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HSD2 in HgCl2-induced nephrotic syndrome in rats
1The Water and Salt Research Center, University of Aarhus, Aarhus C; 2Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju; 3Institute of Anatomy, University of Aarhus, Aarhus C; 4Department of Anatomy, The Catholic University of Korea, Seoul, Korea; 5Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and 6Institute of Clinical Medicine, Aarhus University Hospital, Aarhus N, Denmark
Submitted 2 March 2005 ; accepted in final form 25 September 2005
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ABSTRACT |
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-hydroxysteroid dehydrogenase (11HSD2) is reduced, contributing to the enhanced mineralocorticoid activity. Membranous nephropathy was induced in Brown Norway rats by repeated injections of HgCl2 (1 mg/kg sc), whereas the control group received only vehicle. After 13 days of treatment, the abundance of ENaC subunits, sodium (co)transporters, and 11HSD2 in the kidney was examined by immunoblotting and immunohistochemistry. HgCl2 treatment induced marked proteinuria, hypoalbuminemia, decreased urinary sodium excretion, and ascites. The protein abundance of 
-ENaC was increased in the cortex/outer stripe of outer medulla (OSOM) and inner stripe of the outer medulla (ISOM). The protein abundances of -ENaC and 
-ENaC were decreased in the cortex/OSOM while increased in the ISOM. Immunoperoxidase microscopy demonstrated increased targeting of ENaC subunits to the apical plasma membrane in the distal convoluted tubule, connecting tubule, and cortical and medullary collecting duct segments. Moreover, 11HSD2 abundance was decreased in cortex/OSOM and ISOM. The protein abundances of type 3 Na/H exchanger (NHE3), Na-K-2Cl cotransporter (NKCC2), and thiazide-sensitive Na-Cl cotransporter (NCC) were decreased. Moreover, the abundance of the -1 subunit of the Na-K-ATPase was decreased in the cortex/OSOM and ISOM but remained unchanged in the inner medulla. These results suggest that increased apical targeting of ENaC subunits combined with diminished abundance of 11HSD2 may contribute to sodium retention associated with HgCl2-induced nephrotic syndrome. The decreased abundance of NHE3, NKCC2, NCC, and Na-K-ATPase may play a compensatory role in promoting sodium excretion.
epithelial sodium channel; type 2 11-hydroxysteroid dehydrogenase; collecting duct
25–50% of patients progress to end-stage kidney disease over 10 years (33). Thus MGN has a different and more progressive clinical course compared with the nonprogressive benign character of minimal-change nephrotic syndrome (33). HgCl2 has been known to induce a systemic autoimmune disease, including membranous nephropathy with IgG deposits. This nephropathy is responsible for the development of high-range proteinuria and full-blown nephrotic syndrome associated with generalized edema and ascites (11). Our laboratory demonstrated in a previous study that puromycin aminonucleoside (PAN)-induced nephrotic syndrome was associated with increased abundance and apical targeting of epithelial sodium channel (ENaC) subunits, which was proposed to play a role in the development of sodium retention (20). However, it remains to be elucidated whether the changes in expression and plasma membrane targeting of ENaC subunits in PAN-induced minimal-change nephrotic syndrome (caused by podocyte injury, not immune complex mediated) is unique to the PAN model or may be a more general characteristic of nephrotic syndrome, including immune complex-mediated glomerulonephritis. Furthermore, the physiological role of changes in subcellular distribution of ENaC subunits remains largely undefined in the abnormal sodium retention and ascites formation during disease states, and further studies may aid our understanding of molecular mechanisms of sodium retention.
11-Hydroxysteroid dehydrogenase type 2 (11HSD2) specifically modulates access of aldosterone to the mineralocorticoid receptor (MR) in a physiological environment where there is a molar excess of cortisol, and cortisol and aldosterone have similar affinity for the MR (3). Thus 11HSD2 normally protects the MR from the mineralocorticoid actions of cortisol by converting cortisol to cortisone, which has a low affinity for the MR, allowing aldosterone to be the major mineralocorticoid (17). This hypothesis has received considerable support from in vitro experiments (5, 24) and from the observation that individuals with mutations of the gene encoding 11HSD2 display the syndrome of apparent mineralocorticoid excess, a condition characterized by sodium retention, hypertension, and hypokalemia with decreased plasma aldosterone levels (35). Reduced activity of 11HSD2 causes renal sodium retention, and there is growing evidence that such a downregulation of 11HSD2 accounts, at least partly, for abnormal sodium retention in disease states such as liver cirrhosis (1, 14). If this mechanism is operative in nephrotic syndrome rats, collecting duct sodium reabsorption might well be stimulated. This is also an important subject to study.
