Protein phosphatase 1 inhibitor-1 deficiency reduces phosphorylation of renal NaCl cotransporter and causes arterial hypotension

Abstract

The thiazide-sensitive NaCl cotransporter (NCC) of the renal distal convoluted tubule (DCT) controls ion homeostasis and arterial BP. Loss-of-function mutations of NCC cause renal salt wasting with arterial hypotension (Gitelman syndrome). Conversely, mutations in the NCC-regulating WNK kinases or kelch-like 3 protein cause familial hyperkalemic hypertension. Here, we performed automated sorting of mouse DCTs and microarray analysis for comprehensive identification of novel DCT-enriched gene products, which may potentially regulate DCT and NCC function. This approach identified protein phosphatase 1 inhibitor-1 (I-1) as a DCT-enriched transcript, and immunohistochemistry revealed I-1 expression in mouse and human DCTs and thick ascending limbs. In heterologous expression systems, coexpression of NCC with I-1 increased thiazide-dependent Na(+) uptake, whereas RNAi-mediated knockdown of endogenous I-1 reduced NCC phosphorylation. Likewise, levels of phosphorylated NCC decreased by approximately 50% in I-1 (I-1(-/-)) knockout mice without changes in total NCC expression. The abundance and phosphorylation of other renal sodium-transporting proteins, including NaPi-IIa, NKCC2, and ENaC, did not change, although the abundance of pendrin increased in these mice. The abundance, phosphorylation, and subcellular localization of SPAK were similar in wild-type (WT) and I-1(-/-) mice. Compared with WT mice, I-1(-/-) mice exhibited significantly lower arterial BP but did not display other metabolic features of NCC dysregulation. Thus, I-1 is a DCT-enriched gene product that controls arterial BP, possibly through regulation of NCC activity.

Figure 1.

COPAS allows efficient isolation of EGFP-expressing DCTs. (A and B) Representative immunostainings of renal cryosections from PV-EGFP mice stained for (A) NCC or (B) βENaC. The EGFP signal colocalizes with NCC-positive early DCT (DCT1–D1) but not ENaC-positive late DCT (DCT2–D2), and ENaC-positive connecting tubules (CNs). Arrows indicate the transitions (A) from TAL (T) to D1 and (B) from D1 to D2. (C) EGFP⁺ tubules were sorted using COPAS. Fluorescent emission and relative size (measured by an axial light loss detector and called time of flight) were measured for each tubular segment from the tubular suspension. A selection window for sorting was set to collect EGFP⁺ (green), EGFP⁻ (red), or ALL (blue) tubules. Representative pellets for sorted 400 tubules for each fraction are shown in tubes in the right panel. (D) NCC gene expression levels were assessed by real-time PCR for EGFP⁺, EGFP⁻, and ALL tubules samples (n=4 mice). (E) Immunoblots of EGFP⁺ tubules (400 tubules/lane) and kidney lysates detecting NCC, βENaC, and other nephron segments markers (i.e., NaPi-IIa, proximal tubule; NKCC2, thick ascending limb; AQP2, connecting tubule/collecting duct) confirmed the strong enrichment of NCC-positive DCTs in EGFP⁺ tubules. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

I-1 is highly abundant in mouse DCT and TAL. (A) Immunoblot analysis for I-1 in kidney and brain lysates from WT and I-1^−/− mice. A band of the expected size (28 kD) is detected in kidney and brain of WT mice but not I-1^−/− mice. Actin is used as a loading control. (B) Immunoblot analysis of COPAS-sorted tubules (400 tubules for each lane) for the nonselected (all tubules), EGFP⁻, and EGFP⁺ tubule fractions shows significant enrichment of I-1 in the EGFP⁺ tubules. (C) Immunohistochemistry reveals I-1 in kidney tubules of WT but not I-1^−/− mice. (D) Immunostaining of consecutive cryosections from WT mice shows a cytoplasmic I-1 localization in NCC- and CB28K-positive early DCTs (D1; no or weak CB28K) and late DCTs (D2; strong CB28K) and in the NCC-negative TAL (T). Scale bars, ∼20 μm.

Figure 3.

I-1 regulates NCC activity and phosphorylation. (A) Immunoblot analysis of HEK293 cells with tetracycline-inducible NCC overexpression (Flp-InNCC) reveals endogenous expression of I-1 and PP1a. Small interfering RNA (siRNA) knockdown of I-1 (I-1 siRNA) reduces endogenous I-1 protein abundance compared with no siRNA– and control siRNA–treated cells. Although the reduced I-1 abundance does not affect PP1a and total NCC abundance, it decreases NCC phosphorylation on threonine 53 (pT53 NCC). (B) Thiazide-sensitive ²²Na uptake was measured in oocytes overexpressing NCC. Coexpression of NCC with the catalytic subunit of PP1 (PP1a) does not significantly reduce thiazide-sensitive ²²Na uptake. Coexpression of NCC with constitutively active I-1 (I-1 T35D) profoundly stimulates thiazide-sensitive ²²Na uptake. Together, the data suggest that enhancing the basal level of phosphatase activity in the oocyte has little effect but that inhibiting this activity has a substantial effect; n=3 for each condition (mean±SEM); significance (by ANOVA) is shown (*P<0.05). (C) Immunoblot analysis of kidney membrane fractions from WT and I-1^−/− mice was used to quantify the abundance of total NCC and NCC phosphorylated at threonine 53 (pT53 NCC), threonine 58 (pT58 NCC), serine 71 (pS71 NCC), and serine 89 (pS89 NCC). (D) Densitometric analysis from NCC immunoblots that were normalized to β-actin protein levels and expressed for I-1^−/− mice in percent of control. Mean±SEM (n=7). Statistical significance was calculated with unpaired t test (*P<0.05; ***P<0.001).

