Abstract
Insulin resistance is accompanied by hyperinsulinemia and activation of the renin-angiotensin system, both of which are associated with hypertension. Because the kidney plays a major role in the regulation of blood pressure, we studied the regulation of insulin receptor expression in the kidney during states of insulin resistance. Using two rat models of insulin resistance, Western blot analysis demonstrated a significant reduction in the expression of insulin receptor subunits in the kidney compared to lean control rats. Treatment of insulin resistance in Zucker rats with the insulin-sensitizing drug rosiglitazone partially restored renal insulin receptor levels. Conversely, treatment with the angiotensin II type 1 receptor (AT1) antagonist candesartan increased renal insulin receptor expression compared to untreated rats. Streptozotocin-induced hyperglycemia, which results from hypoinsulinemia, reduced expression of renal insulin receptors. Hyperinsulinemia induced by insulin infusion, however, did not produce a similar effect. In conclusion, insulin receptors are downregulated in the kidneys of insulin resistant rats, possibly mediated by hyperglycemia and angiotensin II.
Insulin is essential for energy management in the body; however, its role in kidney is less defined. Insulin may have a role in glucose reabsorption at the level of the proximal tubule, as well as in sodium reabsorption throughout the length of the renal tubule.1 Moreover, insulin has been demonstrated to increase nitric oxide generation in the kidney2 via activation of nitric oxide synthases, the isoforms of which are expressed in the kidney.3 Thus, insulin may have a relatively unappreciated role in pressure natriuresis and maintenance of normal BP.
Insulin resistance is associated with hyperinsulinemia, as well as activation of the renin-angiotensin-aldosterone system (RAAS). Both of these factors have been independently linked not only to antinatriuresis1,4 but also directly with hypertension.5–7 Because the kidney is a primary determinant of BP control,8,9 understanding how the renal insulin receptor (IR) is regulated, at multiple levels, is essential.
The IR is a disulfide-linked protein composed of two α and two β subunits present in the plasma membrane of target cells.10 The extracellular α subunits contain the ligand-binding domain, and the intrinsic β subunits contain the tyrosine kinase signaling domains.
Decreased IR expression in the tissues that are involved in energy homeostasis (liver, skeletal muscle, and adipose tissue) has been shown to correlate with insulin resistance.11–13 Sechi and colleagues14–16 determined that whereas IR binding and mRNA in the liver and skeletal muscle was decreased in fructose-fed, insulin-resistant rats, there was no difference in the kidney, as compared with control rats. Therefore, the relative absence of renal “insulin resistance” would allow for the greater circulating levels of insulin to bind to renal IR and increase, for example, sodium reabsorption and therefore BP. Nonetheless, although mRNA levels were measured, protein was not. Furthermore, insulin can bind with lower affinity to other receptors in the kidney, such as the insulin-like growth factor receptor.
The aim of these studies was to determine the regulation of renal IR protein in insulin resistance and investigate potential mechanism for this regulation. We used several rat models of insulin resistance and/or hyperinsulinemia; treated the insulin resistance with insulin-sensitizing drug, a peroxisome proliferator–activated receptor-γ (PPAR-γ) agonist, rosiglitazone; and examined the role of angiotensin II type 1 receptor (AT1) in this regulation.
RESULTS
An outline of the study designs is provided in Figure 1. Peptide-blocking experiments confirmed the specificity of the commercial IR antibodies used in this study (Figure 2). The binding of the IR-α and IR-β antibodies was completely inhibited in the presence of their respective peptides (Ab+P), whereas the specific bands corresponding to the expected size for IR-α (125 kD) and IR-β (95 kD) were observed in the absence of blocking peptides (Ab).
Study outline showing types of animal models and experimental designs.
Specificity of insulin receptor antibodies (IR-α and IR-β). For peptide-blocking tests, equal amounts of protein from whole rat kidneys were loaded on two different membranes for immunoblotting. The membranes were incubated with the respective IR-α and IR-β antibodies in the absence (Ab) or presence (Ab+P) of respective blocking peptides. Specific bands were completely ablated by the peptides.
