Translational advances of melanocortin drugs: Integrating biology, chemistry and genetics.

Melanocortin receptors have emerged as important targets with a very unusual versatility, as their widespread distribution on multiple tissues (e.g. skin, adrenal glands, brain, immune cells, exocrine glands) together with the variety of physiological processes they control (pigmentation, cortisol release, satiety mechanism, inflammation, secretions), place this family of receptors as genuine therapeutic targets for many disorders. This review focuses in the journey of the development of melanocortin receptors as therapeutic targets from the discovery of their existence in the early 1990 s to the approval of the first few drugs of this class. Two major areas of development characterise the current state of melanocortin drug development: their role in obesity, recently culminated with the approval of setmelanotide, and their potential for the treatment of chronic inflammatory and autoimmune diseases like rheumatoid arthritis, multiple sclerosis or fibrosis. The pro-resolving nature of these drugs offers the advantage of acting by mimicking the way our body naturally resolves inflammation, expecting fewer side effects and a more balanced (i.e. non-immunosuppressive) response from them. Here we also review the approaches followed for the design and development of novel compounds, the importance of the GPCR nature of these receptors in the process of drug development, therapeutic value, current challenges and successes, and the potential for the implementation of precision medicine approaches through the incorporation of genetics advances.

Diet-induced hypertension in rats is associated with increased renal vasoconstrictor response to angiotensin II after imitated endothelial dysfunction.

The mechanisms behind development of diet-induced hypertension remain unclear. The kidneys play a paramount role in blood volume and blood pressure regulation. Increases in renal vascular resistance lead to increased mean arterial blood pressure (MAP) due to reduced glomerular filtration rate and Na+ excretion. Renal vascular resistance may be increased by several factors, e.g. sympathetic output, increased activity in the renin-angiotensin system or endothelial dysfunction. We examined if a 14-week diet rich in fat, fructose or both led to increased renal vascular resistance and blood pressure. Sixty male Sprague-Dawley rats received normal chow (Control), high-fat chow (High Fat), high-fructose in drinking water (High Fructose), or a combination of high-fat and high-fructose diet (High Fat + Fruc) for 14 weeks from age 4-weeks. Measurements included body weight (BW), telemetry blood pressures, renal blood flow in anesthetized rats, plasma concentrations of atrial natriuretic peptide and glucose, as well as vessel myography in renal segmental arteries. Body weight increased in both groups receiving high fat, whereas MAP increased only in the High Fat + Fruc group. Renal blood flow did not differ between groups showing that renal vascular resistance was not increased by the diets. After inhibiting nitric oxide and prostacyclin production, renal blood flow reductions to Angiotensin II infusions were exaggerated in the groups receiving high fructose. MAP correlated positively with heart rate in all rats tested. Our data suggest that diet-induced hypertension is not caused by an increase in renal vascular resistance. The pathophysiological mechanisms may include altered signaling in the renin-angiotensin system and increases in central sympathetic output in combination with reduced baroreceptor sensitivity leading to increased renal vasoconstrictor responses.

Biased agonism as a novel strategy to harness the proresolving properties of melanocortin receptors without eliciting melanogenic effects.

There is a need for novel approaches to control pathologies with overexuberant inflammatory reactions. Targeting melanocortin (MC) receptors represents a promising therapy for obesity and chronic inflammation, but lack of selectivity and safety concerns limit development. A new way to increase selectivity of biological effects entails the identification of biased agonists. In this study, we characterize the small molecule AP1189 as a biased agonist at receptors MC1 and MC3. Although not provoking canonical cAMP generation, AP1189 addition to MC1 or MC3, but not empty vector, transfected HEK293 cells caused ERK1/2 phosphorylation, a signaling responsible for the proefferocytic effect evoked in mouse primary macrophages. Added to macrophage cultures, AP1189 reduced cytokine release, an effect reliant on both MC1 and MC3 as evident from the use of Mc1r(-/-) and Mc3r(-/-) macrophages. No melanogenesis was induced by AP1189 in B16-F10 melanocytes. In vivo, oral AP1189 elicited anti-inflammatory actions in peritonitis and, upon administration at the peak of inflammation, accelerated the resolution phase by ∼3-fold. Finally, given the clinical efficacy of adrenocorticotropin in joint diseases, AP1189 was tested in experimental inflammatory arthritis, where this biased agonist afforded significant reduction of macroscopic and histological parameters of joint disruption. These proof-of-concept analyses with AP1189, an active oral anti-inflammatory and resolution-promoting compound, indicate that biased agonism at MC receptors is an innovative, viable approach to yield novel anti-inflammatory molecules endowed with a more favorable safety profile.

