Enhanced phosphorylation of Na(+)-Cl- co-transporter in experimental metabolic syndrome: role of insulin

Abstract

In the present study, we investigated the activity of the thiazide-sensitive NCC (Na(+)-Cl(-) co-transporter) in experimental metabolic syndrome and the role of insulin in NCC activation. Renal responses to the NCC inhibitor HCTZ (hydrochlorothiazide), as a measure of NCC activity in vivo, were studied in 12-week-old ZO (Zucker obese) rats, a model of the metabolic syndrome, and in ZL (Zucker lean) control animals, together with renal NCC expression and molecular markers of NCC activity, such as localization and phosphorylation. Effects of insulin were studied further in mammalian cell lines with inducible and endogenous expression of this molecule. ZO rats displayed marked hyperinsulinaemia, but no differences in plasma aldosterone, compared with ZL rats. In ZO rats, natriuretic and diuretic responses to NCC inhibition with HCTZ were enhanced compared with ZL rats, and were associated with a decrease in BP (blood pressure). ZO rats displayed enhanced Thr(53) NCC phosphorylation and predominant membrane localization of both total and phosphorylated NCC, together with a different profile in expression of SPAK (Ste20-related proline/alanine-rich kinase) isoforms, and lower expression of WNK4. In vitro, insulin induced NCC phosphorylation, which was blocked by a PI3K (phosphoinositide 3-kinase) inhibitor. Insulin-induced reduction in WNK4 expression was also observed, but delayed compared with the time course of NCC phosphorylation. In summary, we report increased NCC activity in hyperinsulinaemic rodents in conjunction with the SPAK expression profile consistent with NCC activation and reduced WNK4, as well as an ability of insulin to induce NCC stimulatory phosphorylation in vitro. Together, these findings indicate that hyperinsulinaemia is an important driving force of NCC activity in the metabolic syndrome with possible consequences for BP regulation.

Figure 1

Natriuretic responses to vehicle and hydrochlorothiazide (HCTZ) in Zucker lean and obese rats. Zucker lean (blank columns) and obese (filled columns) rats were administered with a vehicle (200 μl of normal saline) and placed into metabolic cages for 24 hours. The urine was collected during the early 3-hour period (left panels) and for the rest of the 24-hour collection period for determination of urinary sodium excretion (U_NaV), urinary flow (UF) and urinary potassium excretion (U_KV). The following week the measurements were repeated after the administration HCTZ (3.75 mg/kg). The data are expressed in absolute values and as ratio per body weight.

Figure 2

Systolic blood pressure in Zucker lean (empty bars) and obese (filled bars) rats administered with a vehicle (0.9% NaCl, 200 μl i.p.) or hydrochlorothiazide (HCTZ, 3.75 mg/kg i.p. in 200 μl 0.9% NaCl).

Figure 3. Renal cortical protein expression of NCC, SPAK and WNK4 in Zucker lean (ZL) and obese (ZO) rats

A. Total NCC protein abundance and B. Thr53 phosphorylation of the NCC (P-NCC) were analyzed by western blotting in whole cell and crude membrane fractions. C. Graphed results of densitometric analysis of NCC and P-NCC abundance expressed as a ratio of protein/actin (loading control) or as a ratio of phospho- and total NCC (*p<0.05, †p<0.01 vs. ZL). D. The NCC and P-NCC were localized in the DCT by immunofluorescence microscopy. E. Western blot analysis of renal cortical homogenates using primary antibodies against C- and N-termini of SPAK. The graph shows densitometric analysis of all C-SPAK bands and the band with the highest molecular weight (~ 60 kD), corresponding to full size SPAK (FL-SPAK) (†p<0.01 vs. ZL). F. Renal protein expression of WNK4 in ZL and ZO rats. The graph shows the results of densitometric analysis of western blots. Representative blots are shown in insets (*p<0.05 vs. ZL).

Figure 4. Effect of insulin on NCC and Akt kinase phosphorylation in FlpIn NCC cells

A. Immunoblot analysis phospho-Thr53/55 (P-NCC) and total NCC. To study the effect of insulin on NCC phosphorylation in vitro, the FlpIn NCC cells were first stimulated with tetracycline to induce NCC expression. Eighteen hours after NCC induction the cells harvested at baseline (0 minutes) or stimulated with insulin (40 ng/ml), harvested after 10–360 minutes, and analyzed by immunoblotting using primary antibodies raised against P-NCC and total NCC. Uninduced cells (UI) were harvested for western blots as negative controls. Tubulin was used as a loading control. B. Densitometric analysis of P-NCC abundance (P-NCC/tubulin ratio) and a ratio of phospho- and total NCC (*p<0.05, ^†p<0.01 vs. baseline (0′)). C. The Ser473 Akt phosphorylation (P-Akt) in response to insulin stimulation was also determined in FlpIn NCC cells as an established step in insulin signaling cascade. Total Akt protein abundance was analyzed as loading control. D. The effects of insulin on NCC phosphorylation were further investigated in tetracycline-induced cells with or without pretreatment with the phosphatidyl-inositol-3 kinase (PI3K) inhibitor LY294002 (LY, 60 min., 50 μM). The figure shows representative blot after 80–320 min. of insulin stimulation. E. Densitometric analysis of P-NCC abundance (P-NCC/tubulin ratio) in insulin stimulated cells with or without LY pretreatment. Since the baseline NCC phosphorylation was hardly detectable in this experiment, the P-NCC abundance in LY-pretreated cells is for each time point presented as a proportion of P-NCC abundance in insulin-stimulated cells without LY pretreatment (^†p<0.01 vs. insulin treated cells at the same time point). F. Immunoblot of P-Akt and total Akt in insulin-stimulated cells with and without LY pretreatment.

Figure 5. Effect of insulin on Thr 53/55 NCC phosphorylation and WNK4 expression in MDCT cells

The MDCT cells were used as another in vitro mammalian cell model, which displays endogenous NCC and WNK4 expression. The cells were stimulated with insulin (40 ng/ml) for 20, 40 and 240 minutes. At the selected time points, the cells were harvested and analyzed by immunoblotting. A. Immunoblot analysis of phospho-Thr53/55 (P-NCC) and total NCC. B. Densitometric analysis of P-NCC abundance and a ratio of phospho- and total NCC (^†p<0.01 vs. baseline (0′)). C. Immunoblot analysis of WNK4 protein abundance in insulin-stimulated cells. The figure shows representative blot (left) and densitometric analysis (right, *p<0.05 vs. baseline (0′)). D. Representative immunoblot and densitometry of WNK4 protein abundance in insulin-induced cells pretreated with the PI3K inhibitor LY294002 (LY, 60 min., 50 μM). E. Representative immunoblot and densitometric analysis of WNK4 protein abundance in cells grown in insulin-free (C) and insulin-containing media (I) for 24 and 48 hours (*p<0.05, ^†p<0.01 vs. C).