Title: Insulin resistance in chronic kidney disease is ameliorated by spironolactone in rats and humans
Abstract: In this study, we examined the association between chronic kidney disease (CKD) and insulin resistance. In a patient cohort with nondiabetic stages 2–5 CKD, estimated glomerular filtration rate (eGFR) was negatively correlated and the plasma aldosterone concentration was independently associated with the homeostasis model assessment of insulin resistance. Treatment with the mineralocorticoid receptor blocker spironolactone ameliorated insulin resistance in patients, and impaired glucose tolerance was partially reversed in fifth/sixth nephrectomized rats. In these rats, insulin-induced signal transduction was attenuated, especially in the adipose tissue. In the adipose tissue of nephrectomized rats, nuclear mineralocorticoid receptor expression, expression of the mineralocorticoid receptor target molecule SGK-1, tissue aldosterone content, and expression of the aldosterone-producing enzyme CYP11B2 increased. Mineralocorticoid receptor activation in the adipose tissue was reversed by spironolactone. In the adipose tissue of nephrectomized rats, asymmetric dimethylarginine (ADMA; an uremic substance linking uremia and insulin resistance) increased, the expression of the ADMA-degrading enzymes DDAH1 and DDAH2 decreased, and the oxidative stress increased. All of these changes were reversed by spironolactone. In mature adipocytes, aldosterone downregulated both DDAH1 and DDAH2 expression, and ADMA inhibited the insulin-induced cellular signaling. Thus, activation of mineralocorticoid receptor and resultant ADMA accumulation in adipose tissue has, in part, a relevant role in the development of insulin resistance in CKD. In this study, we examined the association between chronic kidney disease (CKD) and insulin resistance. In a patient cohort with nondiabetic stages 2–5 CKD, estimated glomerular filtration rate (eGFR) was negatively correlated and the plasma aldosterone concentration was independently associated with the homeostasis model assessment of insulin resistance. Treatment with the mineralocorticoid receptor blocker spironolactone ameliorated insulin resistance in patients, and impaired glucose tolerance was partially reversed in fifth/sixth nephrectomized rats. In these rats, insulin-induced signal transduction was attenuated, especially in the adipose tissue. In the adipose tissue of nephrectomized rats, nuclear mineralocorticoid receptor expression, expression of the mineralocorticoid receptor target molecule SGK-1, tissue aldosterone content, and expression of the aldosterone-producing enzyme CYP11B2 increased. Mineralocorticoid receptor activation in the adipose tissue was reversed by spironolactone. In the adipose tissue of nephrectomized rats, asymmetric dimethylarginine (ADMA; an uremic substance linking uremia and insulin resistance) increased, the expression of the ADMA-degrading enzymes DDAH1 and DDAH2 decreased, and the oxidative stress increased. All of these changes were reversed by spironolactone. In mature adipocytes, aldosterone downregulated both DDAH1 and DDAH2 expression, and ADMA inhibited the insulin-induced cellular signaling. Thus, activation of mineralocorticoid receptor and resultant ADMA accumulation in adipose tissue has, in part, a relevant role in the development of insulin resistance in CKD. Insulin resistance (IR) is defined as a clinical condition in which there is a reduced biological effect for any given blood concentration of insulin. The presence of IR has been reported in patients with chronic kidney disease (CKD).1.Fliser D. Pacini G. Engelleiter R. et al.Insulin resistance and hyperinsulinemia are already present in patients with incipient renal disease.Kidney Int. 1998; 53: 1343-1347Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar IR in CKD patients is accompanied by hyperinsulinemia and glucose intolerance, as well as by derangement of insulin secretion.2.Feneberg R. Sparber M. Veldhuis J.D. et al.Altered temporal organization of plasma insulin oscillation in chronic renal failure.J Clin Endocrinol Metab. 