The kinetics and dosage dependence of insulin's actions differ depending on the tubule site of action.17 Therefore, in several cases, we examined the IR protein abundance in whole-kidney homogenate, as well as in homogenates prepared from three regions of the kidney separately: cortex (CTX), which is enriched in proximal tubules; inner stripe of outer medulla (OM), which is enriched in thick ascending limbs; and the inner medulla (IM), which is enriched in collecting duct.
Figure 3 shows the results from Western blot analysis of homogenates from the three regions of the kidney, in the lean versus obese Zucker rats (study 1). Protein abundances for both IR-α and IR-β were significantly decreased in obese Zucker rats in all three regions of the kidney relative to lean rats. We previously published the physiologic parameters for these rats,18 where we demonstrated that obese Zucker rats had 11-fold increased final circulating levels of insulin and reduced glucose tolerance, demonstrating their marked insulin resistance.
Insulin receptor subunit (IR-α and IR-β) protein abundance in the kidneys of obese and lean Zucker rats from study 1. Representative immunoblots and the densitometry summary (bottom) for IR-α (A) and IR-β (B) in cortex (CTX), inner stripe of the OM, and IM. The results for densitometry were normalized to β-actin. (C) β-Actin reprobe of blots. For immunoblotting, each lane was loaded with an equal amount of protein from a different rat's sample (confirmed a priori by equal Coomassie staining of representative protein bands). *Significant difference (P < 0.05) from lean rats’ mean by unpaired t test. Obese rats had reduced IR-α and IR-β in all three regions.
Immunoperoxidase labeling of IR (IR-α and IR-β) in the kidneys from lean and obese Zucker rats, shown in Figures 4 and 5, respectively, further confirm our Western blotting results. We found stronger staining for IR in the three regions (CTX, OM, and IM) of kidney from lean rats relative to those from obese Zucker rats. Furthermore, this downregulation seems most apparent in the distal tubule (collecting duct).
Insulin receptor protein (IR-α) expression in the kidneys of obese and lean Zucker rats. Representative pictures of immunoperoxidase labeling for IR-α in rats from study 1. Strong staining was observed in all three regions of the kidney (CTX, OM, and IM) of lean rats relative to obese Zucker rats, especially in the collecting duct. Magnification, ×400.
Insulin receptor protein (IR-β) expression in the kidneys of obese and lean Zucker rats. Representative pictures of immunoperoxidase labeling for IR-β in rats from study 1. Strong staining was observed in all three regions of the kidney (CTX, OM, and IM) of lean rats relative to obese Zucker rats, especially in distal tubule (i.e., collecting duct). Magnification, ×400.
To determine whether the reduced renal IR protein abundance correlated with a reduction in the first step of IR signaling, we analyzed the abundance of phosphorylated IR-β (p-IR). A significantly reduced abundance of p-IR was observed in the IM of obese Zucker rats (Figure 6) but not in the cortex or OM (study 2).
Phosphorylated IR β-subunit (p-IR) protein abundance in the kidneys of obese and lean Zucker rats from study 1. Representative immunoblots and the densitometry summary (bottom) for p-IR in CTX, inner stripe of the OM, and IM. The p-IR blots were reprobed for β-actin (shown below p-IR blots). For immunoblotting, each lane was loaded with an equal amount of protein from a different rat's sample (confirmed a priori by equal Coomassie staining of representative protein bands). *Significant difference (P < 0.05) from lean rats’ mean by unpaired t test. Obese rats had significantly reduced p-IR in the inner medullary region.
We then analyzed the IR protein abundance in another model of insulin resistance (in high-fat–fed F344 × BN rats). Both IR-α and IR-β abundance were significantly decreased in the IM region (Figure 7); however, there was no significant difference in the cortex and OM regions of the kidney between the two groups. We19 have shown that these high-fat–fed rats were mildly insulin resistant, as indicated by their reduced glucose tolerance. This, as well as findings displayed in Figure 6, suggest that the IM is the most sensitive region with regard to downregulation of the renal IR.
