Inhibition of Mammalian Target of Rapamycin Complex 1 Attenuates Salt-Induced Hypertension and Kidney Injury in Dahl Salt-Sensitive Rats

Abstract

The goal of the present study was to explore the protective effects of mTORC1 (mammalian target of rapamycin complex 1) inhibition by rapamycin on salt-induced hypertension and kidney injury in Dahl salt-sensitive (SS) rats. We have previously demonstrated that H₂O₂ is elevated in the kidneys of SS rats. The present study showed a significant upregulation of renal mTORC1 activity in the SS rats fed a 4.0% NaCl for 3 days. In addition, renal interstitial infusion of H₂O₂ into salt-resistant Sprague Dawley rats for 3 days was also found to stimulate mTORC1 activity independent of a rise of arterial blood pressure. Together, these data indicate that the salt-induced increases of renal H₂O₂ in SS rats activated the mTORC1 pathway. Daily administration of rapamycin (IP, 1.5 mg/kg per day) for 21 days reduced salt-induced hypertension from 176.0±9.0 to 153.0±12.0 mm Hg in SS rats but had no effect on blood pressure salt sensitivity in Sprague Dawley treated rats. Compared with vehicle, rapamycin reduced albumin excretion rate in SS rats from 190.0±35.0 to 37.0±5.0 mg/d and reduced the renal infiltration of T lymphocytes (CD3⁺) and macrophages (ED1⁺) in the cortex and medulla. Renal hypertrophy and cell proliferation were also reduced in rapamycin-treated SS rats. We conclude that enhancement of intrarenal H₂O₂ with a 4.0% NaCl diet stimulates the mTORC1 pathway that is necessary for the full development of the salt-induced hypertension and kidney injury in the SS rat.

Keywords: arterial pressure; blood pressure; hypertension; rats, Sprague-Dawley.

Figure 1

A&B, Western blot analysis of pS6^S235/236 and S6 protein level in the renal cortex tissue homogenates prepared from SS and SD rats fed on a 0.4% NaCl diet (n=6) or 4.0% NaCl diet (n=6) for 3, 14 and 21 days. Densitometry was used to determine the mTORC1 activity by measuring the ratio of pS6^S235/236 and S6. Values represent pS6^S235/236/S6 normalized to the 0.4% NaCl fed SS or SD rats. C, Representative photomicrograph of the immunohistochemical profile of pS6 level in the renal cortex and medulla of SS rats fed a 0.4% NaCl or 4.0% NaCl diet. White discontinuous lines designate the boundaries of the cortex (C) and medulla (M) D, pS6^S235/236/S6 was measured in the outer medulla of SD rats with renal interstitial infusion of H₂O₂ for 3 days. Values represent pS6^S235/236/S6 normalized to the saline-treated SD rats. Saline (n=4) and H₂O₂ (n=5). * p<0.05

Figure 2

Mean arterial pressure (MAP) was measured with rats fed a 0.4% NaCl diet for 7 days the final 4 days of which rats were treated with rapamycin or vehicle prior to switching to 4.0% NaCl diet for 21 days. Close circles represent rapamycin (n=9) and open circles represent vehicle (n=7) treated SS rats. Close triangles represent rapamycin (n=6) and open triangles represent vehicle (n=6) treated SD rats. † Significant difference between vehicle and rapamycin treated SS rats (p<0.05) as determined using a two-way analysis of variance (ANOVA) for repeated measures; Holm-Sidak post hoc.

Figure 3

A&B, Albumin and protein were measured in urine samples collected for 24 hours on the last day of the 4.0 % NaCl for the determination of urinary excretion rates. Albumin (U_albV) and protein excretion (U_protV) rates were measured in SS rats treated with vehicle (n=7) or rapamycin (n=9). C, Summary of the outer medullary tubular injury (percentage of tubular cast positive region) and below graph shown here are the representative sections of trichrome-stained kidney from vehicle (n=7) or rapamycin (n=9) treated rats. D, Quantification of glomeruli injury in the cortex of vehicle (n=6) or rapamycin (n=6) treated SS rats. * p<0.05 Vs vehicle.

Figure 4

Western blots of pS6^S235/236 and pAKT^S473 were normalized with total endogenous S6 and AKT proteins, respectively. Graphs represent pS6^S235/236/S6 or pAKT^S473/AKT normalized to the vehicle treated SS rats. Corresponding representative immunoblots are shown below each graph. A, The level of pS6^S235/236/S6 and B, Representative photomicrograph of the immunohistochemical profile of pS6^S235/236 level in the renal cortex and medulla of SS rats treated with vehicle (n=7) or rapamycin (n=9) fed a 4.0% NaCl diet. White discontinuous lines designate the boundaries of the cortex (C) and medulla (M) C, pAKT^S473/AKT in the cortex and outer medulla was measured in these rats kidney. * p<0.05 Vs vehicle.

Figure 5

Quantification of T lymphocytes (CD3⁺) and macrophages (ED1⁺) cells per mm² of kidneys of SS rats treated with vehicle (n=7) or rapamycin (n=9) after 21 days of the 4.0% NaCl diet. A, CD3⁺ cells/mm² in the renal cortex and outer medulla B, ED1⁺ cells/mm² in the renal cortex and outer medulla C, representative kidney sections illustrating the immunohistochemical localization of T cells and macrophage cells (D) in the renal cortex and outer medulla. Blue and brown column on bar graph represent cortex and outer medulla, respectively. * p<0.05 Vs vehicle.

Figure 6

A, hypertrophic index was determined by measuring the ratio of right kidney weight (gm) and total body weight (gm) of the SS rats treated with vehicle (n=7) or rapamycin (n=9) maintained on the 4.0% NaCl diet. B, graph represents the percentage of p27⁺ cells (hypertrophy), C, percentage of Ki67⁺ cells (cell proliferation) D, quantification of apoptotic cells/mm² (TUNEL assay) in the renal cortex and outer medulla of SS rats treated with vehicle (n=6) or rapamycin (n=6) maintained on the 4.0% NaCl diet. Blue and brown column on bar graph represent cortex and outer medulla, respectively. * p<0.05 Vs vehicle.