2002; 87: 1965-1973Crossref PubMed Scopus (24) Google Scholar Recent studies have described IR in CKD as a 'renal IR syndrome' that contributes to the comorbidity of cardiovascular disease, where the authors attributed this syndrome to a higher body mass index (BMI) and increased triglyceride concentrations.3.Becker B. Kronenberg F. Kielstein J.T. et al.Renal insulin resistance syndrome, adiponectin and cardiovascular events in patients with kidney disease: the mild and moderate kidney disease study.J Am Soc Nephrol. 2005; 16: 1091-1098Crossref PubMed Scopus (295) Google Scholar Several factors have been proposed for the pathogenesis of IR in renal dysfunction, including uremic toxins.4.Banerjee D. Recio-Mayoral A. Chitalia N. Kaski J.C. Insulin resistance, inflammation, and vascular disease in nondiabetic predialysis chronic kidney disease patients.Clin Cardiol. 2011; 34: 360-365Crossref PubMed Scopus (39) Google Scholar These factors all inhibit insulin-stimulated glucose disposal in insulin target organs. It was revealed that excess concentrations of mineralocorticoid, aldosterone, or activation of its receptor mineralocorticoid receptor (MR) induce IR.5.Goodfriend T.L. Egan B.M. Kelley D.E. Plasma aldosterone plasma lipoproteins, obesity and insulin resistance in Humans.Prostaglandins Leukot Essent Fatty Acids. 1999; 60: 401-405Abstract Full Text PDF PubMed Scopus (77) Google Scholar,6.Catena C. Lapenna R. Baroselli S. et al.Insulin sensitivity in patients with primary aldosteronesteronism: a follow-up study.J Clin Endocrinol Metab. 2006; 91: 3457-3463Crossref PubMed Scopus (214) Google Scholar In some previous studies, plasma aldosterone concentrations have been shown to increase according to renal function deterioration.7.Bia M.J. DeFronzo R.A. Extrarenal potassium homeostasis.Am J Physiol. 1981; 240: F257-F268PubMed Google Scholar, 8.Berle T. Katz F.H. Henrich W.L. DeTorrente A. et al.Role of aldosterone in the control of sodium excretion in patients with advanced chronic renal failure.Kidney Int. 1978; 14: 228-235Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 9.Reubi F.C. Weidmann P. Relationships between sodium clearance, plasma renin activity, plasma aldosterone, renal hemodynamics and blood pressure in essential hypertension.Clin Exp Hypertens. 1980; 2: 593-612Crossref PubMed Scopus (9) Google Scholar Therefore, increased aldosterone concentrations might contribute to the development of IR in CKD. Other studies have also revealed that one of the uremic toxins, the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA), is involved in the derangements of glucose metabolism in various pathological conditions. A recent study demonstrated that ADMA blocks insulin-induced glucose utilization in the adipocytes.10.Krzyzanowska K. Mittermayer F. Wolzt M. Schernthaner G. ADMA, cardiovascular disease and diabetes.Diabetes Res Clin Pract. 2008; 82: S122-S126Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,11.Yang Z.C. Wang K.S. Wu Y. et al.Asymmetric dimethylarginine impairs glucose utilization via ROS/TLR4 pathway in adipocytes: an effect prevented by vitamin E.Cell Physiol Biochem. 2009; 24: 115-124Crossref PubMed Scopus (15) Google Scholar ADMA is degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which is composed of the two isoforms DDAH1 and DDAH2,12.Zoccali C. Bode-Böger S. Mallamaci F. et al.Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study.Lancet. 2001; 358: 2113-2117Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar,13.Leiper J.M. Santa Maria J. Chubb A. et al.Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases.Biochem J. 1999; 343: 209-214Crossref PubMed Scopus (434) Google Scholar each of which stems from different chromosomes and differs in several aspects. We previously demonstrated that, in Nx dog, DDAH2 expressions are downregulated in the kidney14.Okubo K. Hayashi K. Wakino S. et al.Role of asymmetrical dimethylarginine in renal microvascular endothelial dysfunction in chronic renal failure with hypertension.Hypertens Res. 2005; 28: 181-189Crossref PubMed Scopus (43) Google Scholar and coronary endothelium,15.Tatematsu S. Wakino S. Kanda T. et al.Role of nitric oxide-producing and -degrading pathways in coronary endothelial dysfunction in chronic kidney disease.J Am Soc Nephrol. 