Insulin receptor subunit (IR-α and IR-β) protein abundance in different regions of kidney in the F344 × BN F1 hybrid rats fed with high-fat or control diet in study 2. Representative immunoblots and the densitometry summary (bottom) for IR-α (A) and IR-β (B) in CTX, inner stripe of the OM, and IM. The results for densitometry were normalized to β-actin. (C) β-Actin reprobe of blots. For immunoblotting, each lane was loaded with an equal amount of protein from a different rat's sample (confirmed a priori by equal Coomassie staining of representative protein bands). *Significant difference (P < 0.05) from control rats’ mean by unpaired t test. High-fat–fed rats had reduced IR-α and IR-β in IM.
To determine whether hyperinsulinemia may be an important contributor to IR downregulation in insulin resistance, we examined IR abundance in insulin-sensitive rats that were administered long-term infusions of insulin (study 3). We20 recently published the physiologic parameters and reported two- to three-fold elevated insulin levels in these rats. Figure 8 shows that long-term insulin infusion into normal Sprague-Dawley rats did not reduce IR abundance. In fact, insulin-infused rats had significantly increased IR-α abundance in their cortex and OM regions relative to controls (P = 0.013 and 0.022; Figure 8, A and B, respectively). However, there were no significant differences in IR-β abundance in any of the regions of the kidney between the two groups (Figure 8B). These data suggest that that hyperinsulinemia per se did not downregulate the renal IR.
Effect of long-term insulin infusion on the insulin receptor subunit (IR-α and IR-β) protein abundances in different regions of Sprague-Dawley rats’ kidneys from study 3. Representative immunoblots and the densitometry summary (bottom) for IR-α (A) and IR-β (B) in CTX, inner stripe of the OM, and IM. The results for densitometry were normalized to β-actin. (C) β-Actin reprobe of blots. For immunoblotting, each lane was loaded with an equal amount of protein from a different rat's sample (confirmed a priori by equal Coomassie staining of representative protein bands). *Significant difference (P < 0.05) from vehicle rats’ mean by unpaired t test. Insulin-infused rats had increased IR-α in CTX and OM.
To determine whether hyperglycemia, often present in insulin resistance (at least postprandial), might be a signal for IR downregulation, we analyzed the IR regulation in a rat model of type 1 diabetes, which is characterized by hyperglycemia with hypoinsulinemia (study 4). The physiologic data obtained from these rats such as blood glucose levels and body weights have already been published by us.21 Figure 9 shows that these streptozotocin (STZ)-induced diabetic rats have significantly lower protein abundance for IR-α in CTX and IM and for IR-β in OM relative to nondiabetic (normal) rats. Therefore, hyperglycemia and/or some other factors involved in diabetes may signal for renal IR downregulation.
Effect of hyperglycemia as a result of hypoinsulinemia on the IR protein abundance in the kidneys of STZ-induced diabetic or nondiabetic control rats from study 4. Representative immunoblots and the densitometry summary (bottom) for IR-α (A) and IR-β (B) in CTX, inner stripe of the OM, and IM of rats that were administered an injection of STZ or vehicle (control, n = 6 per treatment). The results for densitometry were normalized to β-actin. (C) β-Actin reprobe of blots. For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat's sample (confirmed a priori by equal Coomassie staining of representative protein bands). *Significant difference (P < 0.05) from control rats’ mean unpaired t test. STZ-injected rats had reduced abundance of IR-α (in CTX and IM) and IR-β (in OM).
To determine whether a commonly prescribed therapy for insulin resistance would upregulate renal IR, we treated Zucker rats with the PPAR-γ agonist rosiglitazone (RGZ; Figures 10 and 11) (study 5). Previously, we demonstrated that RGZ was effective in reducing the blood glucose levels in these rats.18 In obese rats, RGZ significantly increased the abundance of both IR-α and IR-β in OM, relative to nontreated rats (Figure 10). However, in lean rats, IR-α was also increased by RGZ but only in CTX (Figure 11). Other physiologic parameters from these rats, such as plasma insulin, body weight, and BP, were published previously.18
Effect of long-term therapy of insulin resistance by daily administration of RGZ on the protein abundance of the insulin receptor subunits (IR-α and IR-β) in the kidney of obese Zucker rats from study 5. Representative immunoblots and the densitometry summary (bottom) for IR-α (A) and IR-β (B) in CTX, inner stripe of the OM, and IM of obese Zucker rats that were treated with control diet (C) or this diet with added RGZ. The results for densitometry were normalized to β-actin. (C) β-Actin reprobe of blots. For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat's sample (confirmed a priori by equal Coomassie staining of representative protein bands). *Significant difference (P < 0.05) from obese control rats’ mean by unpaired t test. RGZ significantly increased IR-α and IR-β in OM of obese rats.