The vasopressin-induced water permeability as well as the translocation of aquaporin-2 (AQP2) from the intracellular vesicle to the apical plasma membrane in the collecting duct principal cells is well characterized, and there is an extensive axial heterogeneity in the subcellular localization of AQP2 along the connecting tubule (CNT) and collecting duct in different physiological settings (9). Nephrotic syndrome is characterized by an extracellular fluid volume expansion due to renal sodium and water retention. Thus future investigations need to focus on defining the subcellular and segmental localization of AQP2 in nephrotic syndrome, which may play a role in the increased extracellular fluid volume expansion.
The purpose of this study is therefore to investigate whether immune complex-mediated nephrotic syndrome induced by HgCl2 in Brown Norway rats is associated with altered regulation of ENaC subunit and 11HSD2 as well as AQP2 to elucidate the underlying molecular mechanisms responsible for the increased sodium and water retention.
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METHODS |
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The animal protocols have been approved by the boards of the Institute of Anatomy and Institute of Clinical Medicine, University of Aarhus, according to the licenses for use of experimental animals issued by the Danish Ministry of Justice.
Protocol 1 (HgCl2 experiment). Male Brown Norway rats (Elevage Janvier, Le Genest-St.-Isle, France) were used to develop experimental nephrotic syndrome. HgCl2 nephropathy (n = 9) was induced in Brown Norway rats by repeated injections of HgCl2 (Sigma) at a dose of 1 mg/kg body wt, diluted to 1 mg/ml in 0.9% NaCl; subcutaneous injections into the abdominal skin were administered on days 0, 2, 4, 7, 9, and 11. Control rats (n = 7) received the vehicle alone (i.e., sterile 0.9% saline). They were maintained in individual cages on a standard rodent diet (Altromin 1324, Lage, Germany) and allowed free access to drinking water at all times. Control rats were offered the amount of food corresponding to the mean intake of food consumed by nephrotic syndrome rats during the previous day. Thus the food intake was matched between the two groups.
During the final 3 days, the rats were subsequently maintained in metabolic cages to allow urine collections for measurement of protein, sodium, potassium, creatinine, and osmolality. The rats were killed 13 days after onset of HgCl2 treatment, and immunochemical and morphological studies of the kidneys were performed. The rats were anesthetized with isoflurane (Forane, Abbott Laboratories, Gentofte, Denmark), and a large laparotomy was made. Peritoneal fluid volume was quantified in each rat when the fluid was absorbed with preweighed dry papers, and the papers were reweighed afterward. Blood was collected from the inferior caval vein and analyzed for sodium, potassium, creatinine, albumin, osmolality, and plasma aldosterone concentration using methods described previously in detail (20). The right kidney was rapidly removed, dissected into three zones [cortex and outer stripe of outer medulla (cortex/OSOM), inner stripe of outer medulla (ISOM) and inner medulla], and processed for semiquantitative immunoblotting as described below. The left kidney was fixed by retrograde perfusion as described below.
Protocol 2 (PAN experiment).
To investigate whether PAN-induced nephrotic syndrome is associated with altered regulation of 11HSD2 as well, another animal experiment with nephrotic syndrome was carried out. Experiments were performed using male Hannover-Wistar rats (250–300 g, Tacoma-Møllegaard Breeding Centre). PAN nephrotic syndrome (n = 8) was induced by a single intravenous injection of PAN (180 mg/kg body wt, Sigma, St. Louis, MO) via the femoral vein. Control rats (n = 8) received vehicle alone (i.e., sterile 0.9% saline). The rats were killed for immunoblotting analysis and immunocytochemical studies 7 days after PAN treatment.
