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1Division of Endocrinology and Metabolism, Department of Medicine, and 3Center for Sex Differences, Georgetown University, Washington, District of Columbia; and 2Department of Physiology, University of Maryland-Baltimore, Baltimore, Maryland
Submitted 21 March 2005 ; accepted in final form 10 November 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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270 g) underwent one of the following three treatments for 4 wk (n = 6/group): 1) control; 2) insulin-infused plus 20% dextrose in drinking water; or 3) glucose water-drinking (20% dextrose in water). Mean arterial pressures were increased by insulin and glucose (mmHg at 3 wk): 98 ± 1 (control), 107 ± 2 (insulin), and 109 ± 3 (glucose), P < 0.01. Insulin (but not glucose) increased natriuretic response to benzamil (ENaC inhibitor) and hydrochlorothiazide (NCC inhibitor) on average by 125 and 60%, respectively, relative to control rats, suggesting increased activity of these reabsorptive pathways. Neither insulin nor glucose affected the renal protein abundances of NCC or the ENaC subunits (
, 
, and 
) in kidney cortex, outer medulla, or inner medulla in a major way, as determined by immunoblotting. However, insulin and to some extent glucose increased apical localization of these subunits in cortical collecting duct principal cells, as determined by immunoperoxidase labeling. In addition, insulin decreased cortical "with no lysine" kinase (WNK4) abundance (by 16% relative to control), which may have increased NCC activity. Overall, insulin infusion increased blood pressure, and NCC and ENaC activity in rats. Increased apical targeting of ENaC and decreased WNK4 expression may be involved. thiazide; Na-Cl cotransporter; WK4; SGK; telemetry
Hyperinsulinemia has also been linked to hypertension in humans and animals. Semichronic infusion of insulin (anywhere from 5 to 14 days) has been demonstrated to increase blood pressure modestly in male rats (8, 10, 22, 25, 26, 28). Upregulation of the sympathetic nervous system (22), the renin-angiotensin system (8), thromboxane synthesis (28), and endothelin-1 (26) has all been implicated as playing a role. Some of the above factors influence vascular tone, which most likely affects blood pressure. However, in addition, many of these factors may influence sodium reabsorption by the kidney. Furthermore, insulin may directly increase sodium reabsorption via receptor-mediated activation of specific renal transport proteins.
In situ binding studies using 125I-labeled insulin have revealed that insulin receptors are localized along the entire length of the renal tubule, with the density the greatest in the thick ascending limb and distal convoluted tubule (12, 43). In addition, investigators, utilizing renal micropuncture, have reported that insulin is sodium retentive in the thick ascending limb (30) and/or distal tubule (including thick ascending limb through the collecting duct) (14). Insulin, when provided in the perfusion bath for isolated, perfused tubule studies, has been shown to increase sodium reabsorption in the proximal tubule (3, 48), and the thick ascending limb (20, 24, 33, 48). The ability of insulin to enhance collecting duct sodium reabsorption is less clear. However, A6 cells (toad bladder cells), which are a model for distal tubule cells and express the amiloride-sensitive epithelial sodium channel (ENaC), display increased amiloride-sensitive sodium transport in response to insulin (7). No evidence has been reported for an effect of insulin to stimulate sodium reabsorption in the distal convoluted tubule. However, this is a difficult segment to study and ideal cell lines have yet to be established.
Here, we address the question of whether insulin infusion to induce hyperinsulinemia affects protein abundance, cellular distribution, or activity of specific renal sodium transport proteins and channels of the distal convoluted tubule through the collecting duct. We also determine whether chronic infusion of insulin will result in elevated blood pressure in these same animals. We utilize radiotelemetry to continuously monitor blood pressure over a 4-wk period in response to insulin infusion. We use a novel approach to assess specific sodium channel and transporter subunit activities in vivo by measuring the rats' natriuretic responses to selective sodium transporter and channel blockers. Finally, we utilize immunoblotting and immunoperoxidase labeling to examine the renal abundance and cellular distribution of the following proteins: the thiazide-sensitive Na-Cl cotransporter (NCC or TSC) and the -, -, and -subunits of ENaC. We also examine protein abundance of two selected kinases that play important roles in distal tubular sodium reabsorption, i.e., serum- and glucocorticoid-regulated kinase-1 (SGK-1), a regulator of ENaC activity (27), and WNK4 ("with no lysine") kinase, a regulator of NCC activity (53).