Effect of long-term therapy of insulin resistance by daily administration of RGZ on the protein abundance of the insulin receptor subunits (IR-α and IR-β) in the kidney of lean Zucker rats from study 5. Representative immunoblots and the densitometry summary (bottom) for IR-α (A) and IR-β (B) in CTX, inner stripe of the OM, and IM of lean Zucker rats that were treated with control diet (C) or this diet with added RGZ. The results for densitometry were normalized to β-actin. (C) β-Actin reprobe of blots. For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat's sample (confirmed a priori by equal Coomassie staining of representative protein bands). *Significant difference (P < 0.05) from lean control rats’ mean by unpaired t test. RGZ significantly increased IR-α in lean rats.
To determine whether increased activity of the renin-angiotensin system, another factor prevailing in insulin resistance, might contribute to renal IR downregulation, we tested the effect of long-term treatment with candesartan, an AT1 blocker (Figures 12 and 13) (study 6). In obese rats, candesartan increased the abundance of IR-α in the IM and of IR-β in both OM and IM, relative to nontreated rats (Figure 12). In lean rats candesartan significantly increased IR-α in cortex and IR-β in IM of the kidney (Figure 13).
Insulin receptor subunit abundances (IR-β and IR-β) in the kidney of the obese Zucker rats that were treated with or without candesartan, an AT1 antagonist, from study 6. Representative immunoblots and the densitometry summary (bottom) for IR-α (A) and IR-β (B) in CTX, inner stripe of the OM, and IM. The results for densitometry were normalized to β-actin. (C) β-Actin reprobe of blots. For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat's sample (confirmed a priori by equal Coomassie staining of representative protein bands). *Significant difference (P < 0.05) from obese control mean by unpaired t test. Candesartan increased IR-α (in IM) and IR-β (in OM and IM) in obese rats.
Insulin receptor subunit abundances (IR-β and IR-β) in the kidney of the lean Zucker rats that were treated with or without candesartan, an AT1 antagonist, from study 6. Representative immunoblots and the densitometry summary (bottom) for IR-α (A) and IR-β (B) in CTX, inner stripe of the OM, and IM. The results for densitometry were normalized to β-actin. (C) β-Actin reprobe of blots. For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat's sample (confirmed a priori by equal Coomassie staining of representative protein bands). *Significant difference (P < 0.05) from lean control mean by unpaired t test. Candesartan increased IR-α (in CTX) and IR-β (in IM) in lean rats.
DISCUSSION
Our major findings include the following: (1) The protein expression of the renal IR (both subunits) and p-IR is significantly reduced in insulin-resistant rats; (2) treatment of insulin resistance with a PPAR-γ agonist partially but not totally reverses this downregulation of IR; (3) long-term insulin infusion does not decrease the abundance of IR (either subunit) and actually increases IR-α, suggesting that hyperinsulinemia per se is not responsible for renal IR downregulation during the insulin-resistant state; (4) in contrast, hyperglycemic, hypoinsulinemic, type 1 diabetic rats have reduced renal IR expression; and, finally, (5) candesartan, an AT1 blocker, upregulated renal IR in lean and obese rats, demonstrating a role for AT1 activity in renal IR expression. These data provide a potential mechanistic link between angiotensin II activity and insulin resistance in the kidney.