Semiquantitative Immunoblotting
The dissected renal cortex/OSOM, ISOM, and inner medulla were homogenized (Ultra-Turrax T8 homogenizer, IKA Labortechnik, Staufen, Germany) in ice-cold isolation solution containing 0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.2. The homogenates were centrifuged at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria, and the supernatant was pipetted off and kept on ice. The total protein concentration was measured (Pierce BCA protein assay reagent kit, Pierce, Rockford, IL). All samples were adjusted with isolation solution to reach the same final protein concentrations and solubilized at 65°C for 15 min in SDS-containing sample buffer, and then they were stored at –20°C. To confirm equal loading of protein, an initial gel was stained with Coomassie blue. SDS-PAGE was performed on 9 or 12% polyacrylamide gels. The proteins were transferred by gel electrophoresis (Bio-Rad Mini Protean II) onto nitrocellulose membranes (Hybond ECL RPN3032D, Amersham Pharmacia Biotech, Little Chalfont, UK). The blots were subsequently blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4°C with primary antibodies followed by incubation with secondary anti-rabbit (P448, DAKO, Glostrup, Denmark), anti-mouse (P447, DAKO), or anti-sheep (1713–035-147, Jackson Laboratories, Bar Harbor, ME) horseradish peroxidase-conjugated antibodies. The labeling was visualized by an enhanced chemiluminescence system (ECL or ECL+plus) and exposure to photographic film (Hyperfilm ECL, Amersham). ECL films were scanned using an AGFA scanner (ARCUS II).
Immunohistochemistry
A perfusion needle was inserted in the abdominal aorta of isoflurane-anesthetized rats, and the vena cava was cut to establish an outlet. Blood was flushed from the kidneys with cold PBS (pH 7.4) for 15 s before a switch to cold 3% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 min. The kidney was removed and sectioned into 2- to 3-mm transverse sections and immersion fixed for 1 additional hour, followed by 3 x 10-min washes with 0.1 M cacodylate buffer, pH 7.4. The tissue was dehydrated in graded ethanol and left overnight in xylene. After being embedded in paraffin, 2-µm sections of the tissue were cut on a rotary microtome (Leica Microsystems, Herlev, Denmark).
The sections were dewaxed with xylene and rehydrated with graded ethanol. Sections had endogenous peroxidase activity blocked with 0.5% H2O2 in absolute methanol for 10 min. In a microwave oven, the sections were boiled in target retrieval solution (1 mmol/l Tris, pH 9.0, with 0.5 mM EGTA) for 10 min. After the sections were cooled, nonspecific binding was blocked with 50 mM NH4Cl in PBS for 30 min followed by 3 x 10 min with PBS blocking buffer containing 1% BSA, 0.05% saponin, and 0.2% gelatin. The sections were incubated with primary antibody (diluted in PBS with 0.1% BSA and 0.3% Triton X-100) overnight at 4°C. The sections were washed for 3 x 10 min with PBS wash buffer containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit Ig, DAKO) for 1 h at room temperature. After 3 x 10-min rinses with PBS wash buffer, the sites with antibody-antigen reaction were visualized with a brown chromogen produced within 10 min by incubation with 0.05% 3,3'-diaminobenzidine tetrachloride (Kem-en Tek, Copenhagen, Denmark) dissolved in distilled water with 0.1% H2O2. Mayer's hematoxylin was used for counterstaining, and after dehydration coverslips were mounted with hydrophobic medium (Eukitt, Kindler, Freiburg, Germany). Light microscopy was carried out with a Leica DMRE (Leica Microsystems).
Primary Antibodies
Rabbit polyclonal antibodies to the following renal sodium transporters were utilized (20): type 3 Na/H exchanger (NHE3), N-K-2Cl cotransporter (NKCC2), thiazide-sensitive Na-Cl cotransporter (NCC), and ENaC subunits -ENaC, -ENaC, and -ENaC. The polyclonal antibodies used in the study detect the uncleaved forms of the ENaC subunits (22). In addition, we previously used characterized rabbit polyclonal antibodies to AQP2 (26) and pAQP2 (10). A sheep polyclonal antibody to 11HSD2 (AB1296, Chemicon, Temecula, CA) was commercially obtained. A mouse monoclonal antibody against the Na-K-ATPase 1-subunit was kindly provided by Dr. D. M. Fambrough, Johns Hopkins University Medical School (Baltimore, MD).
