A Single Nucleotide Polymorphism in SH2B3/LNK Promotes Hypertension Development and Renal Damage

Abstract

Background: 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 (Trp) for arginine (Arg) 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.

Methods: We used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of this SH2B3 polymorphism. Mice underwent angiotensin II (Ang II) infusion to evaluate differences in blood pressure (BP) elevation and end-organ damage including albuminuria and renal fibrosis. Cytokine production and Stat4 phosphorylation was also assessed in Arg/Arg and Trp/Trp T cells.

Results: Trp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8⁺ T cells from Ang II-infused Trp/Trp mice produce significantly more interferon gamma (IFNg) compared to Arg/Arg controls. Interleukin-12 (IL-12)-induced IFNg production is greater in Trp/Trp compared to Arg/Arg CD8⁺ T cells. In addition, IL-12 enhances Stat4 phosphorylation to a greater degree in Trp/Trp compared to Arg/Arg CD8⁺ T cells, suggesting that Trp-encoding SH2B3 exhibits less negative regulation of IL-12 signaling to promote IFNg production. Finally, we demonstrated that a multi-SNP 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 Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

Keywords: cytokines; hypertension; inflammation; lymphocytes; polymorphism, single nucleotide.

Figure 1.. Creation of mice with Arg- or Trp-encoding Sh2b3 to mimic human major and minor alleles, respectively.

(A) Scores of Sorting Intolerant from Tolerant (SIFT) algorithm predictions of the effect of codon 262 amino acid substitutions on human SH2B3 function. Substitutions in red (score <0.05) are predicted to affect protein function. Letters represent abbreviations for individual amino acids. (B) Representative alignment of amino acids 253–270 of human SH2B3 and amino acids 225–243 of mouse Sh2b3 protein sequences demonstrating presence of Proline (P) at position 234 of mouse Sh2b3 and Arginine (R) at orthologous position 262 of human SH2B3 (boxed). (C) Representation of mouse Sh2b3 DNA and protein sequence in wild type (top) and engineered mouse Sh2b3 gene sequences (bottom) along with restriction enzyme digest sites. DNA sequence changes are in blue and amino acid changes in red. (D) Scores of SIFT algorithm predictions of the effect of codon 234 amino acid substitutions on mouse SH2B3 function. Substitutions in red (score <0.05) are predicted to affect protein function. Letters represent abbreviations for individual amino acids.

Figure 2.. Trp/Trp mice exhibit increased systolic blood pressure (BP) and renal damage with Ang II infusion compared to Arg/Arg controls.

(A) Radiotelemetric systolic BP of Arg/Arg and Trp/Trp mice at baseline and during 4 weeks of Ang II infusion (140 ng/kg/min). Inset to right is magnification of systolic blood pressure (SBP) readings at 3 and 4 weeks of Ang II (n=6 Arg/Arg, 8 Trp/Trp). Two way ANOVA with repeated measures. (B) Urine albumin to creatinine ratio after 4 weeks of Ang II (n=7 Arg/Arg, 8 Trp/Trp). Mann-Whitney test. (C) Urine NGAL to creatinine ratio after 4 weeks of Ang II infusion (n=7 Arg/Arg, 8 Trp/Trp). Mann-Whitney test. (D) Masson’s trichrome staining of kidneys reveals increased perivascular fibrosis (blue) evidenced by representative images (left) and quantification (right) (n=9 Arg/Arg, 8 Trp/Trp). Unpaired t test. Scale bar=50 μm. NGAL=neutrophil gelatinase-associated lipocalin. Data are expressed as mean±SEM of biological replicates.

Figure 3.. Increased F4/80⁺ macrophages in kidneys of Trp/Trp compared to Arg/Arg mice after 4 weeks of Ang II infusion.

(A) Flow cytometry representative gating for CD45⁺ immune cells, CD3⁺ T cells (with CD4⁺ helper and CD8⁺ cytotoxic T cell subsets), and F4/80⁺ macrophages in kidneys of Arg/Arg and Trp/Trp mice. Quantification of (B) CD45⁺ immune cells, (C) CD3⁺ T cells, (D) CD4⁺ helper T cells, (E) CD8⁺ cytotoxic T cells, and (F) F4/80⁺ macrophages in kidneys of Arg/Arg and Trp//Trp mice after 4 weeks of Ang II infusion (n=9 Arg/Arg, 10 Trp/Trp). Unpaired t test. Data are expressed as mean±SEM of biological replicates.

