Expression of Na + /H + exchanger regulatory factor 1 in autosomal-dominant polycystic kidney disease

Abstract

Objective

To explore the role of Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) in autosomal-dominant polycystic kidney disease (ADPKD).

Methods

NHERF1 and β-catenin protein were detected by immunohistochemistry and Western blotting of kidney tissue samples from patients with ADPKD and controls (normal kidney tissue [>5 cm from the foci] collected from patients undergoing unilateral nephrectomy for kidney cancer). NHERF1 and β-catenin protein and mRNA were quantified by Western blot and real-time fluorescent quantitative polymerase chain reaction, respectively, in kidney tissue samples from Han:SPRD (+/+) and (cy/+) rats. The effects of human recombinant NHERF1 on proliferation and cell cycle of ADPKD cyst-lining epithelial cells (WT9–12) were evaluated by MTT assay and flow cytometry, respectively.

Results

Levels of NHERF1 protein and mRNA were significantly lower, and β-catenin levels significantly higher, in patients with ADPKD and Han:SPRD (cy/+) rats, compared with control subjects and (+/+) rats, respectively. Exogenous recombinant NHERF1 significantly inhibited proliferation of WT9–12 cells and increased the proportion of cells in G₀/G₁ phase.

Conclusions

ADPKD is associated with a decrease in NHERF1 protein and mRNA levels. Supplementing exogenous NHERF1 inhibited the proliferation of WT9–12 cells.

Introduction

Autosomal-dominant polycystic kidney disease (ADPKD) is a common hereditary nephropathy and one of the main causes of renal failure. ¹ ADPKD involves abnormal gene expression and protein function, and may therefore be associated with kidney tumour development. ²

Cyst-forming signalling pathways include Wnt/β-catenin, which is involved in cell proliferation, differentiation, apoptosis and adhesion in tumour cells. ³ Since this pathway also controls kidney development and maintenance of normal morphology and function, disturbance can lead to disorders such as polycystic kidney disease. ⁴ Abnormal activation of the Wnt/β-catenin signalling pathway plays a critical role in ADPKD onset.^5,6 Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) regulates signalling molecules (including β-catenin) via phosphorylation, ⁷ and is highly expressed and abnormally located in many types of tumour cells.^8–11

The aim of the present study was to quantify NHERF1 and β-catenin in kidney tissue samples from patients with ADPKD and normal kidney tissue samples from control subjects, and a rat ADPKD model (Han:SPRD). In addition, the effects of recombinant human NHERF1 protein on the proliferation and apoptosis of an ADPKD cyst-lining epithelial cell line were evaluated.

Materials and methods

Study population

The study recruited patients with ADPKD who underwent unilateral nephrectomy due to cyst bleeding or pelvic pressure at the Department of Nephrology, Second Affiliated Hospital of Zhejiang University, Hangzhou, China, between January 2011 and December 2013. Control samples of kidney tissue were provided by patients undergoing unilateral nephrectomy for kidney carcinoma at the same institution: normal kidney tissue samples (>5 cm from the foci) were collected from each control subject.

The study was approved by the ethics committee of the Second Affiliated Hospital of Zhejiang University, Hangzhou, China, and all participants provided written informed consent.

Animals

Male Han:SPRD rats (30 heterozygous [cy/+] and 30 normal [+/+] animals) were purchased from Mayo Clinic (Rochester, MN, USA) and bred in the Experimental Animal Centre, Second Affiliated Hospital of Zhejiang University. Experiments were performed using groups of six rats aged 4, 8, 12, 16 or 24 weeks. Animal welfare and experimental procedures were conducted strictly in accordance with the Guide for the Care and Use of Laboratory Animals (US National Research Council, 1996) and the related ethics regulations of the Second Affiliated Hospital of Zhejiang University.