Statistical Analyses
Values were presented as means ± SE. Comparisons between two groups were made by unpaired t-test. P values < 0.05 were considered significant.
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RESULTS |
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All HgCl2 nephropathic rats developed severe nephrotic syndrome characterized by marked proteinuria, hypoalbuminemia, and moderate amounts of ascites. Urinary protein excretion reached a +4 level from 10 days in HgCl2 nephropathic rats, whereas controls exhibited 0 or +1. Consistent with this, the plasma albumin level was significantly decreased in the HgCl2 nephropathic rats. Plasma urea and creatinine levels were increased, whereas creatinine clearance was decreased in HgCl2 nephropathic rats. In contrast, the urine output was not significantly different between two groups. Importantly, 24-h urinary sodium excretion and fractional excretion of sodium were decreased in HgCl2 nephropathic rats. Accordingly, HgCl2 nephropathic rats displayed a positive sodium balance. The urinary osmolality was not significantly different between the two groups. However, urine/plasma osmolality ratio and the solute-free water reabsorption were decreased, indicating decreased urinary concentration.
The urinary sodium/potassium ratio is widely used for evaluating aldosterone activity at the distal nephron (4). The urinary sodium/potassium ratio was markedly decreased in HgCl2 nephropathic rats, indicating increased aldosterone action in the distal nephron (Table 1). Accordingly, fractional excretion of potassium was increased. Consistent with this, plasma aldosterone levels were increased in HgCl2 nephropathic rats (Table 1).
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Semiquantitative immunoblotting was carried out to investigate whether the abundance of ENaC subunits changes in rats with HgCl2-induced nephrotic syndrome. The protein abundance of -ENaC was increased in the cortex/OSOM and ISOM (Fig. 1). The protein abundances of -ENaC and -ENaC were decreased in the cortex/OSOM but increased in the ISOM (Fig. 1). The analyses of normalized band densities are shown in Table 2.
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In HgCl2 nephropathy, -ENaC labeling was predominantly localized to the apical domains with only marginal labeling of the cytoplasm. This was evident in almost all cross-sectional tubules of distal convoluted tubule (DCT2) (Fig. 2B), CNT (Fig. 2D), cortical collecting duct (CCD) (Fig. 2F), and outer medullary collecting duct (OMCD) (Fig. 2H). In contrast, -ENaC labeling in kidneys of control animals showed predominantly cytoplasmic labeling (Fig. 2, A, C, E, G).
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-ENaC and -ENaC in kidneys from HgCl2 nephropathic rats. There was a marked increase in apical -ENaC immunolabeling in DCT2 (not shown), CNT (Fig. 3, A and B), and collecting duct (not shown). Immunoperoxidase labeling of -ENaC also showed markedly increased apical targeting in DCT2 (not shown), CNT (Fig. 3, C and D), CCD (Fig. 3, E and F), and OMCD (Fig. 3, G and H) in kidneys from HgCl2 nephropathic rats compared with control rats.
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HSD2 in HgCl2- and PAN-Induced Nephropathy
The protein abundance of 11HSD2 was decreased in the cortex/OSOM (52 ± 3 vs. 100 ± 6%, P < 0.05, Fig. 4, A and B) and ISOM (76 ± 3 vs. 100 ± 3%, P < 0.05) in kidneys from rats with HgCl2-induced nephrotic syndrome. Immunoperoxidase microscopy revealed that 11HSD2 is expressed in DCT2 cells, as well as in the CNT and in principal cells of the collecting duct. The results demonstrated that in HgCl2-induced nephrotic syndrome rats, the immunolabeling of 11HSD2 was markedly decreased in CNT (Fig. 4, C and D) and CCD (Fig. 4, E and F).
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HSD2 was decreased in the cortex/OSOM (51 ± 2 vs. 100 ± 5%, P < 0.05, Fig. 5, A and B) and ISOM (74 ± 8 vs. 100 ± 8%, P < 0.05) in kidneys from rats with PAN nephrotic syndrome. Immunoperoxidase microscopy revealed that in PAN rats, the immunolabeling of 11HSD2 was markedly decreased in CNT (Fig. 5, C and D) and CCD (Fig. 5, E and F).