Congestive heart failure in rats is associated with increased collecting duct vasopressin sensitivity and vasopressin type 2 receptor reexternalization.

A number of studies have shown that rats with congestive heart failure (CHF) have increased protein levels of the vasopressin (AVP)-regulated water channel aquaporin-2 (AQP2) even during conditions with unchanged circulating levels of AVP, suggesting an increase in the sensitivity of the AVP type 2 (V2) receptor in experimental CHF. The present study was aimed at investigating AVP signaling in rats with moderate CHF (left ventricular end diastolic pressure >10 mmHg; normal plasma AVP levels) induced by ligation of the left anterior descending coronary artery. Sham-operated rats were used as controls. Western blotting analyses revealed an increased abundance of AQP2 in renal cortex (+33 ± 9% of sham; P < 0.05) and in inner medulla (IM) (+54 ± 15% of sham; P < 0.05) in CHF rats compared with sham-operated controls. Dose-response studies on isolated collecting ducts (CDs) showed an increased accumulation of cAMP in response to AVP in CHF rats compared with controls. V2 receptor surface-binding studies in isolated IMCDs showed a marked and comparable AVP-induced V2 receptor internalization in response to AVP in both CHF and control rats. As expected V2 receptor surface binding remained low after AVP challenge in control rats. In contrast to this, V2 receptor surface binding returned to pre-AVP levels within 30 min in the CHF rats, indicating an obtained recycling ability of the V2 receptor in CHF. Together the results indicate the presence of an increased AVP sensitivity in the CDs from CHF rats, associated with an acquired recycling ability of the V2 receptor.

The melanocortin agonist AP214 exerts anti-inflammatory and proresolving properties.

Synthetic and natural melanocortin (MC) peptides afford inhibitory properties in inflammation and tissue injury, but characterization of receptor involvement is still elusive. We used the agonist AP214 to test MC-dependent anti-inflammatory effects. In zymosan peritonitis, treatment of mice with AP214 (400 to 800 µg/kg) inhibited cell infiltration, an effect retained in MC receptor type 1, or MC(1), mutant mice but lost in MC(3) null mice. In vitro, cytokine release from zymosan-stimulated macrophages was affected by AP214, with approximately 80%, 30%, and 40% reduction in IL-1ß, tumor necrosis factor-α, and IL-6, respectively. Inhibition of IL-1ß release was retained in MC(1) mutant cells but was lost in MC(3) null cells. Furthermore, AP214 augmented uptake of zymosan particles and human apoptotic neutrophils by wild-type macrophages: this proresolving property was lost in MC(3) null macrophages. AP214 displayed its pro-efferocytotic effect also in vivo. Finally, in a model of inflammatory arthritis, AP214 evoked significant reductions in the clinical score. These results indicate that AP214 elicits anti-inflammatory responses, with a preferential effect on IL-1ß release. Furthermore, we describe for the first time a positive modulation of an MC agonist on the process of efferocytosis. In all cases, endogenous MC(3) is the receptor that mediates these novel properties of AP214. These findings might clarify the tissue-protective properties of AP214 in clinical settings and may open further development for novel MC agonists.

Downregulation of aquaporin-1 in alveolar microvessels in lungs adapted to chronic heart failure.

The threshold pressure for lung edema formation is increased in severe chronic heart failure (CHF) due to reduced microvascular permeability. The water channel aquaporin-1 (AQP1) is present in the pulmonary microvascular endothelium, and a number of studies suggest the importance of AQP1 as a molecular determinant of pulmonary microvascular water transport. The present study examined the abundance and localization of AQP1 in lungs from rats with CHF. We used two different models of CHF: ligation of the left anterior descending coronary artery (LAD ligation) and aorta-banding (AB). Sham-operated rats served as controls. Echocardiographic verification of left ventricular dysfunction, enhanced left ventricular end-diastolic pressure, and right ventricular hypertrophy confirmed the presence of CHF. Western blotting of whole-lung homogenates revealed significant downregulation of AQP1 in LAD-ligated rats (24 h: 58 ± 5% of sham; 3 weeks: 8 ± 3% of sham; 9 weeks: 16 ± 6% of sham) and after AB (30 weeks: 37 ± 5% of sham), whereas the protein levels of the specific endothelial cell marker PECAM-1 was increased 3 weeks after LAD ligation (229 ± 20% of sham), but unchanged after 9 weeks and in the AB rats compared to controls. Immunohistochemical examination 3 weeks after LAD ligation showed intact labeling of PECAM-1 but an almost complete absence of AQP1 in the pulmonary alveolar microvessels in the CHF rats. These results suggest that downregulation of AQP1 in the alveolar microvessels may act as a compensatory mechanism to protect against formation of excessive pulmonary edema in CHF.