The cause of insulin resistance is unclear, but the majority of studies suggest that blunted IR signaling, relative to agonist availability, is likely a key mechanism.22 Downregulation of the level of IR protein itself, with insulin resistance, has been demonstrated in various insulin-target tissues central to energy metabolism (e.g., liver, skeletal muscle, adipose tissue)11–13 but not specifically in kidney. Here we demonstrate that kidneys of insulin-resistant rat models (obese Zucker rats and rats fed a high-fat diet for several weeks) have a reduction in IR protein. The reduction occurred in both subunits, and the intensity tended to be correlated with degree of insulin resistance, with the IM seeming to be the most sensitive. This is further supported by our finding of significantly reduced inner medullary p-IR under basal conditions in obese Zucker rats (Figure 6). Furthermore, the immunoperoxidase labeling of IR revealed that the downregulation of IR protein seems most apparent in collecting duct of the kidney in obese Zucker rats. This downregulation could have a major impact on the regulation of collecting duct proteins, such as the amiloride-sensitive epithelial sodium channel and aquaporin 2, which may affect salt and water reabsorption.
Under basal conditions, renal IR signaling seemed blunted in obese Zucker rats, at least in the IM, as indicated by reduced protein levels of p-IR, the first step in insulin signaling. Moreover, IR phosphorylation in response to an acute insulin challenge might be a more sensitive test of IR signaling capacity in these rats. We have planned future studies using this approach to evaluate more comprehensively renal IR signaling Conversely, studies by Sechi and associates16 suggest that there is less insulin desensitization of the renal IR relative to other tissues. They examined the binding of labeled insulin, rather than solely expression, and concluded that there were no differences in IR number in the kidneys of fructose-fed, insulin-resistant rats relative to controls. However, insulin can also bind with lower affinity to the insulin-like growth factor receptor I (IGFI) and the insulin-receptor-like receptor (IRR), which are also expressed in the kidney but coupled to different signaling pathways. Nonetheless, it is equally possible that differences in the models of insulin resistance studied may have accounted for this seeming discrepancy. For example, the fructose-fed rat is a rather unusual model of insulin resistance associated with a higher triglyceride deposition in liver.23 Furthermore, unlike glucose, fructose does not stimulate insulin release from the pancreatic β cells. Moreover, it is interesting to note that in our hands, fructose feeding to Sprague-Dawley rats did not result in a significant increase in BP (as measured by radiotelemetry),24 whereas increased BP was found in obese Zucker rats18 and in high-fat–fed rats.19
Our findings suggest that the mechanism for decreased IR protein in the kidney of insulin-resistant rats is not due to hyperinsulinemia, per se, because long-term insulin infusion into insulin-sensitive Sprague-Dawley rats did not reduce either IR-α or IR-β in any region of the kidney. However, the circulating level of insulin achieved by the infusion20 was considerably less than what we have observed in the obese Zucker rat.18,25,26 Therefore, it is possible that we have not reached the threshold level for observing reduction in the expression of the receptor. However, this is unlikely to be the case, because we actually found an increase in the expression of IR-α in the OM and cortex with the infusion.
Hyperglycemia is another factor that is present in insulin resistance and might have a role in IR protein regulation. In support of this hypothesis, we found a significant decrease in both IR-α and IR-β subunits in the kidneys of rats made type 1 diabetic and thus hyperglycemic (without confounding hyperinsulinemia) by administration of STZ (Figure 9). STZ may have had some toxic effects on the kidney that might have affected renal IR expression. However, STZ toxic effects are generally seen in shorter term studies.27 Nonetheless, β-actin levels were not decreased by STZ. Furthermore, these animals might also be expected to have upregulated (RAAS) activity.
To evaluate better the role of the RAAS, we tested the effect of candesartan, an AT1 antagonist on IR regulation. Insulin resistance and type 2 diabetes are also strongly associated with activation of the RAAS.28–30 Other investigators31,32 have reported increased AT1 activity in the kidneys of the obese Zucker rat relative to age-matched lean rats. We showed that candesartan increased the abundance of IR-α in at least one region of the kidney in both lean and obese rats. The signaling component, IR-β, was significantly increased in both IM and OM in the obese rats but only in the IM in lean rats. This demonstrates sensitivity of renal IR abundance to AT1 blockade in both lean and obese rats but greater sensitivity, at least for the β subunit in the obese rats, perhaps because this pathway was more highly activated in obese rats. Nonetheless, this provides molecular evidence to link high AT1 activity with insulin resistance, at least in the kidney.
