Collectrin Is Involved in the Development of Salt-Sensitive Hypertension by Facilitating the Membrane Trafficking of Apical Membrane Proteins via Interaction With Soluble N-Ethylmaleiamide-Sensitive Factor Attachment Protein Receptor Complex
Background— Collectrin, a homologue of angiotensin converting enzyme 2, is expressed in pancreatic β cells and renal proximal tubular and collecting duct cells under the control of hepatocyte nuclear factors-1α and -1β. Because collectrin interacts with the soluble N-ethylmaleiamide-sensitive factor attachment protein receptor (SNARE) complexes, we investigated whether collectrin is involved in sodium handling in hypertension by vesicle trafficking of apical membrane proteins.
Methods and Results— Collectrin physically interacts with the SNARE complex: snapin, synaptosomal-associated protein 23 kDa, syntaxin-4, and vesicle-associated membrane protein-2 in mIMCD-3 cells. siRNA knockdown of collectrin resulted in a reduction in membrane-associated aquaporin-2, α-epithelial Na+ channel, and H+-ATPase. Collectrin and SNARE proteins were abundantly expressed in collecting ducts of Wistar-Kyoto rats. Wistar-Kyoto rats and spontaneously hypertensive rats 7 weeks of age were subjected to normal-salt (1% NaCl) and high-salt (8% NaCl) chow for 10 weeks. High-salt chow prominently elevated blood pressure, oral intake, and urinary excretion of NaCl and water in both groups. Although urinary excretion of aldosterone was significantly suppressed in both groups, collectrin expression was upregulated and associated with the maintenance of aquaporin-2, α-epithelial Na+ channel, and H+-ATPase in membrane fractions. Collectrin promoter activities and mRNA and protein expressions were upregulated and ubiquitinated collectrin was reduced by high NaCl (175 to 225 mmol/L) and not altered by 1 μmol/L aldosterone in mIMCD-3 cells.
Conclusions— Upregulation of collectrin by high NaCl independent of aldosterone functionally links to the trafficking of apical membrane proteins via the SNARE complex, and collectrin may be responsible for the sodium retention in salt-sensitive hypertension.
Received April 19, 2008; accepted September 17, 2008.
Collectrin, a homologue of angiotensin-converting enzyme 2 (ACE2), is a type I transmembrane protein; we originally reported its localization to the cytoplasm and apical membrane of collecting duct cells.1 The collectrin gene is located in the immediate proximity of the ace2 locus, and ACE2 may be a chimeric protein emerging from the duplication of 2 genes, having homology with ACE at the catalytic domain and homology with collectrin in the membrane proximal domain. Unlike ACE and ACE2, collectrin lacks an N-terminal active dipeptidyl carboxypeptidase catalytic domain; thus, its biology has not been well established.
Clinical Perspective p 2155
Collectrin has been reported to be under the control of transcriptional regulation by hepatocyte nuclear factor (HNF)-1α, which also is expressed in pancreatic β cells2,3 and renal proximal tubules.4,5 Collectrin localizes at the vesicle membrane (VM) and plasma membrane (PM) of pancreatic β cells and at the luminal side of brush border membranes of renal proximal tubular cells. We also described that collectrin is a target of HNF-1β and expressed in apical PMs and primary cilium of renal collecting duct cells.6 Targeted disruption of HNF-1α resulted in diabetes and a renal phenotype with Fanconi syndrome characterized by glucosuria, phosphaturia, calciuria, and aminoaciduria.7 The renal-specific inactivation of HNF-1β causes polycystic kidney disease, and renal cyst formation is accompanied by a drastic defect in the transcriptional activation of HNF-1β-targeted genes such as collectrin and several polycystic kidney disease-related genes, including Umod, Pkhd1, Pkd2, and Tg737/Polaris. Furthermore, the mutations in the HNF-1β gene are seen in the autosomal-dominant disorder MODY5 (maturity-onset diabetes mellitus of the young, type 5). MODY5 patients manifest with type 2 diabetes and develop congenital kidney abnormalities, including simple cysts, polycystic kidneys, cystic dysplasia, and glomerulocystic kidney disease. We reported that collectrin is a primary cilia-associated membrane protein that is involved in ciliogenesis and renal cyst formation in in vitro culture experiments. Collectrin also is localized in the vesicles near the peribasal body region at the base of primary cilia and binds to γ-actin-myosin II-A, the soluble N-ethylmaleiamide-sensitive factor attachment protein receptor (SNARE), and polycystin-2-polaris complexes; all of these are involved in intracellular and ciliary movement of vesicles and membrane proteins.6