2007; 18: 741-749Crossref PubMed Scopus (60) Google Scholar which contributed to blunted vasodilative response. We also demonstrated recently that the DDAH2/ADMA pathway was involved in glucose-stimulated insulin secretion in pancreatic β-cells.16.Hasegawa K. Wakino S. Kimoto M. et al.DDAH2 enhances pancreatic insulin secretion in mice via transcriptional regulation of secretagogin through Sirt1-dependent mechanism.FASEB J. 2013; 27: 2301-2315Crossref PubMed Scopus (29) Google Scholar These data imply that tissue levels of DDAH/ADMA affect the local blood supply or cellular signaling, which is surmised to lead to systemic insulin-resistant state or glucose intolerance.17.Stühlinger M.C. Abbasi F. Chu J.W. et al.Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor.JAMA. 2002; 287: 1420-1426Crossref PubMed Scopus (545) Google Scholar In this study, we have identified plasma aldosterone concentration as an independent risk factor for IR in our CKD cohort. By using Nx rats, we showed the coexistence of MR activation, ADMA accumulation, and impaired insulin signaling in the adipose tissue. We have provided evidence for a novel link between the aldosterone/MR pathway and the DDAH/ADMA pathway in the adipose tissue of CKD that induced the initiation of IR in CKD. The baseline characteristics of the study participants are presented in Table 1. As the CKD stage advanced, fasting insulin concentrations (immunoreactive insulin, IRI) significantly increased without any changes in FBS concentration. Consistently, with the advances in the CKD stage, homeostasis model assessment of IR (HOMA-IR) values significantly increased. Plasma aldosterone concentrations also increased as the CKD stages advanced, although plasma cortisol, adrenocorticotropic hormone (ACTH), and active renin concentration were not significantly altered. The participants were taking various kinds of antihypertensives. However, the population taking each antihypertensive was not different between each stage of CKD patients (Table 2).Table 1Characteristics of the study participants classified into five CKD stagesParameterStage 1 (>90)Stage 2 (60–89)Stage 3 (30–59)Stages 4 and 5 (>30)n19897814Diseases (%) Glomerulonephritis13 (68.4)45 (50.1)49 (62.8)8 (57.1) Nephrosclerosis3 (15.7)26 (29.2)11 (14.1)4 (28.6) Polycystic kidney disease0 (0)5 (5.6)7 (9.0)1 (7.1) Others3 (15.7)13 (14.6)11 (14.1)1 (7.1)Age53.4±3.861.1±1.8*P<0.05 vs. stage 166.1±1.7**P<0.01 vs. stage 1.72.6±1.7**P<0.01 vs. stage 1.Gender (male/female)11/849/4042/367/7BMI (kg/m2)23.6±0.7622.2±0.5223.6±0.4622.7±1.0Systolic BP (mmHg)128.9±3.7133.4±1.6135.5±1.3137.1±1.8Diastolic BP (mmHg)73.0±2.276.1±1.278.3±1.476.1±1.9eGFR (ml/min per 1.73m2)106.1±3.272.3±1.0**P<0.01 vs. stage 1.45.7±1.1**P<0.01 vs. stage 1.17.6±2.2**P<0.01 vs. stage 1.Urinary protein (g/g creatinine)0.072±0.0120.075±0.0090.151±0.010**P<0.01 vs. stage 1.0.627±0.091**P<0.01 vs. stage 1.Hb (g/dl)13.1±0.213.4±0.213.3±0.211.5±0.5**P<0.01 vs. stage 1.LDL-C (mg/dl)121.1±6.4123.9±4.3112.9±1.4115.4±11.5TG (mg/dl)95.6±9.5135.5±10.4131.9±13.1164.4±29.9Blood glucose (mg/dl)101.1±3.4103.4±2.1106.7±2.4108.0±2.7IRI (mmol/l)8.38±0.799.98±1.1010.87±1.70*P<0.05 vs. stage 119.07±4.60*P<0.05 vs. stage 1HOMA-IR2.17±0.212.63±0.313.00±0.54*P<0.05 vs. stage 15.06±1.25**P<0.01 vs. stage 1.Aldosterone (pg/ml)131.5±9.1130.8±9.4156.2±10.7*P<0.05 vs. stage 1168.2±8.9**P<0.01 vs. stage 1.K (mEq/l)4.14±0.184.39±0.15*P<0.05 vs. stage 14.36±0.15*P<0.05 vs. stage 14.60±0.22*P<0.05 vs. stage 1ARC (pg/ml)5.77±1.8212.68±2.0816.24±2.2816.23±5.22ACTH (pg/ml)23.0±3.4929.81±2.5634.82±1.8133.62±5.70Cortisol (pg/ml)10.38±0.7911.4±0.4712.55±0.3511.40±0.67Abbreviations: ACTH, adrenocorticotropic hormone; ARC, active renin concentration; BMI, body mass index; BP, blood pressure; CKD, chronic kidney disease; eGFR, glomerular filtration rate; Hb, hemoglobin; HOMA-IR, homeostasis model assessment of insulin resistance; IRI, immunoreactive insulin; K, potassium; LDL-C, low-density lipoprotein-cholesterol; TG, triglyceride.