The α-MSH analogue AP214 attenuates rise in pulmonary pressure and fall in ejection fraction in lipopolysaccharide-induced systemic inflammatory response syndrome in pigs.

BACKGROUND: The effect of an α-melanocyte stimulating hormone (α-MSH) analogue (AP214) on experimentally endotoxin-induced systemic inflammatory response syndrome (SIRS) was studied, because α-MSH in rodent models has shown promise in attenuating inflammatory response markers and associated organ damage in SIRS. SIRS is associated with considerable morbidity and mortality. Consequently, new treatment modalities are still warranted to address the different aspects of the pathophysiological process. METHODS: SIRS was induced by lipopolysaccharide (LPS) (Escherichia coli endotoxin) infusion in anaesthetized Danish Landrace pigs (20-25 kg). The pigs received an α-MSH analogue (AP214) or saline as a bolus at the initiation of the LPS infusion. The hemodynamic response was registered as well as echocardiographic indices of left ventricular function. RESULTS: The cardiovascular response was recorded together with echocardiographic indices of left ventricular function in control and in intervention animals. AP214 reduced the early peak in pulmonary pressure and pulmonary vascular resistance by approximately 33%. Furthermore, AP214 prevented the decline in left ventricular fractional shortening as observed in the control group. Mean change and standard deviation in fractional shortening (ΔFS) in control group: - 7·3 (4·7), AP214 (low dose): 0·9 (8·2) and AP214 (high dose) 4·1 (6·0), P < 0·05 for both intervention groups versus control. CONCLUSIONS: In the porcine model, the peak increase in pulmonary pressure was attenuated, and the LPS-induced decline in left ventricular function was prevented.

Nociceptin/orphanin FQ peptide receptor agonist Ac-RYYRWKKKKKKK-NH2 (ZP120) induces antinatriuresis in rats by stimulation of amiloride-sensitive sodium reabsorption.

The aim of the present study was to examine the mechanisms responsible for the antinatriuretic effect of the selective, peripherally acting, nociceptin/orphanin FQ peptide (NOP) receptor partial agonist Ac-RYYRWKKKKKKK-NH(2) (ZP120). Using immunohistochemistry, we showed that in the cortex NOP receptors are expressed in the distal convoluted tubules, the connecting tubules, and the collecting ducts. Using clearance techniques, we evaluated renal excretory function during acute administration of ZP120 (1 nmol/kg/min) in chronically catheterized, conscious rats (n = 8/group). To examine the hypothesis that ZP120 induces direct renal effects by modifying the activity of sodium transporters in the distal convoluted tubules or in the collecting ducts, ZP120-induced antinatriuresis was examined during coadministration of an inhibitor of the NaCl cotransporter, bendroflumethiazide, or a blocker of the epithelial sodium channel, amiloride, respectively. ZP120 produced a marked antinatriuresis [fractional excretion of sodium (FE(Na)): ZP120, 0.3 +/- 0.1% versus control, 0.9 +/- 0.1%; p < 0.05] in sodium-replete rats. The natriuretic response to amiloride was significantly increased in ZP120-treated rats compared with controls (DeltaFE(Na): ZP120, 1.1 +/- 0.2% versus control, 0.5 +/- 0.2%; p < 0.01), whereas the effect of BFTZ was equal in ZP120-treated rats and controls. These results suggest that ZP120 exerts a direct renal NOP receptor-mediated stimulatory effect on the epithelial sodium channel in the collecting ducts.

Expression of aquaporin 9 in rat liver and efferent ducts of the male reproductive system after neonatal diethylstilbestrol exposure.