Recently, 2 independent studies reported that the targeted disruption of collectrin in mice resulted in severe and general defects in renal amino acid uptake.4,5 The deficiency of collectrin was associated with a reduction in multiple amino acid transporters on luminal membranes. Collectrin knockout mice are lacking the phenotype of diabetes, hypertension, and renal cystic formation; however, we can speculate that collectrin plays a role in the pathophysiology of pancreatic β cells and collecting duct cells because other genes such as ACE2 may compensate for the action of collectrin in the gene disruption studies. We reported that collectrin binds to the SNARE complex by interacting with snapin, a synaptosomal-associated protein of 25 kDa (SNAP-25)-binding protein, and it facilitates the SNARE complex formation.2,6 In pancreatic β cells, collectrin facilitates the insulin exocytosis by regulating the SNARE complex formation.2 The interaction between collectrin and the SNARE complex may explain the reduction in multiple amino acid transporters on brush borders of renal proximal tubules.
In line with the evidence, we hypothesized that collectrin plays a role in vesicle trafficking of various apical membrane proteins in a polarized manner by interacting with SNARE complexes and that collectrin is critically involved in sodium and water handling in hypertension. Thus, we investigated the role of collectrin in salt-sensitive hypertension in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs) treated with a high-sodium-chloride diet.8 In various salt-sensitive hypertensive rats, it has been reported that high-salt-induced upregulated expression of ACE2 was blunted9 and that the whole picture of renin-angiotensin-aldosterone system (RAAS) regulation in the kidney in hypertension is rather complex. In the present report, we attempted to characterize the function of collectrin besides what is known about RAAS in the regulation of blood pressure homeostasis.
For details, see the online-only Data Supplement.
Polymerase Chain Reaction of SNARE-Associated Proteins in mIMCD-3 Cells
For details, see the online Data Supplement.
Preparation of Stable mIMCD-3 Cell Lines Overexpressing Collectrin and siRNA Experiments
Stable mIMCD-3 cell lines overexpressing collectrin (collectrin stable cell lines) were prepared as described.6 siRNA cocktail (siTrio) targeting mouse collectrin (NM_020626) and siTrio-negative control cocktail were purchased from Dharmacon (Lafayette, Colo), and transfections were carried out with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif). Stable cell lines and siRNA-treated cells were used for immunoprecipitation and immunofluorescence studies as described previously.6
Seven-week-old male SHRs (n=28) and WKY rats (n=28) were used (Japan SLC, Hamamatsu, Japan). Systolic blood pressure was measured by the indirect tail-cuff technique. At 9 weeks of age, SHRs and WKY rats were randomized to 2 groups and were fed MF chow containing 1% or 8% NaCl (Oriental Yeast, Tokyo, Japan), respectively. The 1% NaCl chow was chosen as normal-salt chow because it is similar to standard rat chow. Although 4% NaCl is sufficiently high to elevate blood pressure in SHRs,10 8% NaCl chow induced marked interstitial fibrosis in heart and kidney; thus, 8% NaCl chow was used as high-salt chow.8 The 1% NaCl chow corresponds to the recommended daily average salt intake in the United States,11 and 8% NaCl chow corresponds to a Japanese-style diet with elevated daily salt intake.12 At 17 weeks of age, the rats were placed in metabolic cages to obtain 24-hour urine collections, and their daily albumin and sodium excretion levels were measured. After kidney tissues were collected, they were subjected to the following experiments. Wild and collectrin-deficient male mice were treated with salt-deficient, normal-salt, and high-salt chow, and the blood pressure levels were measured. For details, see the Data Supplement.