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We performed additional immunoblot analyses to examine whether there are changes in the protein abundance of other major sodium transporters in the kidneys from HgCl2-induced nephrotic syndrome compared with control rats (Fig. 6) to investigate whether dysregulation of other sodium transporters are also involved in the sodium retention and decreased urinary sodium excretion.
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1-isoform of Na-K-ATPase abundance in the cortex/OSOM and ISOM, whereas the expression remained unchanged in the inner medulla (Table 3). Compared with these controls, immunolabeling for the Na-K-ATPase in the proximal tubule and thick ascending limb was significantly reduced in HgCl2-treated rats (Fig. 7, A and B). In contrast, immunolabeling of Na-K-ATPase in the cortical and medullary collecting duct had similar labeling intensity in kidneys from HgCl2 nephropathic and control rats (Fig. 7).
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Semiquantitative immunoblotting revealed a marked decrease in AQP2 abundance in the inner medulla and ISOM, whereas in the cortex/OSOM a smaller decrease was seen (Fig. 9). Moreover, semiquantitative immunoblotting with antibodies that selectively recognize phosphorylated AQP2 (p-AQP2), which is phosphorylated in the PKA phosphorylation consensus site (Ser256) (10), demonstrated that the abundance of p-AQP2 was also decreased in the inner medulla, ISOM, and cortex/OSOM. Linear regression analysis revealed that there were significant correlations between AQP2 and p-AQP2 expression levels in cortex/OSOM (r = 0.78, P < 0.01), ISOM (r = 0.91, P < 0.01), and inner medulla (r = 0.88, P < 0.01) in control and HgCl2 nephrotic syndrome rats.
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In addition to the changes in protein abundance of AQP2, potential changes in trafficking of AQP2 and p-AQP2 were also examined by immunoperoxidase microscopy. There was a prominent difference in the subcellular localization of AQP2 in kidneys from HgCl2-induced nephrotic syndrome and control rats in the inner medullary collecting duct (Fig. 10, A and B). In HgCl2-induced nephropathy, AQP2 labeling was mainly associated with the apical plasma membrane domains with only marginal labeling of cytoplasmic domains (Fig. 10B). In contrast, AQP2 labeling in kidneys of control rats showed labeling throughout the cytoplasm and less prominent apical plasma membrane labeling (Fig. 10A). Immunohistochemistry also revealed similar changes in the subcellular distribution of p-AQP2 in kidneys from HgCl2-induced nephropathy rats. There was a marked increase in apical p-AQP2 immunolabeling from HgCl2-induced nephropathy rats (Fig. 10D) compared with control rats (Fig. 10C).
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Figure 11 shows the localization of AQP2 in the CNT, CCD, and OMCD. In CNT, AQP2 was localized in both apical and basolateral plasma membrane domains as well as intracellular vesicles in kidneys from controls (Fig. 11A) and HgCl2 nephrotic syndrome rats (Fig. 11B). In CCD principal cells, immunolabeling of AQP2 is mainly associated with apical plasma membrane domains and very little labeling was observed in the basal part of the cell and the basolateral plasma membrane in controls (Fig. 11C). In contrast, AQP2 labeling was present in both apical and basolateral plasma membrane as well as intracellular vesicles in the principal cells of CCD in HgCl2 nephrotic syndrome rats (Fig. 11D). The immunolabeling of AQP2 in the inner stripe of the OMCD was associated with the apical plasma membrane and intracellular vesicles, with less labeling in the basolateral plasma membranes in the controls (Fig. 11E). In HgCl2-induced nephropathy, AQP2 labeling was mainly associated with the apical plasma membrane domains, with only marginal labeling of cytoplasmic domains (Fig. 11F).