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METHODS |
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In a preliminary study, insulin-infused rats and glucose water-drinking rats were found to consume only about half the amount of chow as did the control rats. Therefore, in this study control rats were fed AIN-93G Purified Diet (TD 94045) and insulin-infused and glucose water-drinking rats were fed Purified Diet (2X, TD 04375) ad libitum (Harlan-Teklad, Madison, WI) to better match mineral, fat, protein, and carbohydrate intakes. In this diet, mineral mix, fat, protein, and fiber content were doubled compared with TD 94045. All rats were killed in the fed state. All animals were maintained at all times under conditions and protocols approved by the Georgetown University Animal Care and Use Committee, an American Association for Accreditation of Laboratory Animal Care-approved facility. An additional set of rats was used (control and insulin-treated only, n = 7/group) to confirm natriuretic test results and provide a larger "n" to more comprehensively evaluate immunohistochemistry.
Specific natriuretic tests. After 2 wk, we began a series of tests of natriuresis in response to two select natriuretic agents. The natriuretic response to benzamil (Sigma, St. Louis, MO) was used as an index of relative ENaC activity (17). This test was performed after 7 days on the study. After a 24-h baseline collection of urine in Nalgene metabolic cages (Harvard Apparatus, Holliston, MA), benzamil (0.7 mg/kg body wt) was administered intraperitoneally (ip) in a single injection based on a previously published therapeutic dose (44). Urine was then collected in the following time increments after the injection: 0–3, 3–6, and 6–24 h. Urinary sodium was measured by an ion-selective electrode system (EL-ISE Electrolyte System, Beckman Instruments, Brea, CA). In a second test performed at 14 days, hydrochlorothiazide (HCTZ; 3.75 mg/kg body wt, Sigma) (38) was administered ip and urine was collected as above. The sodium excretory response to HCTZ was used as an index of NCC activity. During the baseline periods, daily food and water intake records were made.
Plasma analyses. Rats were euthanized after 28 days by decapitation, and trunk blood was collected into both heparinized and K+-EDTA-tubes (Vacutainer, Becton-Dickinson, Franklin Lakes, NJ). Heparinized and K+-EDTA blood was centrifuged at 3,000 rpm (Sorvall RT 6000 D, Sorvall, Newtown, CT) at 4°C for 20 min to separate plasma. Plasma aldosterone levels were measured in heparinized plasma using a Coat-A-Count RIA kit (Diagnostic Products, Los Angeles, CA). Insulin was measured in K+-EDTA-containing plasma using an RIA kit (RI-13K, Linco Research, St. Charles, MO). The antibody in this kit cross-reacts with both human and rat insulin. Triglyceride levels were measured in heparinized plasma utilizing an enzymatic colorimetric assay (Sigma).
Preparation of samples for immunoblotting. Both kidneys were rapidly removed. Whole left kidneys and right kidney cortex, inner stripe of outer medulla, and inner medulla were homogenized using a tissue homogenizer (Tissumizer, Tekmar, Cincinnati, OH) in a chilled isolation solution, as previously described (15). Protein concentrations of the homogenates were measured with a Pierce BCA Protein Assay Reagent Kit (Pierce, Rockford, IL). All samples were then diluted with isolation solution to a protein concentration of between 1 and 3 µg/µl and solubilized at 60°C for 15 min in Laemmli sample buffer. Samples were stored at –80°C until ready to be run on gels.
Electrophoresis and blotting of membranes.
Initially, Coomassie blue-stained loading gels were prepared for all sample sets to assess the quality of the protein by the sharpness of the bands and to confirm the equality of loading, as previously described (15). For immunoblotting, 5–30 µg of protein from each sample were loaded into individual lanes of precast minigels of 7, 10, or 12% polyacrylamide (Bio-Rad, Hercules, CA). Our immunoblotting protocol and the production, affinity purification, and characterization of the polyclonal antibodies against NCC, -, -, and -ENaC have been previously described (45, 46). WNK4 was a rabbit anti-human polyclonal antibody obtained from Alpha Diagnostic (San Antonio, TX), and SGK-1 was a sheep polyclonal obtained from Upstate Biotechnology (Lake Placid, NY).