* P<0.05 vs. stage 1** P<0.01 vs. stage 1. Open table in a new tab Table 2Antihypertensive medications of CKD patients of each stageStage 1Stage 2Stage 3Stages 4 and 5ARBs or ACE-Is9/19 (47.3%)41/89 (46.1%)35/78 (44.9%)6/14 (42.8%)Ca channel blockers9/19 (47.3%)37/89 (41.6%)36/78 (46.2%)10/14 (71.4%)Diuretics2/19 (10.5%)6/89 (6.7%)4/78 (5.1%)1/14 (7.1%)β-Blockers3/19 (15.8%)12/89 (13.4%)9/78 (11.5%)2/14 (14.5%)Abbreviations: ACE-I, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; CKD, chronic kidney disease. Open table in a new tab Abbreviations: ACTH, adrenocorticotropic hormone; ARC, active renin concentration; BMI, body mass index; BP, blood pressure; CKD, chronic kidney disease; eGFR, glomerular filtration rate; Hb, hemoglobin; HOMA-IR, homeostasis model assessment of insulin resistance; IRI, immunoreactive insulin; K, potassium; LDL-C, low-density lipoprotein-cholesterol; TG, triglyceride. Abbreviations: ACE-I, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; CKD, chronic kidney disease. A linear regression analysis was performed between estimated glomerular filtration rate (eGFR) and the various indexes of glucose metabolisms. Both fasting serum insulin concentrations and HOMA-IR levels were correlated with eGFR levels (Figure 1). As renal dysfunction advances, plasma aldosterone concentrations increased and IR progressed (Table 1). In addition, aldosterone has been reported to inhibit insulin signaling and cause IR in vascular smooth muscle cells.18.Hitomi H. Kiyomoto H. Nishiyama A. et al.Aldosterone suppresses insulin signaling via the downregulation of insulin receptor substrate-1 in vascular smooth muscle cells.Hypertension. 2007; 50: 750-755Crossref PubMed Scopus (122) Google Scholar Therefore, we hypothesized that the increase in plasma aldosterone in CKD contributes to the development of IR. The fasting aldosterone concentrations had a significant correlation with fasting glucose concentrations (Figure 2a), fasting insulin concentrations (Figure 2b), and HOMA-IR levels (Figure 2c). Multiple regression analysis using the various anthropometric and biochemical parameters, as described in Materials and Methods, revealed that triglyceride, aldosterone, eGFR levels, and BMI levels were significant determinants of systemic IR in CKD (triglyceride; β=0.347, aldosterone; β=0.149, eGFR; β=-0.178, BMI; β=0.178, each P<0.0001), which demonstrated plasma aldosterone concentrations as one of the independent risk factors of IR in CKD. We next examined the relevant factors for plasma aldosterone concentrations. Simple regression analysis revealed that aldosterone concentrations increase according to decreases in eGFR (Figure 3a). Although plasma potassium concentration had no relevance to aldosterone concentration (Figure 3d), the fasting plasma active renin concentration (ARC), and ACTH were significantly correlated with that of aldosterone (Figure 3b and c, respectively). Multiple regression analysis using these parameters revealed that only eGFR is the independent regulatory factor for plasma concentration of aldosterone (β=-0.874, P=0.0017). Our clinical data provide plausible evidence that as renal function deteriorates, IR progresses with the increase in plasma aldosterone concentration. We finally examined the effects of the aldosterone blocker spironolactone on IR in CKD by conducting the prospective randomized placebo-controlled study described in Research Design and Methods. There was no significant difference in metabolic parameters between the spironolactone group and the control group, and the medications for hypertension were also equivalent between groups (Table 3). After a 6-month treatment with spironolactone, HOMA-IR levels and fasting insulin concentrations in the spironolactone group significantly decreased as compared with those in the control group (HOMA-IR; Figure 4a, % change; control group vs. spironolactone group; 29.0±13.1% vs. -53.7±6.5%, P<0.01, insulin; Figure 4b, % change; control group vs. spironolactone group; 25.7±11.7% vs. -51.2±7.1%, P<0.01). However, the change in ARC and plasma aldosterone concentrations did not differ (% change; control group vs. spironolactone group; ARC; -22.4±16.2% vs. 69.6±34.8%, P=0.0567, serum aldosterone; -5.26±7.18% vs. 