Inhibition of mTORC2 promotes natriuresis in Dahl salt-sensitive rats via the decrease of NCC and ENaC activity

Abstract

We have previously observed that prolonged administration of rapamycin, an inhibitor targeting the mammalian target of rapamycin complex (mTORC)1, partially reduced hypertension and alleviated kidney inflammation in Dahl salt-sensitive (SS) rats. In contrast, treatment with PP242, an inhibitor affecting both mTORC1/mTORC2, not only completely prevented hypertension but also provided substantial protection against kidney injury. Notably, PP242 exhibited potent natriuretic effects that were not evident with rapamycin. The primary objective of this study was to pinpoint the specific tubular sites responsible for the natriuretic effect of PP242 in SS rats subjected to either 0.4% NaCl (normal salt) or 4.0% NaCl (high salt) diet. Acute effects of PP242 on natriuretic, diuretic, and kaliuretic responses were determined in unanesthetized SS rats utilizing benzamil, furosemide, or hydrochlorothiazide [inhibitors of epithelial Na⁺ channel (ENaC), Na-K-2Cl cotransporter (NKCC2), or Na-Cl cotransporter (NCC), respectively] either administered alone or in combination. The findings indicate that the natriuretic effects of PP242 in SS rats stem predominantly from the inhibition of NCC and a reduction of ENaC open probability. Molecular analysis revealed that mTORC2 regulates NCC activity through protein phosphorylation and ENaC activity through proteolytic cleavage in vivo. Evidence also indicated that PP242 also prevents the loss of K⁺ associated with the inhibition of NCC. These findings suggest that PP242 may represent an improved therapeutic approach for antihypertensive intervention, potentially controlling blood pressure and mitigating kidney injury in salt-sensitive human subjects.NEW & NOTEWORTHY This study explored mechanisms underlying the natriuretic effects of mammalian target of rapamycin protein complex 2 inhibition using PP242 and revealed both epithelial Na⁺ channel and Na-Cl cotransporter in the distal tubular segments were potentially inhibited. These observations, with prior lab evidence, indicate that PP242 prevents hypertension via its potent inhibitory effects on these specific sodium transporters and by reducing renal immune responses. This dual action, coupled with potassium sparing effects, suggests an improved approach for managing hypertension and associated kidney damage.

Keywords: ENaC; NCC; kaliuresis; mTORC2; natriuresis.

Graphical abstract

Figure 1.

Experimental protocol for the in vivo natriuretic study. Dahl salt-sensitive (SS) rats were placed in metabolic cages, provided drinking water ad libitum and allowed free access to food 2 days prior to drug or vehicle administration. Urine was collected from 9:00 AM to 1:00 PM and followed by a single intraperitoneal injection of benzamil (B), furosemide (F), hydrochlorothiazide (HCTZ, H), or their combination (B + F + H). Thereafter, urines were collected in 20-min intervals for 80 min. Rats were then allowed to recover for 44 h for drug washout and to again achieve a state of sodium balance. The same protocol was repeated with the injections of the same pharmacological inhibitors but with the addition of PP242. The rats were then fed a high-salt (HS) diet for 19 days before the same drug administration protocol and urine collections were repeated, with and without PP242. Urine volumes were determined by weight, and Na⁺ and K⁺ concentrations of the urine samples were measured by flame photometry as previously described. ENaC, epithelial Na⁺ channel; mTORC1/2, mammalian target of rapamycin (mTOR) protein complexes 1/2; NCC, Na-Cl cotransporter; NKCC2, Na-K-2Cl cotransporter.

Figure 2.

Cumulative urine output and Na⁺ and K⁺ excretion in male Dahl salt-sensitive (SS) rats before and following the diuretic treatment either alone or with PP242. The black line represents values before treatment (baseline), the blue line represents diuretic drug treatment (diuretics alone), and the red line represents diuretic drug coadministered with PP242 (diuretics + PP242). *P < 0.05 (Student’s t test) compared with baseline for the 80-min collection. #P < 0.05 (two-way repeated-measures ANOVA) of cumulated responses to diuretic drug(s) alone vs. PP242 coadministration at the indicated urine collection period (X-axis). A: rats were fed 0.4% NaCl diet (normal salt, NS). B: rats were fed 4.0% NaCl diet (high salt, HS) for 19 or 21 days. P values of ANOVA for the factors of collection time and PP242 effect and their interactions are presented in each graph.

Figure 3.

Effect of PP242 on epithelial Na⁺ channel (ENaC) activity. A: channel open probability (P_o) and active channel number (N) obtained from cell-attached patch-clamp recordings on principal cells of the isolated cortical collecting duct (CCD) obtained from male Dahl salt-sensitive (SS) rats fed 0.4% (white bars) and 4.0% (gray bars) NaCl diet for 21 days. B: acute effect of PP242 (10 μM) on ENaC P_o and N in SS rats fed either 0.4% or 4.0% NaCl diet for 21 days. *P < 0.05 by paired Student’s t test.

Figure 4.

