0022-3565/05/3122-718–725$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 312, No. 2 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 74088/1189055 JPET 312:718–725, 2005 Printed in U.S.A. GI262570, a Peroxisome Proliferator-Activated Receptor ␥ Agonist, Changes Electrolytes and Water Reabsorption from the Distal Nephron in Rats Lihong Chen, Baichun Yang, Judi A. McNulty, Lisa G. Clifton, Jane G. Binz, Angela M. Grimes, Jay C. Strum, W. Wallace Harrington, Zibin Chen, Thomas W. Balon, Stephen A. Stimpson, and Kathleen K. Brown Departments of Molecular Pharmacology (L.C., B.Y., J.A.M., L.G.C., T.W.B., S.A.S.), Metabolic Disease (J.G.B., W.W.H., K.K.B.), Biochemistry & Analytic Pharmacology (Z.C.), and Cellular Genomics (A.M.G., J.C.S.), GlaxoSmithKline, Inc., Research Downloaded from Triangle Park, North Carolina Received July 13, 2004; accepted October 7, 2004 ABSTRACT jpet.aspetjournals.org Peroxisome proliferator-activated receptor-␥ (PPAR␥) agonists treated rats had lower plasma levels of aldosterone compared have been shown to have significant therapeutic benefits such with vehicle-treated controls. mRNA levels for a group of genes as desirable glycemic control in type 2 diabetic patients; how- involved in distal nephron sodium and water absorption are ever, these agents may cause fluid retention in susceptible changed in the kidney medulla with GI262570 treatment. In individuals. Since PPAR␥ is expressed selectively in distal addition, due to a possible rebound effect on epithelial sodium nephron epithelium, we studied the mechanism of PPAR␥ ag- channel (ENaC) activity, a low dose of amiloride did not prevent onist-induced fluid retention using male Sprague-Dawley rats GI262570-induced fluid retention. On the contrary, the rebound treated with either vehicle or GI262570 (farglitazar), a potent effect after amiloride treatment potentiated GI262570-induced at ASPET Journals on January 18, 2020 PPAR␥ agonist. GI262570 (20 mg/kg/day) induced a plasma plasma volume expansion. This is at least partially due to a volume expansion. The plasma volume expansion was accom- synergistic effect of GI262570 and the rebound from amiloride panied by a small but significant decrease in plasma potassium treatment on ENaC␣ expression. In summary, our current data concentration. Small but significant increases in plasma so- suggest that GI262570 can increase water and sodium reab- dium and chloride concentrations were also observed. These sorption in distal nephron by stimulating the ENaC and Na,K- changes in serum electrolytes suggested an activation of the ATPase system. This may be an important mechanism for renal mineralocorticoid response system; however, GI262570- PPAR␥ agonist-induced fluid retention. Peroxisome proliferator-activated receptor-␥ (PPAR␥)isa iglitazone and pioglitazone, are being widely used as thera- member of the PPAR family of the nuclear receptor super- peutic agents for type 2 diabetics. family of ligand-dependent transcription factors (Evans, Most type 2 diabetics treated with rosiglitazone and pio- 1988; Desvergne and Wahli, 1999). PPAR␥ agonists, includ- glitazone tolerated the agents well, despite modest weight ing thiazolidinediones (TZDs), are novel insulin-sensitizing gain in a subpopulation of patients. Although adipogenesis in agents that primarily improve insulin sensitivity and in- the subcutaneous adipose tissue contributed to the weight crease glucose disposal in insulin-sensitive tissues in diabetic gain, it has been documented that some patients also had animals and patients (Evans, 1988; Desvergne and Wahli, increased plasma volume and fluid retention (Wang et al., 1999; Olefsky and Saltiel, 2000). Currently, two TZDs, ros- 2002; Mudaliar et al., 2003). In some cases, patients even developed peripheral edema or congestive symptoms, which is reversible after drug withdrawal (Wang et al., 2002; Ker- Article, publication date, and citation information can be found at mani and Garg, 2003; Tang et al., 2003). Both fluid retention http://jpet.aspetjournals.org. and overt clinical edema typically developed within the first doi:10.1124/jpet.104.074088. ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; TZD, thiazolidinedione; GFR, glomerular filtration rate; GI262570, farglitazar; AVP, arginine vasopressin; q.d., daily; RT, reverse transcription; qPCR, quantitative polymerase chain reaction; ANOVA, analysis of variance; SGK1, serum- and glucocorticoid-regulated kinase 1; GR, glucocorticoid receptor; AQP, aquaporin; ENaC, epithelial sodium channel; NPR-A, type A natriuretic peptide receptor; MR, mineralocorticoid receptor; 11␤-HSD, 11-␤-hydroxysteroid dehydrogenase; AVP-R2, type 2 arginine vaso- pressin receptor. 