10.1±21.1%, P=0.507, Figure 4c and d, respectively). The changes in plasma potassium concentrations were also the same between the two groups (Figure 2e, % change; control group vs. spironolactone group; 0.83±2.13% vs. 2.45±2.91%, P=0.360).Table 3Basic characteristics of CKD participants of spironolactone intervention studyParameterControlInterventionPn1212Age61.7±10.264.1±9.90.72BMI22.7±2.123.5±2.20.38Systolic blood pressure (mmHg)123.3±11.3133.7±12.70.10Diastolic blood pressure (mmHg)72.7±7.077.5±6.20.11eGFR(ml/min per 1.73m2)58.0±9.558.5±140.76Hb (g/dl)14.3±0.7814.3±0.940.72LDL-C (mg/dl)118.0±5.9111.3±140.45TG (mg/dl)150.5±67135.3±65.40.65FBS (mg/dl)104.5±2.9113.7±9.90.13IRI (mU/l)12.6±5.913.1±3.20.20HOMA-IR3.51±1.433.69±1.10.34Aldosterone (pg/ml)252.1±41.1244.5±30.60.35K (mEq/l)4.28±0.124.38±0.350.57Antihypertensive medication ACE-I or ARB5/12 (41.7%)7/12(58.3%)0.48 Ca blocker8/12(66.7%)7/12(58.3%)0.24 Diuretics2/12(16.7%)3/12(25.0%)0.37 β-blocker5/12(41.7%)4/12(33.3%)0.37Abbreviations: ACE-I, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blockers; BMI, body mass index; Ca blocker, calcium blocker; CKD, chronic kidney disease; FBS, fasting blood sugar; eGFR, estimated glomerular filtration rate; HOMA-IR, homeostasis model assessment of insulin resistance; IRI, immunoreactive insulin; LDL-C, low-density lipoprotein cholesterol. Open table in a new tab Abbreviations: ACE-I, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blockers; BMI, body mass index; Ca blocker, calcium blocker; CKD, chronic kidney disease; FBS, fasting blood sugar; eGFR, estimated glomerular filtration rate; HOMA-IR, homeostasis model assessment of insulin resistance; IRI, immunoreactive insulin; LDL-C, low-density lipoprotein cholesterol. Both observation and intervention studies in our CKD cohort suggested that activation of aldosterone/MR system might induce IR in CKD. We investigated the molecular mechanism for IR in CKD by using 5/6Nx rat model, in which spironolactone was given to 5/6Nx rats for 8 weeks. At the time of killing, body weight was not different among the three experimental groups (sham; 601±23g, Nx; 574±15g, Nx+spironolactone; 565±11g). In Nx rats, fasting free fatty acid and insulin concentrations increased, although fasting blood glucose and triglyceride concentrations were not different, suggesting IR in Nx rats. The increases in fasting free fatty acid and insulin concentrations were reversed by treatment with spironolactone for 8 weeks. Plasma aldosterone concentration increased in Nx rats, which further elevated in spironolactone-treated Nx rats (Table 4). Serum potassium levels tended to increase in Nx rats, which levels tended to decrease in spironolactone-treated Nx rats, although these alterations did not reach statistical significant changes and presumed to be affected by the long period treatment with spironolactone for 8 weeks. Oral glucose tolerance test revealed that impaired glucose tolerance was evident in Nx rats, and it was partially ameliorated by spironolactone (Figure 5a, left panel). The area under the curve of oral glucose tolerance test increased in Nx rats, and this increase was attenuated in spironolactone-treated Nx rats (Figure 5a, right panel). Insulin tolerance test revealed that the initial fall in plasma glucose concentrations within 15min was blunted in Nx rats, and this effect was also ameliorated by spironolactone (Figure 5b). After stimulation with insulin, phosphorylated Akt levels in the muscle and liver tissues were not altered between the normal and Nx rat groups, whereas these levels were attenuated in the adipose tissues in Nx rats as compared with those in sham-operated rats (Figure 5c). This decrease in phosphorylated Akt levels in adipose tissues was reversed by spironolactone. These data in combination implied that impaired insulin signaling, at least in part, caused systemic IR in CKD rats. The alterations in insulin signaling in the adipose tissue of insulin-injected Nx rats were further explored. Upon binding to the insulin receptor (IR), insulin catalyzes downstream signaling events, such as phosphorylation of specific tyrosine residues on IR