Aquaporins (AQP) have important solute transport functions in many tissues including the epididymal efferent ducts (ED) and in the liver. We investigated the effect of neonatal exposure to diethylstilbestrol (DES) on AQP9 expressions in the ED and in the liver of rats. DES was administered from day 2 to day 20 postnatally at a dose of 4,8 microg/day, and AQP9 protein and mRNA were measured by immunoblotting and real-time PCR, respectively, along with immunohistochemistry. DES caused hepatic downregulation of AQP9 at both the protein and mRNA level; however, decreased AQP9 labeling was only observed in the periportal zone. In the ED, AQP9 protein expression was increased in the DES-treated animals by 300% that could be ascribed to a widening of the ED lumen, whereas no difference was observed in AQP9 mRNA expression. Immunohistochemical findings revealed that AQP9 expression was confined to the epithelial cells of the ED. In conclusion, neonatal DES exposure appears to upregulate AQP9 channels in the ED in male rats, whereas a downregulation in the hepatic expression was observed, particularly in the periacinous area.

Differential down-regulation of aquaporin-2 in rat kidney zones by peripheral nociceptin/orphanin FQ receptor agonism and vasopressin type-2 receptor antagonism.

We previously showed that aquaresis induced by the peripherally acting nociceptin/orphanin FQ receptor agonist ZP120 is associated with a decreased protein level of aquaporin-2 (AQP2) in whole-kidney homogenates. We now examined the effects of Ac-RYYRWKKKKKKK-NH(2) (ZP120) (1 nmol/kg/min i.v. for 4 h) on renal regional expression (cortex/outer stripe of outer medulla, inner stripe of outer medulla, and inner medulla) and subcellular localization of aquaporin-2. Responses to ZP120 were compared to the effects of an equi-aquaretic dose ( approximately 40% inhibition of distal water reabsorption) of the vasopressin type-2 receptor antagonist 5-dimethylamine-1-[4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro-1H-benzapine (OPC31260) (32 nmol/kg/min). ZP120 decreased the aquaporin-2 protein level in the rat cortex/outer stripe of outer medulla and decreased apical plasma membrane localization of aquaporin-2 in the cortex (P = 0.002) and in the inner medulla (P = 0.06). These effects were not accompanied by a decrease in the aquaporin-2 mRNA level. OPC31260-induced aquaresis was associated with a decreased aquaporin-2 protein level in both the cortex/outer stripe of outer medulla and in the inner stripe of outer medulla. Apical localization of aquaporin-2 was decreased throughout all kidney zones, and OPC31260 decreased the AQP2 mRNA level in the inner medulla. We conclude that equi-aquaretic doses of ZP120 and OPC31260 produce different patterns of aquaporin-2 down-regulation, suggesting different signaling pathways.

Intrarenal octreotide treatment prevents sodium retention in liver cirrhotic rats: evidence for direct effects within the thick ascending limb of Henle's loop.

We have previously shown that systemic treatment with the somatostatin analog octreotide has marked beneficial effects on renal function in rats with liver cirrhosis induced by common bile duct ligation (CBL; Jonassen TEN, Christensen S, Sørensen AM, Marcussen N, Flyvbjerg A, Andreasen F, and Petersen JS. Hepatology 29: 1387-1395, 1999). In the present study, we tested the hypothesis that octreotide has a direct effect on renal tubular function. Rats (CBL or Sham-CBL) were intrarenally treated with low-dose octreotide in a long-acting release formulation, which had no systemic actions (100 microg/kg body wt as a single dose). Rats receiving low-dose octreotide (sc) were used as controls. The rats were chronically instrumented, and renal function was examined 4 wk after CBL or Sham-CBL. Intrarenal octreotide administration (IROA) prevented sodium retention in CBL rats without changes in renal plasma flow, glomerular filtration rate, or circulating levels of aldosterone and vasopressin. Renal clearance studies revealed that IROA normalized the increased natriuretic efficacy of furosemide found in CBL rats. Furthermore, IROA protected against the development of hypertrophy of the inner stripe of the outer medulla and thereby the increased the volume of thick ascending limb of Henle's loop (TAL) epithelium found in CBL rats. Finally, Western blot analyses of outer medullary homogenates showed increased abundance of the furosemide-sensitive Na-K-2Cl (NKCC2) cotransporter. IROA did not affect the abundance of NCKK2 within the outer medulla. Together with the histological findings, these results indicate that IROA reduces the total number of NKCC2 within the outer medulla. In conclusion, the results indicate a direct intrarenal effect of octreotide on TAL function and morphology in cirrhotic rats.