718 GI262570 and Renal Sodium Reabsorption 719 few months of drug administration (Niemeyer and Janney, tional group of rats were treated with vehicle or GI262570 for 10 2002; Kermani and Garg, 2003). The mechanism(s) of PPAR␥ days and then off the drug for 2 days (n ϭ 6). Study 4. Rats were agonist-induced fluid retention/edema is (are) presently unclear treated with vehicle or GI262570 (20 mg/kg/day) for 5 days with/ and may involve multiple factors/systems. A balance between without amiloride (1 mg/kg p.o., q.d.) (n ϭ 6 per group). Study 5. Rats intake and output of water and all electrolytes in the body is were treated with vehicle or GI262570 (20 mg/kg/day) for 4 days with/without amiloride (1 mg/kg p.o., q.d.) (n ϭ 6 per group) for 4 maintained in a large part by the kidney. It is reasonable to days. One GI262570-treated group was given amiloride only on the hypothesize that increased sodium and water retention at the 4th day. Study 6. Rats were treated with a single dose of amiloride ␥ renal level plays an important role in PPAR agonist-induced 6 h before sample collection (n ϭ 5 per group). fluid retention; however, the mechanism and the driving force Plasma Volume Measurement. Rats were treated with either that lead to excessive renal sodium and water retention re- vehicle or GI262570 (2 or 20 mg/kg q.d.) with or without amiloride (1 mains unclear. In our previous studies, we demonstrated that mg/kg q.d.) for 5 days (studies 1 and 4). Rats were anesthetized with GI262570, a potent nonthiozolidinedione PPAR␥ agonist (Will- isoflurane 2 h after the fifth dose. One jugular vein was catheterized son et al., 2000), caused slow accumulation of sodium (sodium for sample collection and dye injection. Blood (0.2 ϳ 0.3 ml) was intake Ͼ sodium excretion) (L. Chen, B. Yang, J. McNulty, L. collected for obtaining baseline plasma samples. Then 1 ml/kg of an Clifton, J. Binz, A. Grimes, J. Strum, W. Harrington, Z. Chen, Evans blue saline solution (2 mg/ml) was administered intrave- nously through the jugular vein cannulation. Five minutes later, 0.5 T. Balon, S. Stimpson and K. Brown, unpublished data) but did ml of blood was collected to measure Evans blue concentration in the not affect glomerular filtration rate (GFR), effective renal plasma. Two milliliters of blood were withdrawn for plasma electro- plasma flow, and renal filtration fraction (Yang et al., 2003). In lytes. Plasma Evans blue concentrations were determined according Downloaded from addition, GI262570-treated rats did not show significant to a standard curve generated by a serial dilution of Evans blue changes in renal hemodynamics compared with vehicle-treated saline solution (2 mg/ml). Plasma volume was calculated using the controls (Gardiner et al., 2004). It is likely that PPAR␥ agonists dilution factors of Evans blue. cause sodium and fluid retention via a direct effect on tubular Blood Sampling and Tissue Collection. Rats were anesthetized sodium reabsorption. with isoflurane 6 h after the administration of the drugs on the last day of treatment for studies 2, 3, 5, and 6. The abdominal cavity was opened Renal expression of PPARs has been investigated in many jpet.aspetjournals.org species (Guan et al., 1997; Yang et al., 1999); all three PPAR via a midline incision, and blood was collected immediately from the inferior vena cava using a heparinized syringe. The medullary portion isoforms have been identified in the nephron. PPAR␣ is pre- of the kidneys was carefully dissected out and placed immediately into dominantly expressed in the proximal tubules and medullary liquid nitrogen and subsequently stored at Ϫ80°C. Plasma was ob- ␦ thick ascending limbs, whereas PPAR is found in all seg- tained from the blood samples via centrifugation (10 min at 1000g). ments of nephron. PPAR␥ is selectively expressed in the Plasma electrolytes (sodium, potassium, and chloride) were measured medullary collecting duct and pelvic urothelium. This expres- using the Instrumentation Laboratory Ilab600 clinical chemistry ana- ␥ sion pattern suggests that PPAR may have effects on the lyzer (Instrumentation Laboratory, Lexington, MA). Plasma aldoste- at ASPET Journals on January 18, 2020 reabsorption of water and electrolytes in the distal nephron, rone concentration was measured using an enzyme immunoassay kit the segment of nephron that responds to the integrated ef- (Cayman Chemical, Ann Arbor, MI) following the manufacturer’s in- fects of multiple hormones such as aldosterone, arginine structions. To avoid the daily differences on plasma electrolytes and vasopressin (AVP), insulin, and atrial natriuretic peptide. aldosterone levels, each GI262570-treated group had its own vehicle control group from the same day. In this study, we characterized the renal effects of Tissue