Pharmacologic inhibition of the renal outer medullary potassium channel causes diuresis and natriuresis in the absence of kaliuresiss

The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter's syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics

GPR40 partial agonist MK-2305 lower fasting glucose in the Goto Kakizaki rat via suppression of endogenous glucose production

<div><p>GPR40 (FFA1) is a fatty acid receptor whose activation results in potent glucose lowering and insulinotropic effects <i>in vivo</i>. Several reports illustrate that GPR40 agonists exert glucose lowering in diabetic humans. To assess the mechanisms by which GPR40 partial agonists improve glucose homeostasis, we evaluated the effects of MK-2305, a potent and selective partial GPR40 agonist, in diabetic Goto Kakizaki rats. MK-2305 decreased fasting glucose after acute and chronic treatment. MK-2305-mediated changes in glucose were coupled with increases in plasma insulin during hyperglycemia and glucose challenges but not during fasting, when glucose was normalized. To determine the mechanism(s) mediating these changes in glucose metabolism, we measured the absolute contribution of precursors to glucose production in the presence or absence of MK-2305. MK-2305 treatment resulted in decreased endogenous glucose production (EGP) driven primarily through changes in gluconeogenesis from substrates entering at the TCA cycle. The decrease in EGP was not likely due to a direct effect on the liver, as isolated perfused liver studies showed no effect of MK-2305 <i>ex vivo</i> and GPR40 is not expressed in the liver. Taken together, our results suggest MK-2305 treatment increases glucose stimulated insulin secretion (GSIS), resulting in changes to hepatic substrate handling that improve glucose homeostasis in the diabetic state. Importantly, these data extend our understanding of the underlying mechanisms by which GPR40 partial agonists reduce hyperglycemia.</p></div

Effects of chronic treatment with MK-2305 in the GK rat.

<p>(A) Morning blood glucose levels in GK rats treated with vehicle, 10, or 30 mg/kg of MK-2305, or 10 mg/kg rosiglitazone for 20 days in feed. (B) Fasted blood glucose levels on days 7 and 14 of the study were significantly reduced with MK-2305 and rosiglitazone treatment compared to vehicle controls. (C) Fasted plasma insulin levels on days 7 and 14. (D) Effects on food intake and (E) body weight during the chronic study. (F) Plasma insulin levels during a OGTT in chronically treated rats on day 13. Changes in blood glucose, food intake or body weight over time with MK-2305 or rosiglitazone vs. vehicle were analyzed by two-way ANOVA with repeated measures followed by Tukeys post hoc analysis. Changes in fasted glucose or insulin of glucose AUC were analyzed by one way ANOVA comparing MK-2305 or rosiglitazone treatments with vehicle followed by Dunnetts post hoc analysis. *p<0.05, **p<0.01.</p

Effects of MK-2305 on glucose metabolism in perfused mouse livers.

<p>Effect of 10 μM MK-2305 or DMSO on the conversion of [2-¹³C] pyruvate to ¹³C-glucose, ¹³C-glycogen, and ¹³C-lactate in perfused db/db mouse livers. MK-2305 treatments were compared to vehicle for each endpoint via students ttest.</p

In vitro and ex-vivo pharmacology of MK-2305.

<p>(A) Structure of the synthetic GPR40 partial agonist MK-2305. (B) Dose-response curves for MK-2305 were generated monitoring IP accumulation in CHO cells expressing rat GPR40. Data are expressed as a percentage of the control response of an in-house partial agonist, and fitted to a standard 4-parameter non-linear regression model. EC₅₀’s were determined for each test compound using a custom in-house developed software package. Each experiment was multiple times with a representative graph shown. The mean parameters of these and other individual experiments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176182#pone.0176182.t001" target="_blank">Table 1</a>. (C) Effect of MK-2305 on GSIS in isolated GPR40 WT and KO islets under high (15 mM) and not basal (2 mM) glucose. Data provided are means +/- SEM. Data were analyzed via ANOVA followed by Bonferroni multiple comparisons test. **p<0.01compared to DMSO treated islets under 15 mM glucose.</p

Uptake and metabolic conversion of [1-¹³C] glucose in liver (A) and muscle (B) following acute treatment with MK-2305 or vehicle.

<p>Data were analyzed via students ttest comparing MK-2305 treatment vs. vehicle for each metabolite in each tissue measured.</p

Effect of (A) acute treatment and (B) chronic treatment with 10 mg/kg MK-2305 or vehicle on endogenous glucose production (EGP) from various substrates in the GK or WKY rats.

<p>Data were analyzed via ANOVA followed by Tukey’s multiple comparisons test comparing MK-2305 treatment to vehicle within GK or WKY rats or comparing WKY rats treated with vehicle to GK rats treated with vehicle. *p< 0.05, **p<0.01, ***p<0.005, ****p<0.001.</p

<i>In vitro</i> pharmacology of MK-2305.

<p><i>In vitro</i> pharmacology of MK-2305.</p

GPR40 partial agonists and AgoPAMs: Differentiating effects on glucose and hormonal secretions in the rodent

<div><p>GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut. Here we describe how GPR40 AgoPAMs stimulate both insulin and incretin secretion in vivo over time in diabetic GK rats. We also describe effects of AgoPAMs in vivo to lower glucose and body weight beyond what is seen with partial GPR40 agonists in both the acute and chronic setting. Further comparisons of the glucose lowering profile of AgoPAMs suggest these compounds may possess greater glucose control even in the presence of elevated glucagon secretion, an unexpected feature observed with both acute and chronic treatment with AgoPAMs. Together these studies highlight the complexity of GPR40 pharmacology and the potential additional benefits AgoPAMs may possess above partial agonists for the diabetic patient.</p></div