Northern and Western Blotting
For the expression studies of collectrin mRNA in rats, total RNAs were isolated from kidney cortex and medulla of SHRs and WKY rats fed MF chow containing 1% and 8% NaCl. Total RNAs (40 μg) were subjected to Northern blot analyses. Human G3PDH cDNA probe (BD Bioscience, Palo Alto, Calif) was used as an internal control. For Western blot analyses, subcellular fractions of WKY rats and SHR kidneys were prepared by differential centrifugation as described.13
mIMCD3 cells were seeded onto 24-well plates and allowed to grow to 80% confluence. mIMCD3 cells were transiently transfected with 1.0 μg pGL4 carrying a 5′-flanking collectrin promoter lesion expanding from −2323 to −1 bp (pGL4–2K) using Transfast Transfection Reagent (Promega Corp, Madison, Wis) per the manufacturer’s instructions. To normalize transfection efficiency, 40 ng pGL4.73[hRluc/SV40], which expresses Renilla luciferase (Promega), was cotransfected. Luciferase activities were measured with a dual-luciferase assay system (Promega). The firefly luciferase activity of the collectrin promoter plasmid (pGL4–2K) was normalized with Renilla luciferase activity.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was purified from mIMCD-3 cells with QIAzol Reagent (Qiagen, Valencia, Calif). mIMCD3 cell lines were cultured by following the manufacturer’s instructions (ATCC, Manassas, Va) and treated with aldosterone (1 μmol/L), fluid flow on an orbital shaker at ≈1 Hz, and NaCl (175, 200, 225, and 275 mmol/L) for 8 hours, respectively. cDNAs were synthesized from 2.5 μg total RNA and analyzed with the LightCycler-FastStart DNA master SYBR Green I system (Roche Diagnostics, Basel, Switzerland) and specific primers. The relative abundance of mRNAs was standardized with β-actin mRNA as the invariant control. The primer sets for mouse collectrin, α-epithelial Na+ channel (α-ENaC), Sgk1, and β-actin were purchased from Nihon Gene Research Laboratory (Sendai, Japan).
Enrichment of Ubiquitinated Protein and Western Blot
mIMCD3 cell and kidney tissues were lysed in radioimmunoprecipitation assay buffer, and the homogenates (100 μg/30 μL) were mixed with 270 μL TBS buffer and incubated with 20 μL polyubiquitin-affinity resin using the Ubiquitin Enrichment Kit (Pierce, Rockford, Ill). Polyubiquitin-affinity resin was loaded to the spin column and washed, and then the polyubiquitinated proteins were eluted with 75 μL SDS-PAGE sample loading buffer. The final products (15 μL) were analyzed with Western blot analysis with rabbit anti-collectrin and mouse anti-monoubiquitinated and anti-polyubiquitinated conjugate antibodies (FK2) (BIOMOL, Plymouth Meeting, Pa).
Data are expressed as mean±SEM. Differences between multiple groups were compared by ANOVA. Two-group analysis was performed with a Student t test.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Collectrin Interacts With SNARE-Associated Proteins in mIMCD-3 Cells
In a previous study, snapin and SNAP-25 binding protein were isolated by the yeast 2-hybrid system with full-length collectrin used as bait.2 Snapin is a component of the SNARE complex, and we further investigated the expression of SNARE-associated proteins in mouse collecting duct cell line (mIMCD-3 cells). mRNA expression of collectrin and SNARE-associated proteins such as snapin, SNAP-23, syntaxin-4, and vesicle-associated membrane protein-2 (VAMP-2) was confirmed by reverse-transcription polymerase chain reaction in mIMCD-3 cells (Figure 1A). In mIMCD-3 cells stably overexpressing collectrin, coimmunoprecipitation studies revealed that collectrin, snapin, SNAP-23, syntaxin-4, and VAMP-2 form the SNARE complex (Figure 1B).
