The association between the expression of aquaporins (AQPs) in kidney tissues and the occurrence of edema in nephrotic syndrome (NS) remains unclear. The current study aimed to investigate this association. A total of 54 patients with primary glomerular disease, diagnosed by renal biopsy, were divided into three groups: Control, NS without edema and NS with edema. The expression of AQP1, AQP2, AQP3 and AQP4 in kidney tissues from these patients was assessed using immunohistochemistry, and urinary AQP concentrations were quantified by ELISA. Comparison of the three groups was conducted using one way analysis of variance, independent samples t-test or the Chi-square test. AQP1 was strongly expressed in the proximal tubules. The proportion of the AQP1-positive area in kidney tissues from patients with NS with edema was significantly reduced, in comparison with the other two groups. By contrast, the proportion of the AQP2-positive area in the NS with edema group was significantly higher than that of the other two groups; significant differences were also observed between the control and NS without edema groups for this parameter. Urinary AQP2 concentrations in patients with NS (with and without edema) were significantly higher than that of the control group, and exhibited a significant positive correlation with kidney tissue AQP2 concentrations. The present study demonstrated the abnormal expression pattern of AQP1-AQP4 in the kidney tissues of patients with NS, providing a basis for an improved understanding of the role of AQP in the pathogenesis of NS.
Nephrotic syndrome (NS), characterized by edema, proteinuria, hypoalbuminemia and hyperlipemia, is nonspecific form of kidney disease. NS occurs as a result of any disorder that damages the nephrons, including diabetes, vasculitis, lupus and nephritis. NS affects the integrity of the glomerular capillary membrane and results in an inflammatory response in the glomeruli (
Aquaporins (AQPs) are low molecular-weight hydrophobic and transmembrane proteins, the monomers of which range between 26 and 34 kDa in size. AQPs facilitate a diffusion rate that is 10-20 times faster than simple diffusion, and thus their selective permeability to water is very high (
Due to the fact that AQP1-4 are the primary types of AQPs in the kidney, the association between AQP1-4 expression and edema was investigated in kidney tissues from patients with primary NS in the current study.
The current study was approved by the Institutional Review Board of the Jinshan Hospital Affiliated to Fudan University (Shanghai, China) and written informed consent was obtained from each participant.
Patients admitted to the hospital and diagnosed with primary glomerular disease [including immunoglobulin A (IgA) nephropathy] by renal biopsy, were eligible for the study. Inclusion criteria were as follows: Patients were >18 years old; diuretics, corticosteroids and renin-angiotensin-aldosterone system blockers were not administered prior to biopsy; no other diseases, such as hepatic cirrhosis and cardiac dysfunction, which may result in edema, were present; no electrolyte imbalance was detected; patients had provided informed consent.
Patients were divided into NS and control groups, according to the following diagnostic criteria for NS: Macroalbuminuria (≥3.5 g/day) and hypoproteinemia (<30 g/l). Patients with NS were further subdivided into NS without edema and NS with edema groups, based on the presence or absence of pitting edema (for example of the eyelids, dorsal and anterior tibial muscles and sacrum).
The EliVision Plus kit and 3,3′-diaminobenzidine (DAB) kit were obtained from Fuzhou Maixin Biotechnical Co., Ltd. (Fuzhou, China). DAB-1031 and hematoxylin staining solution were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Neutral gum was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Polylysine solution, phosphate-buffered saline (PBS) and sodium citrate buffer were obtained from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China).
Blood urea nitrogen (BUN), serum creatinine (Scr), albumin, potassium and sodium were detected using a Beckman CX9 Biochemical Analyzer (Beckman Coulter, Brea, CA, USA) and Beckman designated reagents. Urine protein (24-h) was quantified on a Johnson Vitros-350 Biochemical Analyzer (Johnson & Johnson, Shanghai, China).
The Modification of Diet in Renal Disease equation: eGFR = 170 × Scr−0.999 × Age−0.176 × BUN−0.170 × Alb0.318 × (0.762 Female), was applied to assess the estimated GFR (eGFR).
A portion of renal tissues, obtained from kidney biopsies, were pathologically examined by the Research Institute of Nephrology of Fudan University (Shanghai, China). The remaining tissue samples were preserved in 70% ethanol (Shanghai Jiuyi Chemical Reagent Co., Ltd., Shanghai, China) at 4°C in a refrigerator, following fixation with 4% formaldehyde for 24 h.