Figure 4.

I-1 deficiency lowers arterial blood pressure but has little or no effect on Na⁺, and K⁺ diet adaptation, HCTZ response, and renal ion transporter abundances. (A) Systolic BP was measured with the tail-cuff method in WT and I-1^−/− male and female mice on standard (0.3% Na⁺; open square) or low (0.05% Na⁺; filled circle) Na⁺ diets. Measurements were done for 4 consecutive days. Each data point corresponds to an average of 4 days. Statistical analysis was performed using two-way ANOVA test for each dietary period (variables were the genotype and time). *P<0.05; **P<0.01 (n=10 per group). (B) Urine Na⁺ excretion in WT and I-1^−/− mice before and after the switch from standard to low Na⁺ diet. No statistical difference was found between the two groups (n=6 per group). Urine Na⁺ excretion was normalized to creatinine excretion. (C) Urine K⁺ excretion in WT and I-1^−/− mice before and after the switch from standard (0.8% K⁺) to low (0.05% K⁺) K⁺ diet. No statistical differences were found between the two groups (n=8 per group). Urine K⁺ excretion was normalized to creatinine excretion. (D) Plasma K⁺ was measured after 4 days on low K⁺ diet by blood sampling from the heart. Values are means±SEM. I-1^−/− mice had slightly lower plasma K⁺ values than WT mice, but a significant statistical difference was not reached (P=0.24, t test; n=8 per group). (E) Effect of HCTZ injection (50 mg/kg body wt intraperitoneally) on urinary Na⁺ excretion in WT (open bars) and I-1^−/− (filled bars) mice that were kept for 14 days on either standard or low Na⁺ diet. Urines were collected for 6 hours after injection of either vehicle or HCTZ. The thiazide-sensitive component of urinary Na⁺ excretion is presented as the difference between HCTZ- and vehicle-induced natriuresis within the first 6 hours postinjection. Data are shown as mean±SEM. No statistically significant difference between WT and I-1^−/− was found (P=0.79 for standard Na⁺; P=0.08 for low Na⁺ diet, t test; n=8–9 mice in each group). (F) Immunoblot analysis of kidney membrane fractions from WT and I-1^−/− mice showing the abundance of major apical Na⁺ transporting pathways along the nephron. Densitometric analysis was normalized to β-actin protein levels for each blot (Supplemental Tables 1–4). Statistical significance was calculated with t test (*P<0.05).

Figure 5.

I-1 deficiency does not affect NKCC2 abundance and phosphorylation. (A) Immunoblot analysis of kidney membrane fractions from WT and I-1^−/− mice for total NKCC2 and phosphorylated NKCC2 (pNKCC2). (B) Densitometric analysis was normalized to β-actin protein levels. No statistical difference was detected between groups (t test; n=5 per group). (C) Representative immunostainings for NKCC2 and pNKCC2 on consecutive cryosections of WT and I-1^−/− mouse kidneys. NKCC2 and pNKCC2 are found in the apical membrane of the TAL (T). No differences are seen between WT and I-1^−/− mice. Scale bars, ∼20 μm. (D) Effect of furosemide injection (40 mg/kg body wt intraperitoneally) on urinary Na⁺ excretion in WT (open bars) and I-1^−/− (filled bars) mice. Urines were collected for 6 hours after injection of either vehicle or furosemide. The furosemide-sensitive component of urinary Na⁺ excretion is presented as the difference between furosemide- and vehicle-induced natriuresis within the first 6 hours postinjection. Data are shown as mean±SEM (n=9 for each group). Statistical significance was assessed by t test (*P<0.05).

Figure 6.

I-1 deficiency does not affect SPAK abundance and phosphorylation. (A) Immunoblot analysis of total kidney lysates from WT and I-1^−/− mice for total SPAK and SPAK phosphorylated at Ser373 (pSPAK). Lysates from isolated DCTs were run in parallel and revealed SPAK and pSPAK at 70 kD. (B) Densitometric analysis was normalized to β-actin protein levels. No statistical difference between genotypes was detected with t test (n=5 mice per group). (C) Representative immunostainings for NCC, SPAK, and pSPAK on consecutive cryosections of WT and I-1^−/− mouse kidneys. SPAK and pSPAK are clearly visible in NCC-negative TAL (T) and NCC-positive DCT (D). No differences are seen between WT and I-1^−/− mice. Scale bars, ∼20 μm.