Collectrin Colocalizes With SNAP-23 and Apical Membrane Proteins
Because collectrin interacts with SNARE-associated proteins, it may be involved in trafficking of membrane proteins from VM to PM in renal collecting duct cells. Collectrin showed its immunoreactivities in cytoplasm, and it apparently was also expressed on apical and basolateral membranes in mIMCD-3 and stable cells expressing collectrin (Figure 2A and 2B, red). Collectrin was partially colocalized with SNAP-23, one of the components of SNARE-associated proteins (Figure 2A and 2B). siRNA treatment was associated with diminished expression of both collectrin and SNAP-23 (Figure 2C). Apical and basolateral membranes visualized with the z-axis view constructed with confocal microscopy revealed the immunoreactivities of collectrin and membrane proteins such as aquaporin-2 (Figure 2D and 2E), α-ENaC (Figure 2G and 2H), H+-ATPase (Figure 2J and 2K), and Na+/K+ATPase-α1 (Figure 2M and 2N), although collectrin and membrane proteins did not show completely matched localization. siRNA treatment resulted in diminished expression of collectrin and PM proteins, including aquaporin-2 (Figure 2F), α-ENaC (Figure 2I), H+-ATPase (Figure 2L), and Na+/K+ATPase-α1 (Figure 2O), suggesting that collectrin may be involved in the trafficking of these proteins to PMs.
Collectrin Expression in WKY Rat Kidney Tissues
To investigate the expression and localization of collectrin in vivo, we performed immunochemistry in WKY rats (Figure 3A through 3F) and Western blotting of VM and PM fractions isolated from whole kidney lysates (Figure 3G). Collectrin was expressed in cytoplasm and apical membrane throughout collecting duct cells in the control rat kidney (Figure 3A). Furthermore, the collecting duct cells revealed prominent immunoreactivities for snapin (Figure 3B), SNAP-23 (Figure 3C), syntaxin-4 (Figure 3D), and VAMP-2 (Figure 3E). WKY rat kidneys were minced into cortex and outer and inner medulla, and then PMs and intracellular VMs were prepared by the differential centrifugation method. Collectrin was expressed in both PM and VM fractions throughout the renal tissues. Similarly, SNARE-associated proteins, including snapin, SNAP-23, syntaxin-4, and VAMP-2, were detected in PM and VM fractions in both cortex and medulla (Figure 3G).
Effects of Normal- and High-Salt Chow on SHRs
The effects of normal- (1%) or high- (8%) salt chows on blood pressure and renal physiology were investigated. The systolic and mean blood pressures are higher in SHRs compared with WKY rats receiving normal-salt chow. High-salt chow significantly raised the systolic blood pressure in both WKY rats and SHRs (Figure 4A and 4B). High-salt chow increased daily water intake and urine volume in both SHRs and WKY rats, and a more prominent increase was observed in SHRs compared with WKY rats (Figure 4C and 4D). Similarly, sodium intake and urinary excretion increased with high-salt chow, and a prominent increase in urinary sodium excretion was noted in SHRs (Figure 4E and 4F). Urinary albumin excretion was significantly increased in SHRs treated with high-salt chow, suggesting the presence of glomerular injuries (Figure 4G). Urinary aldosterone excretions were significantly suppressed in both SHRs and WKY rats treated with high-salt chow (Figure 4H). Severe cortical and medullar interstitial fibrosis and glomerular hypertrophy associated with extracellular matrix expansion were observed in SHRs with high-salt chow (Figure 5G and 5H) compared with WKY rats and SHRs with normal-salt chow (Figure 5A, 5B, 5E, and 5F). Mild cortical and medullar interstitial fibrosis and glomerular hypertrophy were observed in WKY rats on high-salt chow (Figure 5C and 5D).
Effects of High-Salt Chow on Collectrin-Deficient Mice
Wild-type male mice did not develop the high-salt (6% NaCl chow) -induced hypertension and were not salt sensitive. Collectrin-deficient males showed blood pressure levels similar to those of wild-type males, and blood pressure was not changed by treatment with high-salt chow. For details, see the Data Supplement.