substrate (IRS) proteins, to initiate the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. Neither serine nor tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was different among the three experimental groups (Figure 5d). Lipid and protein tyrosine phosphatase, phosphatase, and tensin homolog deleted from chromosome 10 (PTEN), is a negative regulator of insulin signaling, whose activity is regulated by phosphorylation status. Neither phosphorylated nor total PTEN expression was altered in Nx rats or by spironolactone treatment (Figure 5e). The 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a mediator of insulin signaling, which is activated by phosphoinositide 3-kinase (PI3-kinase) and phosphorylates Akt. The phosphorylated PDK1 levels were reduced in Nx rats, which was restored by spironolactone (Figure 5f). The Src-homology 2-domain–containing phosphatase (SHP)-1 and SHP-2 are other types of protein tyrosine phosphatases that dephosphorylate IRS and negatively regulate insulin signaling. The expression of either isoform of SHP was unaltered among three experimental groups (Figure 5g). Finally, protein interaction between IRS-1 and PI3-kinase was evaluated by immunoprecipitation, which showed that the expression levels of IRS-1-bound PI3-kinase were not changed in Nx rats or by spironolactone treatment (Figure 5h). These data indicated that insulin signaling was impaired at the level of the activation status of PI3-kinase in the adipose tissue of Nx rats. These changes were not observed in liver or muscle tissues of Nx rats (Supplementary Figure 1 online).Table 4Biochemical parameters of the rats in each groupShamNxNx+SpironolactoneTotal protein (g/dl)5.64±0.165.52±0.195.85±0.14Albumin (g/dl)3.50±0.133.26±0.113.52±0.08Creatinine (mg/dl)0.52±0.021.11±0.19**P<0.05 vs. sham,1.21±0.16**P<0.05 vs. sham,BUN (mg/dl)18.6±1.457.6±9.7**P<0.05 vs. sham,42.8±7.6**P<0.05 vs. sham,FBS (mg/dl)125±21111±20123±21Insulin (ng/ml)1.59±0.792.31±1.83**P<0.05 vs. sham,1.98±1.15#P<0.05 vs. Nx, n=10 in each group.Triglyceride (mg/dl)105±18102±1499.7±17.3Free fatty acid (mg/dl)0.214±0.0190.301±0.028**P<0.05 vs. sham,0.219±0.014#P<0.05 vs. Nx, n=10 in each group.Total cholesterol (mg/dl)55.4±2.5101±21*79.0±24Potassium (mEq/l)3.94±0.335.26±0.484.57±0.19Aldosterone (pg/ml)315±21362±17**P<0.05 vs. sham,421±28#P<0.05 vs. Nx, n=10 in each group.Urinary protein excretion (mg/day)613.2±73.81341.1±305.6**P<0.05 vs. sham,1167.3±147.4#P<0.05 vs. Nx, n=10 in each group.Abbreviations: BUN, blood urea nitrogen; FBS, fasting blood sugar.* *P<0.05 vs. sham,# P<0.05 vs. Nx, n=10 in each group. Open table in a new tab Download .jpg (.07 MB) Help with files Supplementary Figure 1 Abbreviations: BUN, blood urea nitrogen; FBS, fasting blood sugar. We next explored tissue MR activation in rat models of CKD. The nuclear expression levels of MR were elevated in adipose tissue in Nx rats and were attenuated by spironolactone (Figure 6a). The mRNA expressions of the MR target gene serum/glucocorticoid-regulated kinase 1 in the adipose tissue were upregulated in Nx rats, and this effect was also attenuated by spironolactone (Figure 6b). Fractionation of adipose tissue into stromal vascular fraction and adipocyte fraction revealed that MR activation in adipocytes contributed to IR in adipose tissues (Figure 6c). In adipose tissue, corticosterone rather than aldosterone might serve as a ligand of MR,19.Seckl J.R. Walker B.R. Minireview: 11beta-hydroxysteroid dehydrogenase type 1- a tissue-specific amplifier of glucocorticoid action.Endocrinology. 2001; 142: 1371-1376Crossref PubMed Scopus (558) Google Scholar although the tissue corticosterone contents were not different among the three experimental groups (Figure 6c, left panel). On the other hand, tissue aldosterone concentrations increased in Nx rats (Figure 6c, right panel) in parallel with increased plasma aldosterone concentrations (Table 4), which may have contributed to MR activation in the adipose tissue. The expression levels of CYP11B2, which were shown to be expressed in the adipose tissue and to locally synthesize aldosterone,20.Briones A.M. Nguyen Dinh Cat A. Callera G.E. et al.Adipocytes produce aldosterone through calcineurin-dependent signaling pathways: implications in diabetes mellitus-associated obesity and vascular dysfunction.Hypertension. 