Immunohistochemistry.
Separate animals were set up for immunohistochemical examination of the ENaC subunits and NCC. The left kidney was processed in paraffin, and 5-µm sections were cut. Heat-induced target retrieval was performed using pH 6 citrate buffer (Zymed Laboratories) to unmask antigenic sites. Endogenous peroxidase activity was stopped by incubation with 2% H2O2 for 20 min. Tissues were incubated with the primary antibody (1:1,000), i.e., -, -, or -ENaC or NCC, overnight at 4°C. An Envision+ System (DakoCytomation, Carpinteria, CA) goat anti-rabbit antibody was used to conduct peroxidase labeling. Then, 3,3'-diaminobenzidine tetrachloride dihydrate was applied for 10 min and the tissue was counterstained with Mayer's hematoxylin to allow anatomic definition. A positive reaction was identified as a brown stain in the cytoplasm or a dark brown/black nuclear stain as a result of superimposition of the 3,3'-diaminobenzidine tetrachloride dihydrate reaction and the blue counterstain. Pictures were taken with a Photometrics Cool Snap camera (Scanalytics, Fairfax, VA) mounted to a Nikon Eclipse E600 microscope with a x100 oil-immersion lens for a total magnification of x1,000.
Statistics. Data were evaluated by SigmaStat (Chicago, IL). One-way ANOVA followed by Tukey's multiple comparisons test was used to determine significant differences between means.
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RESULTS |
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50% in insulin-infused and glucose-drinking rats (see METHODS), we observed no differences between treatments for mean arterial blood pressure (MAP). However, in this study MAP was significantly elevated in the both the glucose water-drinking group and the insulin-infused group, relative to control rats, after 16 or 20 days, respectively (Fig. 1). The increase was between 5 and 10 mmHg. There were no differences between the glucose-drinking and the insulin-infused groups, however, at any time point.
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-, -, or -ENaC antibodies. A summary of densitometric statistics are found in Tables 3 (- and -ENaC) and 4 (-ENaC). An equal amount of total protein (20–30 µg) was loaded in each lane of each blot. In the cortex and whole kidney homogenates, there were no significant differences between any of the groups for the densities of any of the ENaC channel subunit bands. However, in the inner medulla, -ENaC was modestly but significantly decreased in the insulin-infused rats relative to the glucose water-drinking rats; and in the outer and inner medulla, the lower (70-kDa band) associated with -ENaC was increased in the glucose water-drinking rats relative to control.
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-ENaC in the cortex (top), outer medulla (middle), and inner medulla (bottom) is shown in Fig. 4. Labeling for -ENaC was fairly intense in the collecting duct principal cells of the cortex and outer medulla in all treatments. However, lesser labeling was observed in the inner medullary collecting ducts. Staining for -ENaC was more apical in the rats treated with insulin, especially in the cortex (top, middle column), whereas the staining appeared more diffuse in the control rats (top, left column).
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-ENaC. Like -ENaC, -ENaC labeling appeared most intense near the apical membrane of the principal cells in the cortex in insulin-infused rats. Lesser labeling was observed in the outer medullary collecting duct for all treatments.
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-ENaC is shown in Fig. 6. -ENaC labeling was found in all regions, and the stain was most intense in the apical membrane of insulin-infused and glucose-drinking rats. Again, the labeling pattern was more diffuse in control rats.
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155 kDa (51). We found a modest but significant decrease in this band in cortex homogenates in the insulin-infused rats relative to controls (P < 0.05). The same trend was seen in whole kidney homogenates; however, the difference was not significant (P = 0.076).
