Thomas E. N. Jonassen

AP214, an analogue of α-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality

Sepsis remains a serious problem in critically ill patients with the mortality increasing to over half when there is attendant acute kidney injury. alpha-Melanocyte-stimulating hormone is a potent anti-inflammatory cytokine that inhibits many forms of inflammation including that with acute kidney injury. We tested whether a new alpha-melanocyte-stimulating hormone analogue (AP214), which has increased binding affinity to melanocortin receptors, improves sepsis-induced kidney injury and mortality using a cecal ligation and puncture mouse model. In the lethal cecal ligation-puncture model of sepsis, severe hypotension and bradycardia resulted and AP214 attenuated acute kidney injury of the lethal model with a bell-shaped dose-response curve. An optimum AP214 dose reduced acute kidney injury even when it was administered 6 h after surgery and it significantly improved blood pressure and heart rate. AP214 reduced serum TNF-alpha and IL-10 levels with a bell-shaped dose-response curve. Additionally; NF-kappaB activation in the kidney and spleen, and splenocyte apoptosis were decreased by the treatment. AP214 significantly improved survival in both lethal and sublethal models. We have shown that AP214 improves hemodynamic failure, acute kidney injury, mortality and splenocyte apoptosis attenuating pro- and anti-inflammatory actions due to sepsis.

Glucagon-like peptide-1 (GLP-1): effect on kidney hemodynamics and renin-angiotensin-aldosterone system in healthy men.

INTRODUCTION Glucagon-like peptide-1 (GLP-1) is an incretin hormone with multiple actions in addition to control of glucose homeostasis. GLP-1 is known to cause natriuresis in humans, but the effects on basic renal physiology are still partly unknown. SUBJECTS AND METHODS Twelve healthy young males were examined in a randomized, controlled, double-blinded, single-day, crossover trial to evaluate the effects of 2 hours GLP-1 infusion on kidney functions. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were assessed with (51)Cr-EDTA and (123)I-hippuran, respectively, using a constant infusion renal clearance technique based on timed urine sampling. RESULTS GLP-1 had no significant effect on either GFR [+1.9%, 95% confidence interval (-0.8; 4.6%)] or RPF [+2.4%, 95% confidence interval (-3.6; 8.8%)]. Fractional urine excretion of lithium increased 9% (P = .013) and renal sodium clearance increased 40% (P = .007). Angiotensin II decreased 19% (P = .003), whereas renin, aldosterone, and the urinary excretion of angiotensinogen showed no significant changes. glp-1 did not affect blood pressure but induced a small transient increase in heart rate. CONCLUSION The results indicate that although GLP-1 markedly reduces proximal tubule sodium reabsorption, the acute effects on GFR and RPF are very limited in healthy humans. The finding of GLP-1s ability to reduce angiotensin II concentration is novel and should be further elucidated.

Decreased vasopressin-mediated renal water reabsorption in rats with compensated liver cirrhosis

Experiments were performed to investigate vasopressin type 2 receptor (V2)-mediated renal water reabsorption and the renal expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2) in cirrhotic rats with sodium retention but without ascites. In addition, the expression of the furosemide-sensitive type 1 Na-K-2Cl cotransporter (BSC-1) and the natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) during acute V2-receptor blockade was measured. Acute V2-receptor blockade with the selective nonpeptide antagonist OPC-31260 (800 microg . kg-1 . h-1) was performed during conditions in which volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 150 mM glucose. OPC-31260 produced a significantly smaller increase in urine flow rate (-26%) and free water clearance (-18%) in cirrhotic rats than in control rats. The natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) was significantly increased in cirrhotic rats (+52%), but pretreatment with OPC-31260 did not affect the natriuretic response to furosemide in neither cirrhotic nor in control rats. Semiquantitative immunoblotting showed a significant downregulation of AQP-2 in the renal cortex (-72%) and in the outer medulla (-44%). The relative expression of BSC-1 in the outer medulla was unchanged in cirrhotic rats. The corticopapillary gradient of Na was significantly increased in cirrhotic rats. Since daily urine flow rate was similar in cirrhotic and sham-operated rats, we suggest that non-vasopressin-mediated water reabsorption is increased in cirrhotic rats probably as a result of an increased corticomedullary gradient due to exaggerated NaCl reabsorption in the thick ascending limb of Henles loop.Experiments were performed to investigate vasopressin type 2 receptor (V2)-mediated renal water reabsorption and the renal expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2) in cirrhotic rats with sodium retention but without ascites. In addition, the expression of the furosemide-sensitive type 1 Na-K-2Cl cotransporter (BSC-1) and the natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) during acute V2-receptor blockade was measured. Acute V2-receptor blockade with the selective nonpeptide antagonist OPC-31260 (800 μg ⋅ kg-1 ⋅ h-1) was performed during conditions in which volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 150 mM glucose. OPC-31260 produced a significantly smaller increase in urine flow rate (-26%) and free water clearance (-18%) in cirrhotic rats than in control rats. The natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) was significantly increased in cirrhotic rats (+52%), but pretreatment with OPC-31260 did not affect the natriuretic response to furosemide in neither cirrhotic nor in control rats. Semiquantitative immunoblotting showed a significant downregulation of AQP-2 in the renal cortex (-72%) and in the outer medulla (-44%). The relative expression of BSC-1 in the outer medulla was unchanged in cirrhotic rats. The corticopapillary gradient of Na was significantly increased in cirrhotic rats. Since daily urine flow rate was similar in cirrhotic and sham-operated rats, we suggest that non-vasopressin-mediated water reabsorption is increased in cirrhotic rats probably as a result of an increased corticomedullary gradient due to exaggerated NaCl reabsorption in the thick ascending limb of Henles loop.