2012; 59: 1069-1078Crossref PubMed Scopus (240) Google Scholar increased in the adipose tissue in Nx rats (Figure 6d left panel). In adipose tissue, 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which converts cortisol or corticosterone into cortisone or 11-dehydrocorticosterone, blocks the MR activation by cortisol.21.Milagro F.I. Campión J. Martínez J.A. 11-Beta hydroxysteroid dehydrogenase type 2 expression in white adipose tissue is strongly correlated with adiposity.J Steroid Biochem Mol Biol. 2007; 104: 81-84Crossref PubMed Scopus (37) Google Scholar The expression levels of 11β-HSD2 were not different among the three experimental groups (Figure 6d, right panel). To elucidate how MR activation caused IR in the adipose tissue, we focused on the effects of ADMA accumulation in CKD by MR activation. Although plasma ADMA concentrations did not change (Figure 7a), the concentrations of ADMA in the adipose tissue increased in Nx rats and were decreased by spironolactone (Figure 7b). Consistent with the changes in tissue ADMA concentration, adipose tissue nitric oxide (NO) levels decreased in Nx rats, and this decrease was restored by spironolactone (Figure 7c). Tissue ADMA concentrations were regulated by its degrading enzymes DDAH1 and/or DDAH2. The expression levels of both isoforms in the adipose tissue were downregulated in Nx rats, and these decreases were ameliorated by spironolactone (Figure 7d). In addition, oxidative stress levels increased in adipose tissues of Nx rats, which was ameliorated by spironolactone (Figure 7e). This alteration might affect the DDAH expressions, as previously demonstrated.22.Tain Y.L. Kao Y.H. Hsieh C.S. et al.Melatonin blocks oxidative stress-induced increased asymmetric dimethylarginine.Free Radic Biol Med. 2010; 49: 1088-1098Crossref PubMed Scopus (57) Google Scholar Similarly, in mature adipocytes, both DDAH1 and DDAH2 expressions were decreased by aldosterone (Figure 7d). Insulin-stimulated phosphorylation of Akt decreased after pretreatment with ADMA in mature adipocytes (Figure 7e). Collectively, it is suggested that aldosterone-induced oxidative stress downregulated DDAH, leading to ADMA accumulation in adipose tissue, and the increase in ADMA reduced adipose tissue NO levels, which induced IR in CKD condition. The metabolic effects of MR antagonists in the clinical setting remained to be fully elucidated.23.Lastra-Lastra G. Sowers J.R. Restrepo-Erazo K. et al.Role of aldosterone and angiotensin II in insulin resistance: an update.Clin Endocrinol (Oxf). 2009; 71: 1-6Crossref PubMed Scopus (74) Google Scholar The effects of spironolactone on insulin sensitivity or lipid metabolism were examined in various clinical settings with various results.24.Costa M.B. Andrade Ezequiel D.G. Morais Lovis J.C. et al.Aldosterone antagonist decreases blood pressure and improves metabolic parameters in obese patients with the metabolic syndrome.J Clin Hypertens (Greenwich). 2010; 12: 753-755Crossref PubMed Scopus (8) Google Scholar, 25.Zulian E. Sartorato P. Benedini S. et al.Spironolactone in the treatment of polycystic ovary syndrome: effects on clinical features, insulin sensitivity and lipid profile.J Endocrinol Invest. 2005; 28: 49-53Crossref PubMed Google Scholar, 26.Garg R. Kneen L. Williams G.H. Adler G.K. Effect of mineralocorticoid receptor antagonist on insulin resistance and endothelial function in obese subjects.Diabetes Obes Metab. 2014; 16: 268-272Crossref PubMed Scopus (35) Google Scholar These discrepant results seem to depend on the different clinical settings including patients' backgrounds or treatment duration. Our study was the first to describe its beneficial metabolic effects in CKD patients. On the basis of our clinical observation, we further performed laboratory investigations to elucidate the molecular mechanism for IR in CKD. We first demonstrated MR activation in adipose tissues, a major insulin target organ, in CKD rats. The activation of MR in CKD condition was found in both huma