Expression of Collectrin and Ion/Water Channels in SHRs
Immunofluorescence studies revealed that collectrin expression in renal collecting duct epithelia in the cortex region was upregulated by the treatment of high-salt chow in WKY rats. Similarly, upregulation was observed in SHRs by the treatment of high-salt chow, although the response was rather mild (Figure 6A). Northern blot analyses also supported such observations, and collectrin mRNA isolated from renal cortex and medulla was upregulated in WKY rats and SHRs with high-salt chow (Figure 6B). Western blot analyses using whole lysates from renal cortex revealed that high-salt chow prominently upregulated the protein expression of collectrin, aquaporin-2, α-ENaC, H+-ATPase, and Na+/K+ATPase-α1 in renal cortex, whereas collectrin and aquaporin-2 were upregulated in renal medulla of WKY rats (Figure 6C). In SHRs, treatment with high-salt chow upregulated collectrin expression, whereas expression of other membrane proteins, including aquaporin-2, α-ENaC, H+-ATPase, and Na+/K+ATPase-α1, was not altered in renal cortex and medulla (Figure 6C). In addition, the expression of Sgk1 was enhanced by high-salt chow in the renal cortex of SHRs compared with WKY rats. The kidney whole lysates were further fractionated into VM and PM fractions, and Western blot analyses were performed. In WKY rats and SHRs, collectrin expression was prominent in PM fractions compared with VM fractions under normal- and high-salt chow conditions. Similarly, all apical membrane proteins such as aquaporin-2, α-ENaC, and H+-ATPase were accentuated in PM fractions compared with VM fractions in WKY rats and SHRs in both normal- and high-salt chow conditions (Figure 6D). The expression of basolateral membrane protein, Na+/K+ATPase-α1, was almost similar in the VM and PM fractions in both WKY rats and SHRs (Figure 6D).
Expression Regulation of Collectrin in mIMCD3 Cells
To determine the regulation of collectrin mRNA expression, we performed a collectrin gene promoter assay using mIMCD3 cells treated with 1 μmol/L aldosterone, continuous fluid flow on orbital shaker at 1 Hz, and high-NaCl conditions—175, 200, 225, and 275 mmol/L—for 8 hours. Relative luciferase activities increased by ≈5-fold with the addition of 50 and 75 mmol/L NaCl compared with control media containing 125 mmol/L NaCl. Luciferase activities were progressively reduced in mIMCD3 cells treated with higher concentrations of NaCl, 225 and 275 mmol/L (Figure 7A). The luciferase activities were not altered by the treatment of 1 μmol/L aldosterone or continuous fluid flow. Unexpectedly, collectrin mRNA expression was upregulated only 2-fold in the media containing 175 mmol/L NaCl, and upregulation of collectrin mRNA was not obvious under higher NaCl concentrations (Figure 7B). mRNA expression of α-ENaC was upregulated by treatment with 1 μmol/L aldosterone but was not altered by treatment with higher NaCl concentrations. mRNA expression of Sgk1 was upregulated by the addition of 1 μmol/L aldosterone and sodium chloride in a dose-dependent manner.
Collectrin Protein Expression Is Regulated by Ubiquitination
Because the elevation of collectrin mRNA expressions was rather mild in mIMCD3 cells treated with high-salt conditions, we further investigated collectrin protein expression and its ubiquitination. Western blot analyses of collectrin revealed a prominent increase in mIMCD3 cells grown in the media containing 175, 200, and 225 mmol/L NaCl, whereas collectrin protein expression was not altered in mIMCD3 cells treated with 1 μmol/L aldosterone, continuous fluid flow, and 275 mmol/L NaCl. The protein levels of α-ENaC and Sgk1 were upregulated by the addition of sodium chloride and 1 μmol/L aldosterone; the protein expression remained at control levels in mIMCD3 cells treated with continuous flow (Figure 8A). It has been reported that the increase in α-ENaC protein in renal tubular cells is regulated by decreased ubiquitination rather than transcriptional upregulation of α-ENaC mRNA itself. Furthermore, aldosterone-induced Sgk1 expression is accompanied by decreasing ubiquitin ligase Nedd4-2 phosphorylation and Nedd4-2-dependent ENaC retrieval from the PM. Thus, we further examined ubiquitination of collectrin protein in mIMCD3 cells and kidney tissues. mIMCD3 cell and kidney tissues