Naturally dried tissue samples were immersed in 4% paraformaldehyde (Shanghai Jiuyi Chemical Reagent Co., Ltd.) solution for 30 min in order to improve cell permeability. Following washing three times in PBS for 3 min each time, samples were successively dewaxed by incubation with xylene (Shanghai Jiuyi Chemical Reagent Co., Ltd.) and rehydrated with an ethanol gradient (successively anhydrous; 95, 85 and 70%) for 5 min at each step. The slides were placed into the antigen retrieval buffer (3 g trisodium citrate, 0.4 g citric acid, 1 L distilled water; Shanghai Jiuyi Chemical Reagent Co., Ltd.) and boiled for 20 min. Following 3 washes with PBS, 2 drops of 3% H2O2-methanol (800
Urine specimens were collected over 24 h (6 am to 6 am the next day) in a container, and measured in a dosage cup. The total protein concentrations were then measured using a Johnson Vitros-350 Biochemical Analyzer and AQP concentrations were determined by ELISA. Finally, 24-h total urine protein and AQP quantities were deduced.
Data are expressed as the mean ± standard deviation and statistical analyses were performed using SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA). Continuous variables were compared among groups using one way analysis of variance and the independent samples t-test for two groups, while discrete variables were compared using the Chi-square test. P<0.05 was considered to indicate a statistically significant difference. Single correlation analysis was used to characterize the association between variables.
Among the 54 patients enrolled, 21 who did not have NS, were assigned to the control group, including 1 case of focal segmental glomerulosclerosis (FSGS), 15 individuals with IgA nephropathy and 5 cases of mesangial proliferative glomerulonephritis. The NS without edema group contained 16 patients (7 cases of FSGS, 1 case of membranous nephropathy and 8 cases of IgA nephropathy). The NS with edema group contained 17 patients (3 cases of FSGS, 10 patients with membranous nephropathy, 3 cases of IgA nephropathy and 1 case of minimal change nephropathy). As presented in
AQP1 was strongly expressed in the proximal tubules, and was not detected in the glomerulus (
AQP2 was strongly expressed in the collecting ducts, and was not detected in the glomerulus (
AQP3 was strongly expressed in the collecting ducts, and was not detected in the glomerulus (
AQP4 was strongly expressed in the collecting ducts of certain patients, and was not detected in the glomerulus (
Edema is a key clinical symptom of NS and is considered to be associated with water and sodium retention, in addition to increases in peripheral capillary permeability (
The present study demonstrated that AQP1 expression was significantly reduced in the proximal tubules of patients with NS with edema. In addition, the quantity of AQP1 in the urine in this group was higher than that in the control group. AQP1, also termed prototype water channel, is a six-transmembrane pore protein, and its tetramers on the cell membrane bidirectionally transport water molecules in and out of the cell (
Notably, the expression of AQP2 observed in the kidney, and the quantity of AQP2 in the urine, of the two NS groups was significantly greater than that in the control group, with a greater effect observed in the patients with NS with edema. In addition, it was demonstrated that AQP2 levels in the urine of patients with NS were significantly positively correlated with the AQP2-positive index in kidney tissues. These observations suggest that increased AQP2 expression is associated with the reabsorption of water, which may result in water and sodium retention, and the subsequent development of edema. In addition, the level of AQP2 in the urine may predict the expression of AQP2 in kidney tissues. AQP2 was one of the AQPs in the kidney that were cloned by Fushimi
Furthermore, it was demonstrated that AQP3 expression was higher in patients with edema than in the other two groups, although this results was not statistically significant. AQP3 is permeable to small molecules, such as glycerol and urea, in addition to water. It is expressed in various tissue types, including the trachea, lung, gastrointestinal mucosa and the kidneys (
In the current study, AQP4 was exclusively expressed in the collecting ducts in certain patients, but was not expressed in the glomerulus. AQP4 was distributed predominantly in the inner zone of the outer medulla, outer 1/3 of the inner medulla and the S3 segment of the proximal tubule. AQP4 was observed to participate in water reabsorption and the concentration of urine (
In conclusion, by assessing the expression levels of AQPs in kidney tissues, the present study demonstrated that a correlation exists between edema in NS and AQP levels. Of note, the different AQP subtypes did not share the same pattern of expression. Water and sodium retention may be inhibited by compensatory reductions in AQP1 expression in the kidney. AQP2 was highly expressed in the kidney and participated in the reabsorption of water, which may have resulted in water and sodium retention. Whether AQP3 and AQP4 are associated with edema in NS requires further investigation with a larger sample size or the use of animal experiments. The observations of the current study provide a basis for investigating the roles of AQPs in the pathogenesis of NS.