Immunohistochemistry

Human kidney tissue samples from ADPKD patients and controls were fixed in 100 ml/l neutral formaldehyde solution for 24 h, dehydrated, paraffin-wax embedded and sliced into 2 µm-thick sections. Sections were deparaffinized, microwave recovered in sodium citrate solution for 15 min and washed three times at 37℃ (10 min each wash) with 1 × phosphate buffered saline (PBS; 10 mM, pH 7.2). Endogenous peroxide was inhibited by incubation in 30 ml/l hydrogen peroxide for 10 min, then slides were washed three times with PBS (37℃, 10 min each wash), and blocked in normal goat serum at room temperature for 20 min. Sections were incubated with primary antibody (rat antihuman NHERF1 monoclonal antibody, 1 : 50 dilution or rat anti-human β-catenin monoclonal antibody, 1 : 40 dilution; both Santa Cruz Biotechnology, Santa Cruz, CA, USA) or negative control (PBS) overnight at 4℃, washed three times with PBS (37℃, 10 min each wash) and incubated with horseradish peroxidase conjugated goat antirat immunoglobulin (Ig)G (1 : 1200 dilution; Santa Cruz Biotechnology) at 37℃ for 10 min. After washing three times with PBS (37℃, 10 min each wash), slides were incubated with streptavidin–peroxidase (Sigma–Aldrich; St Louis, MO, USA) at 37℃ for 10 min and washed three times with PBS (37℃, 10 min each wash). Slides were stained with 3,3′-diaminobenzidine (DAB), counterstained with haematoxylin, dehydrated and sealed.

Staining was semiquantitatively analysed using Image-Pro® Plus version 6.0 (Media Cybernetics, Rockville, MD, USA). For each section, five visual fields (magnification × 400) were randomly selected. Cells with yellow-brown particles in the cytoplasm were classified as positive. Image-Pro® Plus software determined the integrated optical density (IOD), and average optical density (AOD) was calculated as IOD/total area of visual field.

Western blotting

Kidney tissue samples were temporarily stored in liquid nitrogen, placed in radioimmunoprecipitation assay buffer (pH 7.4 ± 0.1), homogenized on ice, and centrifuged at 10 000 ×  g at 4℃ for 10 min. The supernatant was collected and protein concentration was determined using BCA protein assay kit (Sigma–Aldrich). Equal quantities of protein per lane were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred to PVDF membrane (Amresco, Solon, OH, USA), then blocked in 5% skimmed milk in 200 nM Tris-HCl with 1.37 M sodium chloride and 10% Tween-20 (TBST) for 1 h at 37℃). Membranes were incubated with rabbit antihuman NHERF1 polyclonal antibody (1 : 1000 dilution), rabbit antihuman β-catenin polyclonal antibody (1 : 900 dilution) or rat antihuman glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1 : 2000 dilution; all Santa Cruz Biotechnology) overnight at 4℃, then washed three times with TBST (37℃, 15 min each wash). Membranes were incubated with horseradish peroxidase conjugated goat antirabbit IgG or goat antirat IgG (1 : 5000 dilution; Both Santa Cruz Biotechnologies) for 1 h at 37℃, washed five times with TBST (37℃, 10 min each wash), and developed in the dark using enhanced chemiluminescence (Sigma ECL™ Prime Western Blotting System; Sigma–Aldrich). Protein bands were scanned with ImageQuant™ TL (GE Healthcare, Little Chalfont, UK), and NHERF1 and β-catenin were semiquantified by reference to GAPDH.

Real-time fluorescent quantitative PCR

Total RNA was extracted from Han:SPRD rat kidney tissue (100 mg, stored temporarily in liquid nitrogen) using 1 ml TRIzol® (Life Technologies, Carlsbad, CA, USA), then (1 µg) reverse transcribed in a 10 μl reaction mix (2 µg RNA, 1 μl oligo(dT), 1 μl dNTP mix, 6 μl RNase free double distilled water. Real-time fluorescent quantitative polymerase chain reaction (PCR) was performed using a 7700 PCR system (Applied Biosystems, Carlsbad, CA, USA) in a 25-μl reaction mix comprising 12.5 μl of 2 × Premix Ex Taq, 1 μl each primer, 1 μl cDNA, and 9.5 μl double distilled water). The cycling programme involved preliminary denaturation at 95℃ for 10 min, followed by 40 cycles of denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, and elongation at 72℃ for 20 s. Primer sequences were NHERF1: sense 5′-GGATTTGGTCGTATTGGG-3′ and antisense 5′-GGATGGAAGATGGTGGAT-3′; β-catenin sense 5′-GGATTTGGTCGTATTGGG-3′ and antisense 5′-GGATGATGAAGAGGGATG-3′; GAPDH sense 5′-AAGGTGGTGAAGCAGGCGGC-3′ and antisense 5′-GAGCAATGC CAGCCCCAGCA-3′. The ratio of target to reference gene (GAPDH) was calculated based on the fluorescence curve and C _t value. ¹²

