ANG II has many biological effects in renal physiology, particularly in Ca handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT and AT receptors (ATR and ATR) to stimulate sarco(endo)plasmic reticulum Ca-ATPase (SERCA) activity. Thus, we investigated whether ANG II-ATR/ATR complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with ATR/ATR heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of β-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of β-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of ATR, ATR, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT/AT complex to target ER, where it might trigger intracellular Ca responses.

The Na+-activated Na+ channel (Nax) and salt-inducible kinase (SIK) are stimulated by increases in local Na+ concentration, affecting (Na+ + K+)-ATPase activity. To test the hypothesis that the triad Nax/SIK/(Na+ + K+)-ATPase contributes to kidney injury and salt-sensitive hypertension (HTN), uninephrectomized male Wistar rats (200 g; n = 20) were randomly divided into 4 groups based on a salt diet (normal salt diet; NSD—0.5% NaCl—or high-salt diet; HSD—4% NaCl) and subcutaneous administration of saline (0.9% NaCl) or deoxycorticosterone acetate (DOCA, 8 mg/kg), as follows: Control (CTRL), CTRL-Salt, DOCA, and DOCA-Salt, respectively. After 28 days, the following were measured: kidney function, blood pressure, (Na+ + K+)-ATPase and SIK1 kidney activities, and Nax and SIK1 renal expression levels. SIK isoforms in kidneys of CTRL rats were present in the glomerulus and tubular epithelia; they were not altered by HSD and/or HTN. CTRL-Salt rats remained normotensive but presented slight kidney function decay. HSD rats displayed augmentation of the Nax/SIK/(Na+ + K+)-ATPase pathway. HTN, kidney injury, and kidney function decay were present in all DOCA rats; these were aggravated by HSD. DOCA rats presented unaltered (Na+ + K+)-ATPase activity, diminished total SIK activity, and augmented SIK1 and Nax content in the kidney cortex. DOCA-Salt rats expressed SIK1 activity and downregulation in (Na+ + K+)-ATPase activity in the kidney cortex despite augmented Nax content. The data of this study indicate that the (Na+ + K+)-ATPase activity response to SIK is attenuated in rats under HSD, independent of HTN, as a mechanism contributing to kidney injury and salt-sensitive HTN.

Salt-inducible kinase (SIK) is a serine/threonine kinase involved in intracellular sensing network. However, its role in the development of salt-sensitive hypertension remains uncertain. We used a mouse model of salt-sensitive hypertension to test the hypothesis that SIK inhibition decreases systolic blood pressure (SBP) and renal injury. Male C57BL/6J mice (20-25g) were randomly fed either normal salt (NS; 0.5% NaCl) or high salt (HS; 4% NaCl) diet and daily treated or not with a SIK inhibitor (iSIK, YKL-05-099, 20 mg/Kg, via IP; n=6/group; CEUA: 013/19). In these mice: 1) SBP was measured by telemetry for 15 days; 2) Urine metabolic collection for 24 h was performed on day 14; and 3) Blood and kidney samples were collected after euthanasia on day 15. HS diet increased SBP during both day and night times (Day: from 114±1 to 136±1 mmHg; Night: from 127±3 to 141±4 mmHg; p<0.0001). Treatment with iSIK prevented increases in SBP (Day: 112±1 and Night: 123±2 mmHg). iSIK did not change SBP nor renal Na + handling in NS diet. In HS mice, iSIK prevented the augmentation of urine osmolality (HS: 1692±14 vs. HS+iSIK: 1104±34 mOsm/Kg H 2 O; p=0.0019) but did not change increases in urine volume and Na + excretion neither decreases in urine creatinine. Moreover, HS mice displayed glomerular segmentation, tubular interstitial disruption, infiltration of inflammatory cells and collagen accumulation. These responses were attenuated by iSIK. Increased kidney SIK activity in HS mice were sensitive to iSIK (HS: 222.8±45 vs. HS+iSIK: 19.10±15 pmol.mg -1 .min -1 ; p=0.0026). The augmentation of (Na + +K + )ATPase activity in HS mice was exacerbated during iSIK (HS: 517±112 and HS+SIKi: 763±18 mmol.mg -1 ;.min -1 p<0.0001). Equally important, SIK inhibition in HS mice: 1) Decreased plasma MCP-1 levels (HS: 79±7 vs. HS+iSIK: 51±2 pg/dL; p=0.0019); 2) Prevented HS-dependent macrophages renal infiltration; 3) Decreased kidney ROS; and 4) Decreased kidney TGF-β protein expression (HS: 1.9±0.01 vs. HS+iSIK: 1.4±0.3 au. p=0.0007). Thus, the data indicate that SIK inhibition attenuates salt-sensitive hypertension and prevents kidney injury, without altering Na + handling in response to HS. In perspective, SIK might be a pharmacological target for salt-sensitive hypertension treatment.