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-1β, tumor necrosis factor-α, and IL-6, respectively. Inhibition of IL-1β 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-1β 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.

Increased Apical Targeting of Renal Epithelial Sodium Channel Subunits and Decreased Expression of Type 2 11β-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.

Changes of renal AQP2, ENaC, and NHE3 in experimentally induced heart failure: response to angiotensin II AT1 receptor blockade

Heart failure (HF) was induced by ligation of the left anterior descending artery (LAD). Left ventricular end-diastolic pressure (LVEDP) >25 mmHg (at day 23 after LAD ligation) was the inclusion criterion. The rats were divided into three groups: sham-operated (Sham, n = 23, LVEDP: 5.6 +/- 0.6 mmHg), HF (n = 14, LVEDP: 29.4 +/- 1.4 mmHg), and candesartan (1 mg.kg(-1).day(-1) sc)-treated HF (HF + Can, n = 9, LVEDP: 29.2 +/- 1.2 mmHg). After 7 days (i.e., 29 days after LAD ligation) semiquantitative immunoblotting revealed increased abundance of inner medulla aquaporin-2 (AQP2) and AQP2 phosphorylated at Ser(256) (p-AQP2) in HF. There was also markedly enhanced apical targeting of AQP2 and p-AQP2 in inner medullary collecting duct (IMCD) in HF compared with Sham rats, shown by immunocytochemistry. Candesartan treatment significantly reversed the increases in both AQP2 and p-AQP2 expression and targeting. In contrast, there were only modest changes in other collecting duct segments. Semiquantitative immunoblots revealed increased expression of type 3 Na(+)/H(+) exchanger (NHE3) and Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) in kidneys from HF compared with Sham rats: both effects were reversed or prevented by candesartan treatment. The protein abundance of alpha-epithelial sodium channel (alpha-ENaC) was increased while beta-ENaC and gamma-ENaC expression was decreased in the cortex and outer stripe of the outer medulla in HF compared with Sham rats, which was partially reversed by candesartan treatment. These findings strongly support an important role of angiotensin II in the pathophysiology of renal water and sodium retention associated with HF.

Renal extraction and acute effects of glucagon-like peptide-1 on central and renal hemodynamics in healthy men

The present experiments were performed to elucidate the acute effects of intravenous infusion of glucagon-like peptide (GLP)-1 on central and renal hemodynamics in healthy men. Seven healthy middle-aged men were examined on two different occasions in random order. During a 3-h infusion of either GLP-1 (1.5 pmol·kg⁻¹·min⁻¹) or saline, cardiac output was estimated noninvasively, and intraarterial blood pressure and heart rate were measured continuously. Renal plasma flow, glomerular filtration rate, and uptake/release of hormones and ions were measured by Ficks Principle after catheterization of a renal vein. Subjects remained supine during the experiments. During GLP-1 infusion, both systolic blood pressure and arterial pulse pressure increased by 5±1 mmHg (P=0.015 and P=0.002, respectively). Heart rate increased by 5±1 beats/min (P=0.005), and cardiac output increased by 18% (P=0.016). Renal plasma flow and glomerular filtration rate as well as the clearance of Na⁺ and Li⁺ were not affected by GLP-1. However, plasma renin activity decreased (P=0.037), whereas plasma levels of atrial natriuretic peptide were unaffected. Renal extraction of intact GLP-1 was 43% (P<0.001), whereas 60% of the primary metabolite GLP-1 9-36amide was extracted (P=0.017). In humans, an acute intravenous administration of GLP-1 leads to increased cardiac output due to a simultaneous increase in stroke volume and heart rate, whereas no effect on renal hemodynamics could be demonstrated despite significant extraction of both the intact hormone and its primary metabolite.

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

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.

Model explaining the relation between distal nephron Li + reabsorption and urinary Na + excretion in rats

Li+ may be reabsorbed via an amiloride-sensitive mechanism in the collecting ducts of rats administered a low-Na+ diet. This was investigated by measuring the increase in fractional urinary excretion of Li+(FELi) in response to amiloride in conscious rats at two different levels of plasma Li+ concentration and after administration of bendroflumethiazide (BFTZ), angiotensin III (ANG III), and aldosterone (Aldo). The results confirmed that amiloride increased (FELi) in rats on a low-Na+ diet (20 ± 1 to 35 ± 1%, means ± SE), whereas no increase was observed in rats on a normal Na+ diet (37 ± 1 to 38 ± 1%). The lithiuretic effect of amiloride was 1) abolished by preadministration of BFTZ (32 ± 1 to 33 ± 2%) to Na+-deprived rats and 2) increased by ANG III (27 ± 3 to 33 ± 2%) and Aldo (25 ± 2 to 37 ± 2%) in Na+-replete rats. Amiloride-induced changes in FELiwere independent of plasma Li+concentration but inversely related to the fractional excretion of Na+ and the amiloride-sensitive excretion of K+. These results are compatible with the hypothesis that a low tubular Na+ concentration reduces end-tubular Na+ reabsorption and results in hyperpolarization of the apical membrane, thus favoring Li+ uptake into the cells.