T cell infiltration in kidney induces salt‐retention via NCC up‐regulation

The key role of the kidney in the development of salt‐sensitive hypertension has been demonstrated by many laboratories, and excessive sodium (Na + ) reabsorption by the distal nephron is a key contributor to this disease. Recent studies suggest T cells, especially CD8+ T cells (CD8Ts), play a major role in the development of salt‐sensitive hypertension. However, whether T cells enhance sodium retention in kidney still remains unclear. This question if answered may reveal a critical relationship between the immune system and the pathogenesis of salt‐sensitive hypertension. In the present study, we propose a novel mechanism of salt‐sensitive hypertension via distal nephron: that under impaired immune‐homeostasis, renal infiltrating T cells (CD8+) interact with distal convoluted tubule cells (mDCTs), which leads to up‐regulation of the sodium‐chloride‐co‐transporter (NCC), and results in enhancement of sodium retention. In vivo , we used immuno‐staining to detect infiltration of T cells (CD3 or CD8), up‐regulation of NCC and their co‐localization in the kidneys of DOCA‐salt mice and Dahl‐salt‐sensitive (Dahl‐SS) rats, two classic salt‐sensitive hypertensive animal models. Results confirmed that there are significantly more T cells infiltrated in the kidneys of DOCA‐salt & Dahl‐SS animals compared to their controls. It is noteworthy that a great number of CD8Ts in the renal tubulointerstitium surround DCT segments which demonstrate higher levels of NCC expression. These results lead us to speculate that CD8Ts can interact with DCTs to increase NCC expression and function in the distal nephron of salt‐sensitive hypertensive animals. In‐vitro , we co‐cultured mDCTs with CD8Ts, and found this co‐culture significantly increased NCC expression in mDCTs via “cell‐cell direct contact”. Furthermore, we detected abundant NCC protein localized to the cell membrane of the mDCTs that been treated by CD8Ts, but not of control cells ( Fig 1). To evaluate the increased NCC induced sodium retention in mDCTs, an intracellular fluorescent sodium indicator‐ CoroNa Green, was preloaded to cells and sodium uptake was measured after 45 mins. Data showed much more mDCTs demonstrating high sodium retention in the CD8T treated group compared to the control group. This result was further confirmed by measuring the fluorescence intensity ( Fig 2). Moreover, we found most CD8T‐induced enhancement of sodium uptake in mDCTs can be inhibited by NCC siRNA or NCC inhibitors such as hydrochlorothiazide (HCTZ), thiazide metolazone (MTZ) or chlorothiazide (CTZ) ( Fig 3), suggesting that CD8T‐induced NCC up‐regulation in mDCTs is responsible for sodium retention in the distal nephron. Taken together, our work illustrates a novel role of the immune system in the development salt‐sensitive hypertension in kidney: intra‐renal CD8T cells could directly interact with DCT cells, lead to NCC up‐regulation, result in sodium retention via the distal nephron and consequently develop salt‐sensitive hypertension. Support or Funding Information AHA grant 15BGIA25730047 1co‐culture with T cells increased NCC protein expression in both total lysate (left panel) and membrane extraction (right panel) of mDCT cells.2measuring fluorescence from intracellular fluorescent sodium indicator. Co‐culture with T cells enhance sodium retention in mDCT cells. 5×10 5 mDCT cells of each group were analyzed by flow cytometer (left panel); about 2×10 6 mDCT cells of each group were lysate for fluorescence measurement, fluorescence data normalized by total amount of protein (mg) (right panel).3co‐culture with T cells induced increase of sodium uptake (Δ sodium uptake) in mDCTs is higher in high‐salt (+40mM NaCl) incubation environment; and can be inhibited by NCC siRNA or NCC blockers, hydrochlorothiazide (HCTZ), thiazide metolazone (MTZ) or chlorothiazide (CTZ).