Effects of PP242 on ribosomal protein S6 (S6) and AKT (protein kinase B). A: Western blot analysis showing effects of PP242 (P; light gray bars) or vehicle (V; dark gray bars) upon total S6, phosphorylated S6 (pS6 Ser235/6), and the ratio of pS6 Ser235/6 to total S6 determined in cortical tissue homogenates of normal salt (NS) and high salt (HS) fed SS rats. B: effects of PP242 (P; light gray bars) or vehicle (V; dark gray bars) upon total AKT, phosphorylated AKT (pAKT S473), and the ratio of pAKT S473 to total AKT. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way ANOVA in multiple comparisons. #P < 0.05 by Student’s t test.

Figure 5.

Effect of PP242 (light gray bars) on epithelial Na⁺ channel (ENaC) in cortex tissue homogenate and membrane fractions from 0.4% (normal salt, NS) and 4.0% (high salt, HS) NaCl-fed salt-sensitive (SS) rats treated with PP242 (P) or vehicle (V). A and B: Western blot images (A) and quantitation of band intensities (B) showing the effects of PP242 on ENaC α-subunit on tissue homogenate and membrane fractions. P < 0.05 by Student’s t test. C and D: Western blot images (C) and quantitation of band intensities (D) showing the effects of PP242 on ENaC γ-subunit on tissue homogenate and membrane fractions. *P < 0.05, ***P < 0.001, and ****P < 0.0001 by two-way ANOVA multiple comparisons. All blots in A are from the same tissue sample with the pAKT S473 blot repeated for orientation purposes.

Figure 6.

Effect of PP242 on total and phosphorylated Na-Cl cotransporter (NCC). A and B: Western blot images (A) of NCC total protein and phosphorylated NCC in kidney cortex tissue homogenate from normal salt (NS) and high salt (HS) fed rats treated with PP242 (P; light gray bars) or vehicle (V; dark gray bars) and quantification analysis (B). *P < 0.05 by Student’s t test. C and D: Western blot images (C) of NCC total protein and phosphorylated NCC in kidney cortex tissue membrane fractions from NS-fed Dahl salt-sensitive (SS) rats and quantification analysis (D). **P < 0.01 and ***P < 0.001 by Student’s t test. All blots in A are from the same tissue sample with the pAKT S473 blot repeated for orientation purposes.

Figure 7.

Effect of PP242 (light gray bars) on total and phosphorylated Na-K-2Cl cotransporter (NKCC2) in cortical tissues of normal salt (NS)-fed rats. A and B: Western blot images of NKCC2 total protein in cortical tissue homogenate and quantification. C: Western blot images of NKCC2 total protein and phosphorylated NKCC2 in kidney cortex tissue membrane fractions from NS-fed Dahl salt-sensitive (SS) rats treated with PP242 (P) or vehicle (V). D: quantitation of the band intensities. *P < 0.05 by Student’s t test. All blots in A are from the same tissue sample with the pAKT S473 blot repeated for orientation purposes.

Figure 8.

Effect of PP242 on total and phosphorylated Na-K-2Cl cotransporter (NKCC2) in outer medulla (OM) tissues of normal salt (NS) fed rats. A and B: Western blot images of mammalian target of rapamycin (mTOR) protein complexes 1/2 (mTORC1/2) pathway markers and total NKCC2 and detectable phosphorylated NKCC2 isoforms (T96, T101) in outer medulla (OM) tissue homogenate and quantifications. *P < 0.05, **P < 0.01, and ****P < 0.0001 by two-way ANOVA multiple comparisons. C and D: Western blot images of NKCC2 total protein and three phosphorylated isoforms (T96, T101, S126) in OM tissue membrane fractions and quantification. **P < 0.01 by Student’s t test.

Figure 9.

Effect of PP242 (light gray bars) on serum and glucocorticoid-regulated kinase 1 (SGK1), N-myc downstream regulated gene 1 (NDRG1), and NEDD4 like ubiquitin protein ligase (Nedd4l). A and B: Western blot images and band quantitation of SGK1 and its target proteins Nedd4l and NDRG1 in kidney cortex tissue homogenate from 0.4% (normal salt, NS) and 4.0% (high salt, HS) NaCl-fed Dahl salt-sensitive (SS) rats treated with PP242 (P) or vehicle (V). *P < 0.05, **P < 0.01 and ***P < 0.001 by two-way ANOVA multiple comparisons. C: Western blot images of SGK1 and its target proteins Nedd4l and NDRG1 in kidney cortex tissue cellular cytosol fractions from NS-fed SS rats treated with PP242 (P) or vehicle (V). The migration patterns of SGK1 and NDRG1 were clearly changed by PP242. D: densitometry quantifications of Nedd4l. *P < 0.05 by Student’s t test. All blots in A are from the same tissue sample with the pAKT S473 blot repeated for orientation purposes.

Figure 10.

The comparisons of 80 min total urine excretion from high salt (HS) treated rats upon the treatment of benzamil (n = 8), hydrochlorothiazide (HCTZ, n = 8), furosemide (n = 8), and PP242 (n = 4). The sum of averaged urine excretion from benzamil and HCTZ is presented.