice, respectively. Trp/Trp mice exhibited increased systolic blood pressure (SBP) by radiotelemetry during weeks 3-4 of angiotensin II (Ang II) infusion compared to Arg/Arg mice (nighttime SBP 168 vs 158 mm Hg, respectively). Renal dysfunction was also exacerbated in Ang II-treated Trp/Trp compared to Arg/Arg mice, as evidenced by significantly increased urinary albumin/creatinine ratio (0.41 vs 0.17), renal perivascular fibrosis (fibrosis score 2.0 vs 1.0), and renal macrophages (8,341 vs 6,413 per kidney). In addition, renal CD8 + T cells from Ang II-treated Trp/Trp mice produced significantly more IFNγ compared to Arg/Arg controls (median fluorescence intensity 20,455 vs 14,134), and ex vivo stimulated splenic CD8 + T cells from Trp/Trp compared to Arg/Arg mice made 2.7-fold more IFNγ. To determine a mechanism for increased T cell IFNγ production, dendritic cells and naïve T cells from Trp/Trp and Arg/Arg mice were co-cultured in different combinations. The greatest increase in GM-CSF-induced IFNγ production occurred when both dendritic cells and T cells came from Trp/Trp mice (3.4-fold greater than both cell types from Arg/Arg mice). This effect appears to be due to loss of SH2B3-mediated suppression of GM-CSF signaling, as overexpression of Trp-encoding SH2B3 in HEK cells exhibited significantly less repression of GM-CSF-induced Stat5 activation compared to Arg-encoding SH2B3. Taken together, these findings suggest that the Trp encoding allele of rs3184504 is a causal variant promoting blood pressure elevation and renal dysfunction, at least in part through loss of SH2B3-mediated repression of T cell IFNγ production.