Figure 4.. Trp/Trp mice have increased circulating leukocytes and splenomegaly after Ang II infusion compared to Arg/Arg controls.

Circulating (A) white blood cell, (B) monocyte, (C) lymphocyte, and (D) neutrophil counts after 4 weeks of Ang II infusion (n=7 Arg/Arg,8 Trp/Trp). Mann Whitney test. (E) Representative images of spleen size in Trp/Trp and Arg/Arg mice (left) with quantification of spleen weight normalized to body weight (right) (n=9 Arg/Arg,12 Trp/Trp). Mann Whitney test. Scale bar=0.5 cm. Data expressed as mean±SEM of biological replicates.

Figure 5.. Splenic T cells from Ang II-infused Trp/Trp mice produce more IFNγ and IL-17A compared to Arg/Arg mice.

(A) Schematic of CD4⁺ and CD8⁺ T cell isolation from spleens of Arg/Arg or Trp/Trp mice after 4 weeks of Ang II infusion. Equal cell numbers were cultured for 3 days with anti-CD3 and anti-CD28 antibodies for T cell activation followed by ELISA for inflammatory cytokines. Parts of the figure were from Servier Medical Art licensed under a Creative Commons Attribution 3.0 Unported License. (B) IFNγ from CD4⁺ T cells (n=6 Arg/Arg, 6 Trp/Trp) and (C) CD8⁺ T cells (n=8 Arg/Arg, 7 Trp/Trp) along with (D) IL-17A from CD4⁺ T cells (n=7 Arg/Arg, 6 Trp/Trp) and CD8⁺ T cells (n=7 Arg/Arg, 6 Trp/Trp). Mann-Whitney test. Data are expressed as mean±SEM of biological replicates.

Figure 6.. Increased IFNγ from renal CD8⁺ T cells of Ang II-infused Trp/Trp mice.

(A) CD8⁺IFNγ⁺ T cell numbers and (B) median fluorescence intensity (MFI) of IFNγ in CD8⁺ T cells isolated from kidneys of Arg/Arg or Trp/Trp mice after 4 weeks of Ang II infusion (n=8). (C) RT-PCR for Ifnγ from kidneys of Arg/Arg and Trp/Trp mice after 4 weeks of Ang II infusion (n=11 Arg/Arg, 8 Trp/Trp). Unpaired t test. Data are expressed as mean±SEM of biological replicates.

Figure 7.. Increased IFNγ production and Stat4 phosphorylation by IL-12 in Trp/Trp compared to Arg/Arg splenic CD8⁺ T cells.

(A) Splenic CD8⁺ T cells were cultured with anti-CD3 and CD28 antibodies along with vehicle or IL-12 (10 ng/mL) for three days with detection of IFNγ by ELISA in the culture supernatants. Data shown are technical replicates (n=3 Arg/Arg IL-12, n=4 in other groups) of cells from one mouse per genotype which are representative of three experiments from separate biological replicates. Results of the other 2 biological replicate experiments are shown in Figure S18. Statistics based on align-and-rank nonparametric factorial ANOVA with the Holm’s post hoc test. (B) Representative images of percent phospho-Stat4⁺ cells (left) from flow cytometric analysis of CD8⁺ T cells stimulated for three days with anti-CD3 and CD28 antibodies followed by treatment for 15 minutes with vehicle or IL-12 (10 ng/mL). Quantitation of the percentage of phospho-Stat4 positive cells from live singlets (right). Data shown are three experiments, each with CD8⁺ T cells from a separate mouse of each genotype, with four technical replicates of cells in each experiment. Data are expressed as mean±SEM. Statistics based on align-and-rank nonparametric factorial ANOVA with Holm’s post hoc test incorporating experiment and mouse cluster. pStat4=phosphorylated Stat4. (C) Working model of the effect of the tryptophan (Trp) encoding minor allele of the rs3184504 polymorphism in SH2B3 on hypertension and end-organ damage. Trp-encoding SH2B3 results in less inhibition of Stat4 phosphorylation with resultant enhanced T cell IFNγ production to promote increased blood pressure and renal injury in mice. IL-12=interleukin-12; IL-12R=interleukin-12 receptor. Created with Biorender.com.