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DISCUSSION |
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HSD2. Increased Apical Targeting of ENaC Subunits in HgCl2-Induced Nephrotic Syndrome
The most important finding is the striking increase in apical targeting of ENaC subunits observed as increased immunolabeling in apical plasma membrane domains in the sodium-retaining nephrotic syndrome rats, suggesting that this contributes significantly to the increased renal tubular sodium reabsorption. This was seen in the DCT, CNT, CCD, and medullary collecting duct. These observations, therefore, strongly support the view that the renal sodium retention associated with HgCl2-induced nephrotic syndrome is caused by increased sodium reabsorption in the distal nephron, including the CNT and DCT. These results are consistent with the results of our previous studies of nephrotic syndrome induced by PAN (20). Thus a straightforward interpretation of these observations would be that increased apical targeting of ENaC subunits plays a role in the development of sodium retention in HgCl2 nephrotic syndrome as well, and it could be argued that this mechanism in fact could be a general characteristic of nephrotic syndrome, including both minimal-change disease and immune complex-mediated glomerulonephritis.
Increased Abundance of -ENaC in HgCl2-Induced Nephrotic Syndrome
In the present study, the protein abundance of -ENaC was increased whereas expression of -ENaC and -ENaC was decreased in the cortex/OSOM. The changes in expression in the cortex/OSOM are consistent with the previously described pattern seen in response to aldosterone infusion (22), and because plasma aldosterone levels were significantly increased in HgCl2-induced nephrotic syndrome, the observed changes in the subcellular distribution and protein expression of ENaC subunits would be consistent with this view. Curiously, we find that the regulation of ENaC subunit abundance is different in the outer medulla relative to the cortex. The protein expression of all ENaC subunits was increased in ISOM from HgCl2-induced nephrotic rats. This observation is similar to the changes in ENaC protein expression in response to chronic vasopressin infusion rather than aldosterone (12). Plasma vasopressin levels are increased in PAN nephrotic syndrome in rats (2, 31) and are presumably increased in HgCl2-induced nephrotic syndrome as well. The underlying mechanism for the segmental dysregulation of ENaC subunits remains obscure. However, it can be speculated that differences in sensitivity may be caused by differences in receptor expression patterns of aldosterone (7, 32), vasopressin (28) and 11HSD2 (7, 32). The localization of aldosterone receptors indicates that high-affinity aldosterone receptors are most densely located in the distal tubule and CCD (15), and immunolabeling studies for 11HSD2 demonstrated that the expression is most prominent in the CNT and CCD, with decreased labeling in medullary collecting duct segment (7, 32). Thus segmental differences in receptor expression could explain increased sensitivity to changes in circulating aldosterone levels in the cortex relative to the medulla, which could be more influenced by plasma vasopressin. We cannot rule out that the increased trafficking may be in part independent of aldosterone activation by the classic mineralocorticoid receptor. Nielsen et al. (25) recently demonstrated that the increased targeting of ENaC but not expression of ENaC in response to sodium restriction was insensitive to cotreatment with the mineralocorticoid receptor blocker spironolactone. The nature of such a receptor and the potential role for increased ENaC trafficking in nephrotic syndrome remains to be established.
Decreased Protein Expression of 11HSD2 in HgCl2-Induced Nephrotic Syndrome
Apical targeting of ENaC subunits and protein abundance of -ENaC can be regulated by aldosterone (21, 22). Indeed, plasma aldosterone levels were significantly increased in HgCl2 nephropathy, and thus it can be speculated that aldosterone stimulates sodium reabsorption in HgCl2 nephropathy. However, substantial evidence argues against a major role for aldosterone in nephrotic syndrome: 1) ascites and edema occur in rats with nephrosis, attributable to rabbit anti-rat kidney serum injection, without elevated aldosterone levels (19); 2) inhibition of angiotensin-converting enzyme by captopril failed to induce natriuresis in rats with PAN nephrosis despite decreased aldosterone levels (29); and 3) sodium retention was observed only in the affected proteinuria kidney in unilateral rat models of PAN nephrosis (18). These observations suggest that aldosterone may not alone be involved in sodium retention and presumably in ENaC regulation in nephrotic syndrome.