Proliferation and apoptosis assays

The human ADPKD cyst-lining epithelial cell line WT9–12 ¹³ was cultured in 1 : 1 Dulbecco’s modified Eagles’ medium (DMEM)/F12 (Life Technologies), containing 10% fetal bovine serum, 1000 IU/ml penicillin and 100 IU/ml streptomycin, at 37℃ in 5% carbon dioxide in air. Cells were passaged every 2–3 days until they adhered to the culture vessel.

Cell proliferation assays were performed in quadruplicate. Adherent cells were inoculated into 96-well plates at 4000 cells/well, synchronized in serum-free DMEM/F12 culture medium for 24 h, then incubated with 50, 100, 200 or 400 ng/ml recombinant human NHERF1 (OriGene, Rockville, MD, USA) for 24 h, 48 h or 72 h. MTT solution (10 μl of 5 g/l) was added and cells were incubated for a further 4 h, the supernatant was discarded, 100 μl of dimethyl sulphoxide was added and the plates were shaken for 10 min. Optical density at 490 nm was measured using a microplate reader, and the cell proliferation rate (%) was calculated as [1 – (OD_control – OD_experiment)/OD_control] × 100%.

Apoptosis assays were performed in quadruplicate. WT9-12 cells were inoculated onto 6-well plates at 2 × 10⁵ cells/well, synchronized in serum-free DMEM/F12 culture medium for 24 h after 60–70% cell fusion, then incubated with 50, 100, 200 or 400 ng/ml recombinant human NHERF1 (OriGene) for 72 h. The culture medium was discarded, and cells were digested with 0.25% trypsin/EDTA, pelleted by centrifugation (conducted at 4℃, three cycles of 8 min per cycle, 12000  g ), then dissociated into single-cell suspensions. Cells were added to cold 70% ethanol, fixed overnight at 4℃, stained in the dark with 50 μl of 50 mg/l propidium iodide, and evaluated using flow cytometry (Coulter® Epics® XL™; Beckman Coulter, Pasadena, CA, USA). Flow cytometry data were analysed with MultiCycle AV-OCX-De Novo Software (Phoenix Flow Systems, San Diego, CA, USA).

Statistical analyses

All experiments were performed in quadruplicate, and data were expressed as mean ± SD. Intra- and inter-group comparisons were conducted using analysis of variance and Student’s t-test, respectively. Data were analysed using SPSS® version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. P-values < 0.05 were considered statistically significant.

Results

The study included kidney tissue samples from six patients with ADPKD (3 male and 3 female; average age 52.2 ± 2.3 years) and six control subjects (4 male and 2 female; average age 55.7 ± 1.3 years). Immunohistochemical staining of control human kidney tissue found that NHERF1 was located in the cytoplasm of epithelial cells from the distal convoluted tubule, but not the glomerulus. Control samples had visibly higher levels of NHERF1 staining than samples from patients with ADPKD (Figure 1); AOD analysis confirmed this finding (1.41 ± 0.27 in controls versus 0.42 ± 0.13 in patients; P < 0.01). Levels of β-catenin were visibly higher in the cytoplasm of distal convoluted tubular epithelial cells from patients with ADPKD (Figure 2(b)) and WT9–12 cells (Figure 2(c)), compared with controls (Figure 2(a)). The β-catenin AOD was significantly higher in ADPKD patients than in controls (1.36 ± 0.23 versus 0.26 ± 0.24; P < 0.01).

Figure 1.

Representative light photomicrographs of immunohistochemical staining (yellow/brown staining) for Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1). (a) Normal human kidney tissue, and (b) kidney tissue from a patient with autosomal-dominant polycystic kidney disease. Original magnification × 400. The colour version of this figure is available at: http://imr.sagepub.com

Figure 2.