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DISCUSSION |
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Also, in agreement with others (23, 40) we showed that the consumption by rats of a diet high in a simple sugar, i.e., glucose in the drinking water, also raises blood pressure, in our case, to a similar degree as the insulin infusion. This is likely due to the development of insulin resistance in these rats (4, 18, 23). We did not plan to address the degree of insulin resistance in this study. Nevertheless, it is important to consider the possibility that the hypertension caused by the insulin infusion may have also been due to the development of insulin resistance rather than to a direct response to the insulin. Insulin has been demonstrated to increase Ca2+-ATPase activity (54) and endothelial nitric oxide release (47) from endothelial tissue, thus resulting in vasodilation in sensitive tissues, phenomena expected to lower, not raise, blood pressure. In line with this, Hall and associates (11, 21) have consistently demonstrated that dogs, which do not as readily develop insulin resistance when infused with physiological levels of insulin, fail to show an increase in blood pressure, even in the presence of reduced kidney mass and high sodium intake, which should render the animal highly susceptible to hypertensive stimuli.
These data in combination with our preliminary work also suggest that the increase in blood pressure observed with insulin infusion was "salt sensitive," because no differences in blood pressure were observed between groups when dietary sodium intake of the insulin-infused and glucose-drinking rats was only half that of the control group (see RESULTS, Blood pressure changes). This was contrary to Brands et al. (9), who showed no differences in the rise in MAP in rats given anywhere from 0.6 to 11.4 meq Na+/day while being infused intravenously for 7 days with 2.5 U insulin·kg body wt–1·day–1. However, it is in agreement with Tomiyami et al. (49), who showed increased MAP with insulin infusion in salt-sensitive, but not salt-resistant, Dahl rats, and only when they were fed a high-salt diet. Differences in study length, achieved plasma insulin levels, blood glucose levels, and other unknown variables may have resulted in these different findings.
Despite similarly increased MAP between insulin-infused and glucose-drinking rats, only rats receiving the insulin infusion had an increased natriuretic response to benzamil (a selective ENaC inhibitor) and HCTZ (a selective Na-Cl cotransporter inhibitor). This may imply that the mechanisms underlying the increase in blood pressure between these two treatments are different. Perhaps insulin infusion increases distal tubule sodium reabsorption associated with it and glucose drinking does not. A variation of this approach, termed "pharmacological blockade or knockout" of sodium channel or transporter activity, was employed by Frindt et al. (19) using amiloride (rather than benzamil) to block ENaC and polythiazide to block NCC. In those studies, the drugs were given to assess the contribution of ENaC vs. NCC reabsorption of sodium in rats that were on a sodium-deprived diet. However, the use of such agents to gauge the activity of these specific transport routes is quite novel and thus may be subject to skepticism. The fact that the natriuretic response to each of these agents was positive (relative to baseline), different (between the treatment groups), appropriate to expectations (with regard to degree of response), and reproducible among different groups of rats indicates that pharmacological blockade may be a valuable means of identifying sites of altered transport in the kidney. Nevertheless, it is important to also consider the possibility that differences in volume status, rate of drug absorption from the intraperitoneal cavity, and/or distribution throughout the body might exist between treatments and thus might also affect the response.
Insulin has been shown to increase sodium reabsorption through ENaC in A6 cells and cortical collecting duct cell lines via activation, likely phosphorylation, of SGK-1 (7, 39, 41, 50). However, whether insulin may also change the protein abundances of individual subunits of ENaC, as do aldosterone (34) and vasopressin (15), is not known. In this study, when sodium levels were matched, we found that a chronic yet modest (2x) elevation in circulating levels of insulin did not dramatically affect the protein abundances of any of the subunits, nor did it affect the ratio of the 70- to 85-kDa band of -ENaC as does aldosterone (34). Furthermore, NCC abundance, also shown to be increased by aldosterone (29), was not significantly increased by insulin infusion or glucose drinking in this study.
On the other hand, we did see increased apical residency of -, -, and -ENaC in the cortical collecting ducts from the insulin-infused rats relative to control. This occurred despite no measurable change in the final circulating level of plasma aldosterone in these rats (Table 2). Aldosterone has also been shown to increase apical residency of ENaC (32, 34). Therefore, the increase in ENaC on the apical membrane may have been the cause of the increased benzamil responsivity in these rats. In addition, it might have played a role in the elevated blood pressure; however, an absolute causal relationship between these variables could not be determined from this study. However, inappropriately high ENaC or NCC activity has been associated with hypertension in humans with Liddle's or Gordon's syndrome, respectively (31, 51).