It has been suggested that the activity of 11HSD2 can regulate sodium reabsorption in aldosterone-responsive renal tubules by glucocorticoid-induced activation of MR (17, 34). We also demonstrate that there is downregulation of 11HSD2 protein expression in the kidney in HgCl2 nephrotic syndrome as well as in PAN nephrosis. This finding is in line with recent studies in nephrotic patients (38) and in experimental nephrotic syndrome induced by PAN or adriamycin (37). The investigators observed a rise in the plasma corticosterone/11-dehydrocorticosterone ratio as well as in the urinary (tetrahydrocorticosterone+5-tetrahydrocorticosterone)/11-dehydro-tetrahydrocorticosterone ratio in nephrotic syndrome (37) and interpreted this change as a result of decreased activity of 11HSD2. Here, we propose that this may occur via downregulated protein expression of 11HSD2 in nephrotic syndrome. These findings suggest that downregulation of 11HSD2 provides access of glucocorticoids to the MR, resulting in increased activity of MR and collecting duct sodium retention in rats with nephrotic syndrome.
The underlying mechanisms for the regulation of 11HSD2 are still unclear. Reduced activity of 11HSD2 in vivo may possibly be caused by the presence of inhibitors, such as in bile acids in liver cirrhosis (30) or glycyrrhetinic acid-like factor in hypertension (23). The decreased activity of 11HSD2 has also been demonstrated in response to potassium and acid loading (27, 36), and in the present study the nephrotic syndrome rats revealed high plasma potassium levels. Thus it is possible that hyperkalemia and/or potentially associated acidosis may explain the decreased activity of 11HSD2. Further studies are needed to address which mechanisms are controlling the downregulation of 11HSD2 in nephrotic syndrome.
Decreased Abundances of NHE3, NKCC2, NCC, and Na-K-ATPase in HgCl2-Induced Nephrotic Syndrome
Immunoperoxidase microscopy for the Na-K-ATPase in the kidney revealed that there was a dramatic decrease in labeling in the proximal tubule and thick ascending limb, whereas labeling in the collecting duct appeared to be conserved in HgCl2 nephrotic syndrome. The decreased abundance of Na-K-ATPase demonstrated by immunoblotting, therefore, appears to be mainly attributed to the decreased expression of the proximal tubule and the thick ascending limb. In contrast to the reduced expression of multiple channels and transporters in the proximal tubule and thick ascending limb, we here demonstrate an increased apical targeting of ENaC subunits and confirm the absence of reduced expression of Na-K-ATPase in the collecting duct. Thus this is likely to cause, at least in part, the sodium retention associated with HgCl2-induced nephrotic syndrome.
An overall decrease in sodium excretion and a concomitant expansion of extracellular fluid volume are seen in this experimental model despite a decrease in expression of apical and basolateral sodium transporters in nephron segments at a site proximal to the connecting tubule (i.e., NHE3, NKCC2, Na-K-ATPase, and NCC), consistent with the previous findings seen in PAN (20)- and adriamycin-induced nephrotic syndrome (16). These findings suggest that downregulation of sodium transporters in the proximal nephron is a feature of nephrotic syndrome per se rather than being an idiosyncrasy of any particular model, and this may play a compensatory role against the sodium retention observed in HgCl2-induced nephrotic syndrome.
Enhanced Apical Plasma Membrane Targeting of AQP2 and p-AQP2 in the Medullary Collecting Duct Despite Decreased Abundance
The decreased protein abundance of AQP2 in HgCl2 nephropathy is consistent with the results of previous studies in adriamycin (16)- and PAN-induced nephrosis (2). The finding that the collecting duct water transport pathways are downregulated is consistent with the generally held view that the maintenance of water retention is not dependent on increased water reabsorption in the collecting duct but rather depends on increased NaCl absorption along the nephron. We speculate that the downregulation of AQP2 expression could represent a physiologically appropriate response to extracellular volume expansion, perhaps similar to the suppression of AQP2 that accounts for the vasopressin escape phenomenon (13).