Biphasic effects of ANP infusion in conscious, euvolumic rats: roles of AQP2 and ENaC trafficking.

Atrial natriuretic peptide (ANP) acutely promotes water and sodium excretion, whereas subchronic effects involve water retention. Renal hemodynamics, water and sodium excretion, and aquaporin-2 (AQP2) and epithelial Na channel (ENaC) subcellular trafficking were determined in response to continuous ANP infusion in conscious rats, where body sodium and fluid balance was constantly maintained. ANP (0.5 microg x kg(-1) x min(-1)) evoked a transient (peak at 10 min) fivefold diuresis followed by reduced urine production to control levels (30- to 90-min period). The fractional distal water excretion was significantly increased initially and then decreased in response to ANP. There was no change in the subcellular localization of AQP2 and AQP2 phosphorylated in PKA consensus site S256 (p-AQP2) 10 min after ANP infusion. In contrast, after 90 min a marked increase in apical labeling of AQP2 and p-AQP2 was observed in the inner and outer medullary collecting ducts but not in cortical collecting ducts. In support of this, ANP induced plasma membrane targeting of AQP2 in transiently AQP2-transfected cells. ANP infusion evoked an instant increase in renal sodium excretion, which persisted for 90 min. Ten minutes of ANP infusion induced no changes in the subcellular localization of ENaC subunits, whereas a marked increase in apical targeting of alpha- and gamma-subunits was observed after 90 min. In conclusion, 1) ANP infusion induced a sustained natriuresis and transient diuresis; 2) there were no changes in the subcellular localization of AQP2 and ENaC subunits after 10 min of ANP infusion; and 3) there was a marked increase in apical targeting of AQP2, p-AQP2, and alpha- and gamma-ENaC after 90 min of ANP infusion. The increased targeting of ENaC and AQP2 likely represents direct or compensatory effects to increase sodium and water reabsorption and to prevent volume depletion in response to prolonged ANP infusion.

Increased apical targeting of renal epithelial sodium channel subunits and decreased expression of type 2 11beta-hydroxysteroid dehydrogenase in rats with CCl4-induced decompensated liver cirrhosis.

It was hypothesized that dysregulation of renal epithelial sodium channel (ENaC) subunits and/or 11beta-hydroxysteroid dehydrogenase (11betaHSD2) may play a role in the increased sodium retention in liver cirrhosis (LC). Experimental LC was induced in rats by CCl(4) (1 ml/kg, intraperitoneally, twice a week) for 12 wk (protocol 1) or for 11 wk (protocol 2). In both protocols, one group of rats with cirrhosis showed significantly decreased urinary sodium excretion and urinary Na/K ratio (group A), whereas a second group exhibited normal urinary sodium excretion (group B) compared with controls, even though extensive ascites was seen in both groups of rats with cirrhosis. In group A, protein abundance of alpha-ENaC was unchanged, whereas beta-ENaC abundance was decreased in the cortex/outer stripe of outer medulla compared with controls. The gamma-ENaC underwent a complex change associated with increased abundance of the 70-kD band with a concomitant decrease in the main 85-kD band, corresponding to an aldosterone effect. In contrast, no changes in the abundance of ENaC subunit were observed in group B. Immunoperoxidase microscopy revealed an increased apical targeting of alpha-, beta-, and gamma-ENaC subunits in distal convoluted tubule (DCT2), connecting tubule (CNT), and cortical and medullary collecting duct segments in group A but not in group B. Immunolabeling intensity of 11betaHSD2 in the DCT2, CNT, and cortical collecting duct was significantly reduced in group A but not in group B, and this was confirmed by immunoblotting. In conclusion, increased apical targeting of ENaC subunits combined with diminished abundance of 11betaHSD2 in the DCT2, CNT, and cortical collecting duct is likely to play a role in the sodium retaining stage of liver cirrhosis.

Uncoupling of vasopressin signaling in collecting ducts from rats with CBL-induced liver cirrhosis.