In some cases (e.g., insulin infusion), we found differential regulation of the protein levels of IR-α from those of IR-β. The explanation for this is not clear, because both IR subunits are transcribed and translated together from a single gene. However, it is possible that different rates of degradation are important. With insulin infusion, we found an increase in both cortical and outer medullary IR-α, likely predominantly reflective of proximal tubule and thick ascending limb IR, respectively, with no change in IR-β. An increase in the abundance of the ligand-binding subunit (IR-α) with insulin is in agreement with findings of Corin and Donner33,34 of increased hormone affinity of the hepatic IR upon insulin stimulation. Furthermore, insulin-induced proteolysis selectively of the IR-β subunit has been described, which might result in increased turnover of IR-β relative to IR-α with insulin infusion.35,36 In our insulin-resistant rat models (the obese Zucker rat and the high-fat–fed rat), we found decreased abundance of both subunits, although there was a tendency for the percentage of decrease to be greater for IR-β, the signaling subunit.
We have shown downregulation of IR protein (both subunits) and inner medullary p-IR in the kidneys of insulin-resistant rat models. Our studies further suggest that activation of the RAAS and/or hyperglycemia may play a role in renal IR downregulation. Downregulation of receptor protein, if associated with decreased signaling, may be important in dysregulation of key insulin-related cellular events, such as glucose or sodium handling by the renal tubule and/or nitric oxide generation for BP control.
CONCISE METHODS
Animals and Study Design
Six different animal models were used. In the first study, we used the obese Zucker rat, a well-characterized model of insulin resistance.37 Male lean and obese Zucker rats (9 wk old; n = 6 per body type) were obtained from Charles River Laboratories (Wilmington, MA) and fed standard rodent chow (Purina 5001 Rodent Chow; Purina Mills, St. Louis, MO) for 12 wk.
To study the effect of more modest insulin resistance on renal IR regulation, in study 2, we developed a high-fat–fed model of insulin resistance in the male F344 × BN F1 hybrid rats.19 These rats (approximately 9 wk old; n = 12) were obtained from Harlan (Indianapolis, IN) and fed a control diet (4% fat by weight) or a high-fat diet (35% fat; Research Diets, New Brunswick, NJ) for 8 wk (n = 6 per diet).
Study 3 was designed to study the effect of hyperinsulinemia, a common component of insulin resistance, on IR regulation. Sprague-Dawley rats (approximately 250 to 280 g; Taconic Farms, Germantown, NY) either received subcutaneous osmotic minipump implants (Alzet Model 2002; Alza Corp., Palo Alto, CA) to infuse insulin or received a sham operation (n = 6 per treatment). Insulin (Humulin-R; Eli Lilly, Indianapolis, IN) was infused at the rate of 20 U/kg body wt per d for 28 d. The insulin-infused group was also given 20% dextrose water to drink in lieu of plain water to maintain euglycemia. Diets and treatments have been detailed previously.20
For studying the effect of hyperglycemia, in study 4, 12 Sprague-Dawley rats weighing approximately 210 g were obtained from Taconic Farms. For inducing type 1 diabetes, STZ (55 mg/kg body wt, dissolved in 0.2 ml of 0.1 M citric acid) was injected intraperitoneally into half of the rats (n = 6). The remaining rats (n = 6) received intraperitoneal injections of the vehicle. In the STZ groups, blood glucose levels were measured twice weekly by glucometer (TheraSense; Freestyle, Alameda, CA) reading of tail blood. When the level was >25 mmol/L, insulin glargine (2 to 6 U; Lantus; Aventis Pharmaceuticals, Kansas City, MO) was administered (at most every other day) so that rats did not lose weight and survived the 12-wk study. All animals had free access to water and standard rodent chow for 12 wk.