Representative light photomicrographs of immunohistochemical staining (yellow/brown staining) for β-catenin. (a) Normal human kidney tissue, (b) kidney tissue from a patient with autosomal-dominant polycystic kidney disease (ADPKD), (c) ADPKD cyst-lining epithelial cell line, WT9–12. Original magnification × 400. The colour version of this figure is available at: http://imr.sagepub.com

Levels of NHERF1 protein were significantly lower in patients with ADPKD compared with controls (P < 0.05), and in 4-week-old Han:SPRD (cy/+) rats compared with normal animals of the same age (P < 0.01; Figure 3). Levels of β-catenin were significantly higher in patients with ADPKD compared with controls (P < 0.05), and in 4-week-old Han:SPRD rats compared with normal animals of the same age (P < 0.01; Figure 4).

Figure 3.

(Upper Panel) Representative Western blot for NHERF1 (Na⁺/H⁺ exchanger regulatory factor 1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in kidney tissue samples from (1) normal human; (2) patient with autosomal-dominant polycystic kidney disease; (3)4-week old Han:SPRD +/+ rat; (4)4-week old Han:SPRD cy/+ rat. (Lower Panel) Semiquantification of NHERF1 by reference to GAPDH. #P < 0.05 vs normal human kidney; **P < 0.01 vs Han:SPRD (+/+) rat kidney; Student’s t-test; n = 6

Figure 4.

(Upper Panel) Representative Western blot for β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in kidney tissue samples from: (1) normal human; (2)patient with autosomal-dominant polycystic kidney disease; (3)4-week old Han:SPRD +/+ rat; (4)4-week old Han:SPRD cy/+ rat. (Lower Panel) Semiquantification of β-catenin by reference to GAPDH. #P < 0.05 vs normal human kidney; **P < 0.01 vs Han:SPRD (+/+) rat kidney; Student’s t-test; n = 6

Levels of NHERF1 and β-catenin mRNA in kidney tissue samples from Han:SPRD rats are shown in Table 1. At 4 weeks old, there were no significant differences between Han:SPRD (+/+) and (cy/+) rats in NHERF1 and β-catenin mRNA levels. Levels of NHERF1 mRNA were significantly lower and β-catenin mRNA levels were significantly higher in (cy/+) rats than (+/+) animals aged 8, 12, 16 and 24 weeks (P < 0.05 for each comparison; Table 1).

Table 1.

Levels of Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) and β-catenin mRNA in kidney tissue samples from Han:SPRD rats (animal model of polycystic kidney disease), quantified via real-time quantitative polymerase chain reaction

Age, weeks	NHERF1 mRNA		β-catenin mRNA
	Han:SPRD (+/+)	Han:SPRD (cy/+)	Han:SPRD (+/+)	Han:SPRD (cy/+)
4	1.23 ± 0.23	1.61 ± 0.04	1.41 ± 0.07	1.40 ± 0.04
8	1.64 ± 0.19	1.37 ± 0.11*	1.47 ± 0.13	1.65 ± 0.07*
12	2.11 ± 0.33	1.64 ± 0.17*	1.53 ± 0.22	1.77 ± 0.11*
16	3.22 ± 0.25	1.71 ± 0.21*	1.62 ± 0.21	3.34 ± 0.05*
24	3.31 ± 0.27	1.62 ± 0.22*	1.65 ± 0.16	5.21 ± 0.02*

Data presented as mean ± SD of six animals/group.

*

P < 0.05 vs age-matched Han:SPRD (+/+) rats; Student’s t-test.

Data regarding the effect of recombinant NHERF1 on proliferation of WT9–12 cells are shown in Figure 5. There were no significant effects on cell proliferation after treatment for 24 h. Treatment with 100, 200 or 400 ng/ml NHERF1 significantly inhibited cell proliferation at 48 and 72 h, compared with untreated cells at the same timepoint (P < 0.05 for each comparison). Treatment with 50 ng/ml NHERF1 had no significant effect on cell proliferation.

Figure 5.