In addition, WNK4 kinase has been shown to inhibit NCC by phosphorylating the transporter (52). It may also suppress its delivery to the plasma membrane (53). We found a significant decrease in the density in WNK4 on the immunoblot for the insulin-infused rats relative to the glucose water-drinking rats. This may explain the differential response of these two groups to HCTZ. However, we did not find clear discernible differences in cellular distribution of NCC with immunoperoxidase-based labeling.
Furthermore, it is possible that if we had achieved a relatively higher level of circulating insulin via the infusion, we might have observed a significant effect on ENaC and NCC abundances. However, this study would necessitate coinfusion of glucose to control hypoglycemia. Based on our preliminary work, this is difficult to titrate, stressful to the animals in a chronic scenario, and results in weight loss in the animals. Unlike aldosterone or vasopressin, which we can manipulate over fairly high ranges in the circulation to study their physiological roles in renal transport, high levels of insulin, in an insulin-sensitive rat can rapidly lead to hypoglycemia and death.
On the other hand, insulin-resistant rat models such as the obese Zucker rat have 2- to 10-fold higher circulating levels of insulin (5, 6). In these animals, we found an increase in the abundance of the -subunit of ENaC as well as NCC. However, additional studies will need to be done to determine whether the insulin receptors in the kidney become "resistant" as do peripheral insulin receptors in these rats.
Overall, chronic insulin infusion as well as drinking of a glucose solution were shown to increase blood pressure in rats when dietary sodium intakes were carefully matched. This increase corresponded to, and thus may have been the result of, increased activity of distal renal tubular sodium transport pathways including NCC and ENaC, possibly via trafficking into the apical membrane.
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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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REFERENCES |
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-ENaC abundance in obese Zucker rats. Am J Physiol Renal Physiol 281: F639–F648, 2001., , and subunit proteins in rat kidney. J Clin Invest 104: R19–R23, 1999.[Medline]This article has been cited by other articles:
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  A. R. Subramanya, J. Liu, D. H. Ellison, J. B. Wade, and P. A. Welling WNK4 Diverts the Thiazide-sensitive NaCl Cotransporter to the Lysosome and Stimulates AP-3 Interaction J. Biol. Chem., July 3, 2009; 284(27): 18471 - 18480. [Abstract] [Full Text] [PDF]
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 E. J. Hoorn, N. van der Lubbe, and R. Zietse The renal WNK kinase pathway: a new link to hypertension Nephrol. Dial. Transplant., April 1, 2009; 24(4): 1074 - 1077. [Full Text] [PDF]
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 M. B. Butterworth, R. S. Edinger, R. A. Frizzell, and J. P. Johnson Regulation of the epithelial sodium channel by membrane trafficking Am J Physiol Renal Physiol, January 1, 2009; 296(1): F10 - F24. [Abstract] [Full Text] [PDF]
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 C. E Hills, P. E Squires, and R. Bland Serum and glucocorticoid regulated kinase and disturbed renal sodium transport in diabetes J. Endocrinol., December 1, 2008; 199(3): 343 - 349. [Abstract] [Full Text] [PDF]
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F. J. McDonald A new SGK1 knockout mouse Am J Physiol Renal Physiol, June 1, 2008; 294(6): F1296 - F1297. [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. Tiwari, S. Riazi, and C. A. Ecelbarger Insulin's impact on renal sodium transport and blood pressure in health, obesity, and diabetes Am J Physiol Renal Physiol, October 1, 2007; 293(4): F974 - F984. [Abstract] [Full Text] [PDF]
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 S. Tiwari, V. K.M. Halagappa, S. Riazi, X. Hu, and C. A. Ecelbarger Reduced Expression of Insulin Receptors in the Kidneys of Insulin-Resistant Rats J. Am. Soc. Nephrol., October 1, 2007; 18(10): 2661 - 2671. [Abstract] [Full Text] [PDF]
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S. Tiwari, L. Nordquist, V. K. M. Halagappa, and C. A. Ecelbarger Trafficking of ENaC subunits in response to acute insulin in mouse kidney Am J Physiol Renal Physiol, July 1, 2007; 293(1): F178 - F185. [Abstract] [Full Text] [PDF]
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) by 1-way ANOVA followed by Tukey's multiple comparisons test.