For determination of the effect of attenuation of the insulin resistance, in study 5, obese and lean Zucker rats (approximately 9 wk old) were treated long term (12 wk) with a PPAR-γ agonist, RGZ. Rats were fed either ground standard rodent chow in agar with 70% water or the same base diet with RGZ maleate (0.0145 mg incorporated per gram wet weight diet; GlaxoSmithKline, London, England), as described previously.18,38 This resulted in an approximate dosage of 3 mg/kg body wt per d RGZ (n = 6 rats per treatment per body type).
Study 6 was done to determine whether relatively upregulated activity of the renin-angiotensin system, a factor common in insulin resistance, might play a role in renal IR downregulation. Lean and obese Zucker rats were fed either chow diet (as in study 5) or the same diet with an AT1 antagonist, candesartan (25 mg/kg diet; AstraZeneca, London, England) for 12 wk, which resulted in an approximate dosage of 5 mg/kg body wt per d candesartan (n = 6 rats per treatment per body type). This dosage was approximately half of the losartan dosage used by Alonso-Galicia et al.31 in their studies showing marked BP reduction (approximately 25 to 35 mmHg) in both lean and obese Zucker rats that were treated for 7 d.
Rats were maintained according to protocols approved by the Georgetown Animal Care and Use Committee, an Association for Assessment and Accreditation of Laboratory Animal Care (International)–approved facility. At the end of the studies, animals were killed by rapid decapitation, and kidneys were collected. The right kidney was dissected into CTX, OM and IM, and whole homogenates were processed separately for immunoblotting according to previously published protocols.39
Immunohistochemistry
Perfusion-fixed, paraffin-processed 5-μm sections of the left kidneys were used for immunohistochemical examination of the IR in lean and obese Zucker rats. Heat-induced target retrieval was performed using a citrate buffer, pH 6 (Zymed Laboratories, South San Francisco, CA). Endogenous peroxidase activity was removed by incubation with 2% H2O2 for 20 min. Tissues were incubated with the rabbit polyclonal antibodies against IR-α or IR-β (1:200; catalog nos. sc-710 and sc-711, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. The Envision+System (DakoCytomation, Carpinteria, CA) was used to conduct peroxidase labeling. 3,3′-Diaminobenzidine tetrachloride dihydrate was applied for 2 min, and the tissue was counterstained with Mayer's hematoxylin. A positive reaction was identified as a brown stain. Pictures were taken with a Photometrics Cool Snap camera (Scanalytics, Fairfax, VA) mounted to a Nikon Eclipse E600 microscope with a ×40 oil-immersion lens for a total magnification of ×400.
Electrophoresis and Blotting of Membranes
Coomassie-stained “loading gels” were done to assess the quality of the protein by sharpness of the bands and to adjust protein concentrations before blotting, if necessary, as described previously.39,40 For immunoblotting, 20 to 30 μg of protein from each sample was loaded into individual lanes of 10% polyacrylamide (precast; Bio-Rad, Hercules, CA). Blots were probed with commercial rabbit polyclonal anti–IR-α, anti–IR-β, or p-IR (catalog no. sc-25103 for p-IR; Santa Cruz Biotechnology) antibodies at a final concentration of 0.2 μg/ml. Some of the IR-α blots were obtained by stripping and reprobing of the IR-β blots. The IR blots were stripped and reprobed for β-actin (Sigma, St. Louis, MO), an internal control, for protein loading. In peptide-blocking tests, 1 μg/ml blocking peptide for IR-α antibody or IR-β antibody was added to the incubation solution containing anti–IR-α or anti–IR-β antibodies, respectively.
Statistical Analyses
Data were evaluated by SigmaStat (Chicago, IL). Unpaired t test was used to determine significant differences (P < 0.05) between pairs of means.
DISCLOSURES
None.
Acknowledgments
This work was supported by National Heart, Lung, and Blood Institute grants HL073193, HL074142, and DK064872 to C.E.
A portion of these data were published previously in abstract form (FASEB J 20: A1169, 2006).
We thank GlaxoSmithKline for supplying RGZ.
Footnotes
Published online ahead of print. Publication date available at www.jasn.org.
- © 2007 American Society of Nephrology
References
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