Vasopressin (AVP) stimulates collecting duct water reabsorption through cAMP-mediated membrane targeting and increased expression of the aquaporin-2 (AQP2) water channel. Rats with liver cirrhosis induced by common bile duct ligation (CBL) show decreased protein expression of AQP2 despite increased plasma concentrations of AVP. The present study was conducted to investigate possible mechanisms behind this uncoupling of AVP signaling. The rats were examined 4 wk after CBL or sham operation. The CBL rats had increased plasma AVP concentrations (CBL: 3.2 +/- 0.2 vs. sham: 1.4 +/- 0.4 pg/ml, P < 0.05) and reduced AQP2 (0.62 +/- 0.11) and phosphorylated AQP2 (0.50 +/- 0.06) protein expression compared with sham-operated rats. However, examination of subcellular AQP2 localization by immunohistochemistry showed unchanged plasma membrane targeting in CBL rats, indicating a sustained ability of AQP2 short-term regulation. In a separate series of animals, thirsting was found to normalize AQP2 expression, indicating that AVP uncoupling in CBL rats is a physiological compensatory mechanism aimed at avoiding dilutional hyponatremia. Studies on microdissected collecting ducts from CBL rats showed decreased cAMP accumulation in response to AVP stimulation. The presence of the nonspecific phosphodiesterase inhibitor IBMX normalized the cAMP accumulation, indicating that cAMP-phosphodiesterase activity is increased in CBL rats. However, in contrast to this, Western blotting showed a decreased expression of several phosphodiesterase splice variants. We conclude that CBL rats develop an escape from AVP to prevent the formation of dilutional hyponatremia in response to increased plasma AVP concentrations. The mechanism behind AVP escape seems to involve decreased collecting duct sensitivity to AVP as a result of increased cAMP-phosphodiesterase activity.

Opioid receptor-like 1 stimulation in the collecting duct induces aquaresis through vasopressin-independent aquaporin-2 downregulation.

Nociceptin, the endogenous ligand of the inhibitory G protein-coupled opioid receptor-like 1 receptor, produces aquaresis (i.e., increases the excretion of solute-free urine) in rats. However, the mechanism underlying this effect has not yet been explained. Using immunohistochemistry, we found the opioid receptor-like 1 receptor in the rat kidney colocalized with the vasopressin-regulated water channel aquaporin-2 in inner medullary collecting ducts. We investigated the aquaretic effect of opioid receptor-like 1 receptor stimulation by infusing the selective nociceptin analog ZP120C; volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 50 mM glucose. ZP120C induced a marked and sustained aquaresis in normal and congestive heart failure rats in the absence of changes in vasopressin plasma concentrations. The ZP120C-induced aquaresis was associated with downregulation of the aquaporin-2 protein level in both rat groups, suggesting that opioid receptor-like 1 receptor stimulation produces aquaresis by inhibiting the vasopressin type-2 receptor-mediated stimulation on collecting duct water reabsorption. However, substantial amounts of PKA-mediated serine 256 phosphorylated aquaporin-2 were still present after 4 h of ZP120C treatment. Furthermore, neither preincubation with nociceptin nor ZP120C inhibited vasopressin-mediated cAMP accumulation in isolated collecting ducts. We conclude that renal opioid receptor-like 1 receptor stimulation in normal and congestive heart failure rats produces aquaresis by a direct renal effect, via aquaporin-2 downregulation, through a mechanism not involving inhibition of vasopressin type-2 receptor-mediated cAMP production.

Identification of a core set of genes that signifies pathways underlying cardiac hypertrophy.

Although the molecular signals underlying cardiac hypertrophy have been the subject of intense investigation, the extent of common and distinct gene regulation between different forms of cardiac hypertrophy remains unclear. We hypothesized that a general and comparative analysis of hypertrophic gene expression, using microarray technology in multiple models of cardiac hypertrophy, including aortic banding, myocardial infarction, an arteriovenous shunt and pharmacologically induced hypertrophy, would uncover networks of conserved hypertrophy-specific genes and identify novel genes involved in hypertrophic signalling. From gene expression analyses (8740 probe sets, n = 46) of rat ventricular RNA, we identified a core set of 139 genes with consistent differential expression in all hypertrophy models as compared to their controls, including 78 genes not previously associated with hypertrophy and 61 genes whose altered expression had previously been reported. We identified a single common gene program underlying hypertrophic remodelling, regardless of how the hypertrophy was induced. These genes constitute the molecular basis for the existence of one main form of cardiac hypertrophy and may be useful for prediction of a common therapeutic approach. Supplementary material for this article can be found at: http://www.interscience.wiley.com/jpages/1531-6912/suppmat.