Shuttling of AQP2 water channels into and out of the plasma membrane via exocytosis and endocytosis is the major means by which water transport in the collecting duct is regulated by vasopressin on a short-term basis (26). There was a decrease in the intensity of AQP2 and p-AQP2 labeling in the medullary collecting ducts from HgCl2 nephrotic rats compared with controls. However, the residual AQP2 and p-AQP2 can be seen to be located chiefly in the apical region of the principal cells, suggesting that trafficking mechanisms that deliver AQP2 to the plasma membrane are increased, not impaired. Therefore, we speculate that the short-term and long-term regulation of AQP2 occurs, in part, by independent mechanisms. The increased apical targeting of AQP2 may help to maintain the urinary concentration despite the decreased AQP2 abundance in nephrotic syndrome. It is noteworthy that there is a significant correlation between AQP2 and p-AQP2 expression levels, and p-AQP2 subcellular distribution tracks total AQP2 fairly well in both control and HgCl2 nephrotic syndrome rats. Thus the effect of HgCl2 nephropathy may be due to changes in the expression of AQP2 rather than effects from PKA phosphorylation-dependent regulation.
Our results demonstrated that there is an axial heterogeneity in the changes in AQP2 subcellular localization in HgCl2 nephropathy compared with controls. Little AQP2 labeling of the basolateral plasma membrane was observed in CCD principal cells in controls. In this segment, a major increase in AQP2 expression in the basolateral plasma membrane was observed in HgCl2 nephropathy, whereas in the CNT AQP2 subcellular localization was not affected. These changes in segmental and subcellular localization of AQP2 fit the previously described pattern seen in response to vasopressin infusion (9). From these observations, we hypothesize that vasopressin levels are high in this model, as in other models of nephrotic syndrome (2, 31) and that the observed downregulation of abundance of AQP2 without impairment of trafficking could be due to a vasopressin escape phenomenon (13). The underlying signaling mechanisms and its physiological roles remain uncertain, but the axial heterogeneity strongly suggests that cell-specific mechanisms are involved.
Summary
The results demonstrate an increased apical targeting of ENaC subunits combined with diminished abundance of 11HSD2 in the DCT2, connecting tubule, and collecting duct. This is likely to play a role in sodium retention associated with HgCl2-induced nephrotic syndrome in rats. Although it cannot be ruled out that the changes in 11HSD2 are secondary to hyperkalemia and/or potentially associated acidosis, the results raise the possibility that downregulation of 11HSD2 may, in part, play a role in increased activity of MR and collecting duct sodium retention in rats with nephrotic syndrome. The decreased abundance of sodium transporters expressed in the nephron at a site proximal to the CNT (i.e., NHE3, NKCC2, and NCC) may play a compensatory role in promoting sodium excretion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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  E. T. B. Olesen, S. de Seigneux, G. Wang, S. C. Lutken, J. Frokiaer, T.-H. Kwon, and S. Nielsen Rapid and segmental specific dysregulation of AQP2, S256-pAQP2 and renal sodium transporters in rats with LPS-induced endotoxaemia Nephrol. Dial. Transplant., August 1, 2009; 24(8): 2338 - 2349. [Abstract] [Full Text] [PDF]
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 C. Kastner, M. Pohl, M. Sendeski, G. Stange, C. A. Wagner, B. Jensen, A. Patzak, S. Bachmann, and F. Theilig Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis Am J Physiol Renal Physiol, April 1, 2009; 296(4): F902 - F911. [Abstract] [Full Text] [PDF]
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 V. Bhalla and K. R. Hallows Mechanisms of ENaC Regulation and Clinical Implications J. Am. Soc. Nephrol., October 1, 2008; 19(10): 1845 - 1854. [Abstract] [Full Text] [PDF]
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R. P. Hughey and T. R. Kleyman Functional cross talk between ENaC and pendrin Am J Physiol Renal Physiol, November 1, 2007; 293(5): F1439 - F1440. [Full Text] [PDF]
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S. de Seigneux, J. Nielsen, E. T. B. Olesen, H. Dimke, T.-H. Kwon, J. Frokiaer, and S. Nielsen Long-term aldosterone treatment induces decreased apical but increased basolateral expression of AQP2 in CCD of rat kidney Am J Physiol Renal Physiol, July 1, 2007; 293(1): F87 - F99. [Abstract] [Full Text] [PDF]
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S. de Seigneux, S. W. Kim, S. C. Hemmingsen, J. Frokiaer, and S. Nielsen Increased expression but not targeting of ENaC in adrenalectomized rats with PAN-induced nephrotic syndrome Am J Physiol Renal Physiol, July 1, 2006; 291(1): F208 - F217. [Abstract] [Full Text] [PDF]
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