were lysed in radioimmunoprecipitation assay buffer; ubiquitinated proteins were isolated by polyubiquitin-affinity resin. Western blot analysis with mouse anti-monoubiquitinated and anti-polyubiquitinated conjugate antibodies revealed that almost equal amounts of ubiquitinated proteins were purified in mIMCD3 cells and kidney tissues (Figure 8B). The ubiquitination of collectrin was reduced in mIMCD3 cells grown in the media containing 175, 200, and 225 mmol/L NaCl. In renal tissues in WKY rats, the ubiquitination of collectrin protein was reduced by treatment with high-salt chow in both the cortex and medulla, whereas such a downregulation was not observed in SHRs (Figure 8C). Taken together, these results show that upregulation of collectrin protein in both mIMCD3 cells and renal tissues by treatment of high-sodium-chloride conditions was mediated by reduced ubiquitation of the collectrin in both WKY rats and SHR.14
ACE is a key enzyme in the RAAS and converts angiotensin I to the vasoconstrictor angiotensin II, which is thought to be responsible for most of the physiological and pathophysiological effects of the RAAS. This classic view of the RAAS was challenged with the discovery of the enzyme ACE2, which degrades angiotensin II and leads to the formation of the vasodilatory and antiproliferative peptide angiotensin(1-7).14 The identification of ACE2 in the heart and kidney adds further complexity to the RAAS. It is believed that ACE2 acts in a counterregulatory manner to ACE to modulate the balance between vasoconstrictors and vasodilators within the heart and kidney and thus may play a significant role in the pathophysiology of cardiac and renal diseases. Collectrin is a member of the ACE family; however, it lacks N-terminal active dipeptidyl carboxypeptidase catalytic domain, unlike ACE and ACE2, and shares collectrin domain with ACE2. Thus, it has not been well established how and where collectrin is functionally integrated into RAAS, especially in the context of blood pressure regulation.
We reported that collectrin physically interacts with snapin and further demonstrated that collectrin forms the SNARE complex with snapin, SNAP-23, syntaxin-4, and VAMP-2 in collecting duct cells. The specific localization of apical membrane proteins such as aquaporin-2, H+-ATPase, and α-ENaC in a polarized manner was reported to be mediated by SNAP-23,15 syntaxin-4,13 and other SNARE complex proteins. In the present study, overexpression of collectrin and siRNA knockdown experiments in mIMCD3 cells revealed that the sorting and distribution of aquaporin-2, α-ENaC, H+-ATPase, and Na+/K+ATPase-α1 on PM are functionally mediated by collectrin. The specific localization of collectrin in apical membrane of collecting duct cells suggests that collectrin mediates the vesicle trafficking of various apical membrane proteins in a polarized manner by interacting with the SNARE complexes and that collectrin may play a role in sodium and water handling in hypertension.
Aldosterone controls the final sodium reabsorption and potassium secretion in the kidney by regulating the activity of the ENaC in the aldosterone-sensitive distal nephron. α-ENaC is an aldosterone-induced transcript by direct binding of the ligated mineracorticoid receptor (MR) to hormone binding elements in the α-ENaC promoter in collecting duct cells.16 In addition, the aldosterone-induced regulatory protein Sgk117 increases the PM abundance of ENaC by the phosphorylation of Nedd4-2, blockade of its binding to ENaC, and subsequent reduction of ubiquitin ligase activities. Thus, the lack of Sgk1 in mice revealed protective effects on the salt-sensitive hypertension induced by a high-fat/high-salt diet.18 High-salt diet, or sodium loading, induced hypertension and renal tissue injuries in both humans and rodents such as WKY rats and SHRs.8 Although ACE inhibitors, angiotensin-receptor blockers, and selective MR antagonists exert blood pressure-lowering effects and renoprotective properties, circulating and urinary aldosterone levels are prominently suppressed under high-salt diet conditions. It has been reported that salt loading reduced circulating aldosterone but increased nuclear MR and Sgk1 in the kidney of SHR/NDmcr-cp. Such paradoxical activation of MR and Sgk1 may be explained by the oxidative stress