SH2B3 Mutation Attenuates T Lymphocyte Intracellular Ca 2+ Release in Response to Direct Angiotensin‐II Stimulation in Dahl S Hypertensive Rats

T lymphocytes were shown to enhance the progression of salt‐sensitive hypertension and the associated kidney damage. These cells express SH2B3 (LNK), an adapter protein that was linked to hypertension by a Gene Wide Association Study, and is involved in regulation of several ligand‐induced transduction pathways. Angiotensin II (AngII) is a peptide hormone of the renin‐angiotensin system and is a major effector of blood pressure. AngII was found to stimulate the production and release of T‐cell inflammatory mediators via the AT1 receptor. Additionally, SH2B3 was shown to be involved in AngII‐induced hypertension in mice, where the knockout of SH2B3 increased T‐cells cytokine production and potentiated the development of hypertension and kidney damage. The lack of direct measurements of T‐cell response to AngII precludes conclusions of how AngII and the SH2B3 adaptor protein signaling cascade operate together to affect T‐cell function and further modulate the production of inflammatory mediators during the development of hypertension. To explore the interaction of AngII and T‐cells, we used SH2B3 mutant rats (SH2B3 em1Mcwi ) created on the Dahl salt sensitive (SS) rat background. The mutation, produced by zinc‐finger nuclease technology, resulted in a predicted 3 amino acid change in the evolutionarily conserved Src homology 2 (SH2) domain. This mutation in the SH2B3 gene was shown to cause attenuated development of high salt‐induced hypertension and kidney damage that was mediated by T cells. SH2B3 mutant and SS littermate control rats were kept on a 0.4% NaCl diet. At age 12 weeks, splenocytes from each group were isolated by sieving and centrifugation, labeled with anti‐CD5 FITC antibody, and loaded with the intracellular Ca 2+ indicator Fura‐2 AM for further epifluorescence imaging experiments. Splenic T‐cells were then treated with Concanavalin A to engage the T Cell Receptor for cell activation. Bath applications of 10 μM of AngII rapidly produced an intracellular Ca 2+ transient in both types of T‐cells. However, SH2B3 mutant CD5 + T‐cells had significantly attenuated response to AngII (0.41 ± 0.13 Fura340/380 ratio, n = 81) compared to SS littermates (0.92 ± 0.05 Fura340/380 ratio, n = 62) (P < 0.05). This result indicates that AngII is directly involved in the signal transduction pathway of CD5 + T‐cells and this pathway can be significantly modulated by SH2B3 adapter protein function. We conclude that the SH2 domain of SH2B3 may be a potential target for the control of T‐cell mediated oxidative damage and inflammation, and control salt‐sensitive hypertension. Further studies are required to define the role of SH2B3 in signaling and molecular mechanisms underlying T‐cells function and the development of hypertension.