The current study was supported by a grant from the Fudan University Shanghai Medical College Youth Fund (grant no. 11L-8).
Expression of AQP1 in kidney tissues in the control, NS without edema and NS with edema groups (magnification, ×400). Representative micrographs in each group demonstrate the immunostaining results for AQP1 in renal tubules (bottom panel) and glomeruli (top panel). AQP, aquaporin; NS, nephrotic syndrome.
Expression of AQP2 in kidney tissues in the control, NS without edema and NS with edema groups (magnification, ×400). Representative micrographs in each group demonstrate the immunostaining results for AQP2 in renal tubules (bottom panel) and glomeruli (top panel). AQP, aquaporin; NS, nephrotic syndrome.
Expression of AQP3 in kidney tissues in the control, NS without edema and NS with edema groups (magnification, ×400). Representative micrographs in each group demonstrate the immunostaining results for AQP3 in renal tubules (bottom panel) and glomeruli (top panel). AQP, aquaporin; NS, nephrotic syndrome.
Expression of AQP4 in kidney tissues in the control, NS without edema and NS with edema groups (magnification, ×400). Representative micrographs in each group demonstrate the immunostaining results for AQP4 in renal tubules (bottom panel) and glomeruli (top panel). AQP, aquaporin; NS, nephrotic syndrome.
Clinical data, urinary protein and serum markers of the three groups.
Variable | Control group (n=21) | NS without edema group (n=16) | NS with edema group (n=17) |
---|---|---|---|
Age (years) | 41.5±14.4 | 42.4±13.3 | 42.4±16.6 |
Gender (M/F) | 9/12 | 7/9 | 8/9 |
GFR (ml/min) | 98.85±23.27 | 95.06±29.24 | 85.27±21.43 |
Albumin (g/l) | 42.10±3.86 | 26.38±3.14 |
25.59±3.22 |
Urinary protein (g/24 h) | 0.56±0.28 | 4.57±1.05 |
5.46±1.74 |
Sodium (mmol/l) | 140.33±3.07 | 140.44±3.58 | 139.06±2.46 |
Potassium (mmol/l) | 3.97±0.33 | 4.06±0.35 | 4.00±0.30 |
Data are presented as the mean ± standard deviation.
P<0.01, compared with the control group,
P<0.01, compared with the control group. NS, nephrotic syndrome; GFR, glomerular filtration rate.
Expression of AQPs in kidney tissues.
AQP | Positive index (%)
| ||
---|---|---|---|
Control group | NS without edema group | NS with edema group | |
1 | 0.683±0.311 | 0.652±0.300 | 0.414±0.201 |
2 | 0.512±0.213 | 0.665±0.228 |
0.823±0.002 |
3 | 0.585±0.111 | 0.592±0.119 | 0.614±0.114 |
P<0.05, compared with the control group,
P<0.01, compared with the control group,
P<0.05, compared with the NS without edema group. AQPs, aquaporins; NS, nephrotic syndrome.
Urinary AQP levels.
AQP | Quantity in 24 h urine sample (ng/ml)
| ||
---|---|---|---|
Control group | NS without edema group | NS with edema group | |
1 | 39.189±12.448 | 41.492±14.766 | 43.078±17.923 |
2 | 30.320±9.528 | 38.621±13.187 |
45.309±16.921 |
Data are presented as the mean ± standard deviation.
P<0.05, compared with the control group,
P<0.01, compared with the control group. AQP, aquaporin; NS, nephrotic syndrome.
Correlation between AQP expression in kidney tissues and urinary AQP levels in patients with NS.
AQP | R | P-value |
---|---|---|
1 | 0.1558 | 0.2605 |
2 | 0.6198 | <0.001 |
AQP, aquaporin; NS, nephrotic syndrome.