induced by a high-salt diet.19 In the present study, we demonstrated that collectrin mRNA and protein expression in renal tissues is increased by treatment with a high-salt diet in both WKY rats and SHRs. In WKY rats, the expression of apical membrane proteins such as collectrin, aquaporin-2, α-ENaC, and H+-ATPase is maintained in the PM fractions, although the animals were treated with a high-salt diet and urinary aldosterone levels were significantly reduced. Furthermore, the salt intake was prominently increased in SHRs, and these apical membrane proteins were still maintained in the PM fractions, although collectrin upregulation was rather mild compared with WKY rats. The expression of ENaC may be partly sustained by the activation of MR and Sgk1; however, the maintenance of ENaC in PM is facilitated by upregulation of collectrin via the protein trafficking with SNARE proteins. Certainly, we should further examine other salt-sensitive rat models or collectrin knockout mice of a salt-sensitive strain. Dahl salt-sensitive rats exhibit high sensitivity to aldosterone, and it may be difficult to dissect out the function of collectrin under a high-salt diet. Thus, WKY rat and SHR models with mild to moderate salt sensitivity were used in the present study. In addition, we demonstrated that the mice on a mixed background of 129×C57BL/6 may not be a suitable model to reveal protection from salt-induced hypertension by deletion of the collectrin gene.
Next, we investigated the mechanism of high-salt-induced upregulation of collectrin. Luciferase reporter gene assay revealed that transcriptional activities were not altered by the stimulation of aldosterone. Because we reported that collectrin is colocalized with primary ciliary membranes of renal tubular cells and that the primary cilia are recognized as a mechanical sensor, we stimulated the mIMCD3 cells by continuous flow of the media. However, luciferase activity is not changed by the continuous-flow stimulation. In contrast to these stimuli, sodium chloride treatment ranging from 175 to 225 mmol/L significantly upregulated luciferase activity; however, the upregulation of collectrin mRNA was not as impressive in mIMCD3 cells. Thus, we next investigated the ubiquitination of collectrin protein. It was reduced in mIMCD3 cells treated with sodium chloride ranging from 175 to 225 mmol/L and in renal cortex and medulla tissues in WKY rats and SHRs treated with high-salt chow.
Taken together, these results show that in salt-sensitive hypertension the retention of sodium and water seems to be partly mediated by the upregulation of collectrin. Transcriptional activities of collectrin were upregulated by high-salt simulation but not altered by aldosterone or mechanical-flow stimulation. In addition to transcriptional regulation, collectrin protein was rather upregulated, with the increasing concentration of sodium chloride associated with reduced ubiquitination of collectrin. With the high-salt diet, aldosterone is prominently suppressed, and the recruitment of ENaC to apical membrane fractions is explained partly by the paradoxical upregulation of Sgk1 and MR. Here, we postulate that upregulation of collectrin by high sodium chloride independent of aldosterone functionally links to the trafficking of apical membrane proteins via the SNARE complex and that collectrin may be responsible for the sodium and water retention in salt-sensitive hypertension. Now, we would like to add collectrin, a new ACE gene member, to the RAAS in the context of ion and water handling in the kidney.
Sources of Funding
This work was supported by Grants-in-Aid for Scientific Research (B), Ministry of Education, Science and Culture, Japan, to Drs Wada (20390257) and Makino (18390249).
Zhang H, Wada J, Hida K, Tsuchiyama Y, Hiragushi K, Shikata K, Wang H, Lin S, Kanwar YS, Makino H. Collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ACE2 and is developmentally regulated in embryonic kidneys. J Biol Chem. 2001; 276: 17132–17139.
Fukui K, Yang Q, Cao Y, Takahashi N, Hatakeyama H, Wang H, Wada J, Zhang Y, Marselli L, Nammo T, Yoneda K, Onishi M, Higashiyama S, Matsuzawa Y, Gonzalez FJ, Weir GC, Kasai H, Shimomura I, Miyagawa J, Wollheim CB, Yamagata K. The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation. Cell Metab. 2005; 2: 373–384.