Chronic nitric oxide synthase inhibition exacerbates renal dysfunction in cirrhotic rats.

The present study investigated sodium balance and renal tubular function in cirrhotic rats with chronic blockade of the nitric oxide (NO) system. Rats were treated with the nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) starting on the day of common bile duct ligation (CBL). Three weeks of daily sodium balance studies showed that CBL rats developed sodium retention compared with sham-operated rats and that l-NAME treatment dose dependently deteriorated cumulative sodium balance by reducing urinary sodium excretion. Five weeks after CBL, renal clearance studies were performed, followed by Western blotting of the electroneutral type 3 sodium/proton exchanger (NHE3) and the Na-K-ATPase present in proximal tubules. Untreated CBL rats showed a decreased proximal reabsorption with a concomitant reduction of NHE3 and Na-K-ATPase levels, indicating that tubular segments distal to the proximal tubules were responsible for the increased sodium reabsorption. l-NAME-treated CBL rats showed an increased proximal reabsorption measured by the lithium clearance method and showed a marked increase in NHE3 and Na-K-ATPase protein levels. Our results show that chronic l-NAME treatment exacerbates the sodium retention found in CBL rats by a significant increase in proximal tubular reabsorption.

Effects of renal denervation on tubular sodium handling in rats with CBL-induced liver cirrhosis.

This study was designed to examine the effect of bilateral renal denervation (DNX) on thick ascending limb of Henle's loop (TAL) function in rats with liver cirrhosis induced by common bile duct ligation (CBL). The CBL rats had, as previously shown, sodium retention associated with hypertrophy of the inner stripe of the outer medulla (ISOM) and increased natriuretic effect of furosemide in vivo, and semiquantitative immunoblotting showed increased expression of the furosemide-sensitive Na-K-2Cl cotransporter type 2 (NKCC2) in ISOM from CBL rats. DNX significantly attenuated the sodium retention in the CBL rats, which was associated with normalization of the natriuretic effect of furosemide, as well as a significant reduction in the expression of NKCC2 in the ISOM. However, the marked hypertrophy of the ISOM found in CBL rats was not reversed by DNX. Together, these data indicate that increased renal sympathetic nerve activity known to be present in CBL rats plays a significant role in the formation of sodium retention by stimulating sodium reabsorption in the TAL via increased renal abundance of NKCC2.

Lipopolysaccharide-induced acute renal failure in conscious rats: effects of specific phosphodiesterase type 3 and 4 inhibition.

In conscious, chronically instrumented rats we examined 1) renal tubular functional changes involved in lipopolysaccharide (LPS)-induced acute renal failure; 2) the effects of LPS on the expression of selected renal tubular water and sodium transporters; and 3) effects of milrinone, a phosphodiesterase type 3 (PDE3) inhibitor, and Ro-20-1724, a PDE4 inhibitor, on LPS-induced changes in renal function. Intravenous infusion of LPS (4 mg/kg b.wt. over 1 h) caused an immediate decrease in glomerular filtration rate (GFR) and proximal tubular outflow without changes in mean arterial pressure (MAP). LPS-induced fall in GFR and proximal tubular outflow were sustained on day 2. Furthermore, LPS-treated rats showed a marked increase in fractional distal water excretion, despite significantly elevated levels of plasma vasopressin (AVP). Semiquantitative immunoblotting showed that LPS increased the expression of the Na(+),K(+),2Cl(-)-cotransporter (BSC1) in the thick ascending limb, whereas the expression of the AVP-regulated water channel aquaporin-2 in the collecting duct (CD) was unchanged. Pretreatment with milrinone or Ro-20-1724 enhanced LPS-induced increases in plasma tumor necrosis factor-alpha and lactate, inhibited the LPS-induced tachycardia, and exacerbated the acute LPS-induced fall in GFR. Furthermore, Ro-20-1724-treated rats were unable to maintain MAP. We conclude 1) PDE3 or PDE4 inhibition exacerbates LPS-induced renal failure in conscious rats; and 2) LPS treated rats develop an escape from AVP in the CDs, which could be aimed to protect against water intoxication in septic conditions associated with decreased GFR and high levels of AVP.