Effect of recombinant human Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) on proliferation of the human autosomal-dominant polycystic kidney disease (ADPKD) cyst-lining epithelial cell line WT9–12. #P < 0.05 vs 0 ng/ml NHERF1; Student’s t-test. Data presented as mean ± SD of quadruplicate experiments

Table 2 shows data regarding the effect of recombinant NHERF1 on the cell cycle of WT9–12 cells. Treatment with 400 ng/ml NHERF1 for 72 h significantly increased the proportion of cells in G₀/G₁-phase and decreased those in S-phase, compared with control cells (P < 0.05 for both comparisons). Treatment with 200 ng/ml NHERF1 had no significant effect on cell cycle.

Table 2.

Effect of treatment with recombinant human Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1 for 72 h on cell-cycle phase in the human ADPKD cyst-lining epithelial cell line, WT9–12

Cell-cycle phase	Control	NHERF1
		200 ng/ml	400 ng/ml
G0/G1	58.91 ± 1.34	59.28 ± 2.67	67.87 ± 3.01*
S	40.12 ± 2.11	41.31 ± 4.04	30.27 ± 2.56*
G2/M	10.56 ± 1.85	11.95 ± 4.29	9.98 ± 2.71

Data presented as mean ± SD of quadruplicate experiments.

*

P < 0.05 vs control; Student’s t-test.

Discussion

Polycystic kidney disease is a monogenic hereditary disease that is classified into autosomal-dominant and autosomal-recessive forms. ADPKD accounts for ∼85% of cases and is typified by the formation of bilateral progressive renal fluid-filled cysts, resulting from tumour-like proliferation and abnormal apoptosis of cyst-lining epithelial cells.^14,15

As a tumour-like disease, ADPKD may be closely associated with carcinogenesis. Clear ectopic expression of β-catenin has been observed in kidney tissues from a murine model of ADPKD; this activated the Wnt signalling pathway and facilitated abnormal cell proliferation. ¹⁶ In addition, functional changes in the PCI/E-cadherin/β-catenin complex altered cell polarity and induced formation of renal cysts. ¹⁶ Mice that overexpress the β-catenin gene in kidney epithelial cells have been shown to have symptoms similar to ADPKD, ¹⁷ providing a possible link between cyst formation, and excessive cell proliferation and apoptosis. Mutation and depletion of APC (adenomatous polyposis coli) in the β-catenin destruction complex (GSK3D–Axin–APC) has been shown to induce cyst formation, ⁶ and abnormal activation of the Wnt signalling pathway is known to play a critical role in ADPKD onset. ⁴

The 358-amino acid protein NHERF1 contains an amino-terminal end tandem PDZ domain (i.e. PDZ-1 and PDZ-2) and a carboxyl-terminal end domain that binds proteins in the ERM family via typical protein–protein interactions. ¹⁸ Located on the membrane of normal epithelial cells, NHERF1 is distributed in the cytoplasm or nucleus of tumour cells, such as hepatoma HepG2 cells. ¹⁹ It is thought that NHERF1 may have different roles, depending on its cellular location.^20–22 In addition, NHERF1 affects β-catenin, which in turn participates in the onset and progression of ADPKD.^5,6,17

The present study examined the differences in NHERF1 and β-catenin protein and mRNA levels between diseased and healthy (paracancerous) kidney tissue, and found that patients with ADPKD and Han:SPRD (cy/+) rats had significantly less NHERF1 protein and mRNA than control subjects and animals, respectively. In contrast, β-catenin protein and mRNA levels were significantly higher in patients with ADPKD and Han:SPRD (cy/+) rats than in control subjects and animals. In addition, levels of NHERF1 mRNA were significantly lower, and β-catenin mRNA levels were significantly higher, in (cy/+) rats than (+/+) animals aged 8–24 weeks in the present study. It is possible that abnormal NHERF1 and β-catenin expression in ADPKD kidney tissue may promote the expression of cell proliferation genes, and lead to renal cysts.

Treating WT9–12 cells with human recombinant NHERF1 inhibited proliferation and arrested cells in the G₀/G₁ phase, in the present study. This finding may provide new avenues for ADPKD treatment.

In conclusion, ADPKD is associated with a decrease in NHERF1 protein and mRNA levels; supplementing exogenous NHERF1 inhibited the proliferation of WT9–12 cells. These findings provide experimental evidence for clarifying the pathogenesis of ADPKD and for developing new treatment options.