Malakauskas SM, Quan H, Fields TA, McCall SJ, Yu MJ, Kourany WM, Frey CW, Le TH. Aminoaciduria and altered renal expression of luminal amino acid transporters in mice lacking novel gene collectrin. Am J Physiol Renal Physiol. 2007; 292: F533–F544.
Zhang Y, Wada J, Yasuhara A, Iseda I, Eguchi J, Fukui K, Yang Q, Yamagata K, Hiesberger T, Igarashi P, Zhang H, Wang H, Akagi S, Kanwar YS, Makino H. The role for HNF-1beta-targeted collectrin in maintenance of primary cilia and cell polarity in collecting duct cells. PLoS ONE. 2007; 2: e414.
Yu HC, Burrell LM, Black MJ, Wu LL, Dilley RJ, Cooper ME, Johnston CI. Salt induces myocardial and renal fibrosis in normotensive and hypertensive rats. Circulation. 1998; 98: 2621–2628.
Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga SE, Oliveira-dos-Santos AJ, da Costa J, Zhang L, Pei Y, Scholey J, Ferrario CM, Manoukian AS, Chappell MC, Backx PH, Yagil Y, Penninger JM. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature. 2002; 417: 822–828.
McBride SM, Culver B, Flynn FW. Prenatal and early postnatal dietary sodium restriction sensitizes the adult rat to amphetamines. Am J Physiol Regul Integr Comp Physiol. 2006; 291: R1192–R1199.
Pittman DW, Contreras RJ. Dietary NaCl influences the organization of chorda tympani neurons projecting to the nucleus of the solitary tract in rats. Chem Senses. 2002; 27 (4): 333–341.
Banerjee A, Li G, Alexander EA, Schwartz JH. Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells. Am J Physiol Cell Physiol. 2001; 280: C775–C781.
Huang DY, Boini KM, Osswald H, Friedrich B, Artunc F, Ullrich S, Rajamanickam J, Palmada M, Wulff P, Kuhl D, Vallon V, Lang F. Resistance of mice lacking the serum- and glucocorticoid-inducible kinase SGK1 against salt-sensitive hypertension induced by a high-fat diet. Am J Physiol Renal Physiol. 2006; 291: F1264–F1273.
Nagase M, Matsui H, Shibata S, Gotoda T, Fujita T. Salt-induced nephropathy in obese spontaneously hypertensive rats via paradoxical activation of the mineralocorticoid receptor: role of oxidative stress. Hypertension. 2007; 50: 877–883.
Salt-sensitive hypertension is a common clinical problem, particularly in certain subsets of hypertensive patients, including blacks, the elderly, and patients with chronic kidney disease. Recent long-term follow-up data suggest that even a short period of dietary salt restriction significantly improves cardiovascular risk. Accordingly, identifying mechanisms of salt sensitivity is clinically important. The renin-angiotensin-aldosterone system plays a central role in salt handling in the kidney, and aldosterone upregulates Sgk1 and recruits α-epithelial Na+ channel on the apical membrane, which results in sodium retention. However, aldosterone levels are significantly reduced under a high-salt diet, and the mechanism of salt-sensitive hypertension is not well understood. The present studies link collectrin, a transmembrane protein localized to the apical membrane of collecting duct cells, to sodium retention in rats exposed chronically to high dietary salt. Collectrin binds to the soluble N-ethylmaleiamide-sensitive factor attachment protein receptor (SNARE) complex, and upregulation of collectrin by a high-salt diet independent of aldosterone functionally links to the facilitation of vesicle fusion and trafficking of apical membrane proteins such as α-epithelial Na+ channel. In clinical settings, blockade of the renin-angiotensin-aldosterone system by a renin inhibitor, angiotensin-converting enzyme inhibitor, angiotensin receptor antagonist, and selective aldosterone blockers is a major therapeutic option to prevent cardiovascular events and the progression of chronic kidney disease. Upregulation of collectrin in collecting duct cells by a high-salt diet may blunt the efficacy of renin-angiotensin system inhibitors in salt-sensitive hypertension. Salt restriction is further warranted in the clinic, and collectrin may be the new therapeutic target in hypertension.
Guest Editor for this article was Aruni Bhatnagar, MD.
The online Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.787259/DC1.