Elke Persohn

Effects of Aliskiren on Blood Pressure, Albuminuria, and (Pro)Renin Receptor Expression in Diabetic TG(mRen-2)27 Rats

The aim of this study was to explore the effects of the renin inhibitor aliskiren in streptozotocin-diabetic TG(mRen-2)27 rats. Furthermore, we investigated in vitro the effect of aliskiren on the interactions between renin and the (pro)renin receptor and between aliskiren and prorenin. Aliskiren distributed extensively to the kidneys of normotensive (non)diabetic rats, localizing in the glomeruli and vessel walls after 2 hours exposure. In diabetic TG(mRen-2)27 rats, aliskiren (10 or 30 mg/kg per day, 10 weeks) lowered blood pressure, prevented albuminuria, and suppressed renal transforming growth factor-&bgr; and collagen I expression versus vehicle. Aliskiren reduced (pro)renin receptor expression in glomeruli, tubules, and cortical vessels compared to vehicle (in situ hybridization). In human mesangial cells, aliskiren (0.1 &mgr;mol/L to 10 &mgr;mol/L) did not inhibit binding of 125I-renin to the (pro)renin receptor, nor did it alter the activation of extracellular signal-regulated kinase 1/2 by renin (20 nmol/L) preincubated with aliskiren (100 nmol/L) or affect gene expression of the (pro)renin receptor. Evidence was obtained that aliskiren binds to the active site of prorenin. The above results demonstrate the antihypertensive and renoprotective effects of aliskiren in experimental diabetic nephropathy. The evidence that aliskiren can reduce in vivo gene expression for the (pro)renin receptor and that it may block prorenin-induced angiotensin generation supports the need for additional work to reveal the mechanism of the observed renoprotection by this renin inhibitor.

Brain Penetration of the Oral Immunomodulatory Drug FTY720 and Its Phosphorylation in the Central Nervous System during Experimental Autoimmune Encephalomyelitis: Consequences for Mode of Action in Multiple Sclerosis

FTY720 [2-amino-2-[2-(4-octylphenyl) ethyl]propane-1,3-diol hydrochloride] is an oral sphingosine-1-phosphate receptor modulator under development for the treatment of multiple sclerosis (MS). The drug is phosphorylated in vivo by sphingosine kinase 2 to its bioactive form, FTY720-P. Although treatment with FTY720 is accompanied by a reduction of the peripheral lymphocyte count, its efficacy in MS and experimental autoimmune encephalomyelitis (EAE) may be due to additional, direct effects in the central nervous system (CNS). We now show that FTY720 localizes to the CNS white matter, preferentially along myelin sheaths. Brain trough levels of FTY720 and FTY720-P in rat EAE are of the same magnitude and dose dependently increase; they are in the range of 40 to 540 ng/g in the brain tissue at efficacious doses and exceed blood concentrations severalfold. In a rat model of chronic EAE, prolonged treatment with 0.03 mg/kg was efficacious, but limiting the dosing period failed to prevent EAE despite a significant decrease in blood lymphocytes. FTY720 effectiveness is likely due to a culmination of mechanisms involving reduction of autoreactive T cells, neuroprotective influence of FTY720-P in the CNS, and inhibition of inflammatory mediators in the brain.

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinsons disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD.

Blockade of the Activin Receptor IIB Activates Functional Brown Adipogenesis and Thermogenesis by Inducing Mitochondrial Oxidative Metabolism

ABSTRACT Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.

Imatinib does not induce cardiotoxicity at clinically relevant concentrations in preclinical studies

Cytotoxic concentrations of imatinib mesylate (10-50 microM) were required to trigger markers of apoptosis and endoplasmic reticulum stress response in neonatal rat ventricular myocytes and fibroblasts, with no significant differences observed between c-Abl silenced and nonsilenced cells. In mice, oral or intraperitoneal imatinib treatment did not induce cardiovascular pathology or heart failure. In rats, high doses of oral imatinib did result in some cardiac hypertrophy. Multi-organ toxicities may have increased the cardiac workload and contributed to the cardiac hypertrophy observed in rats only. These data suggest that imatinib is not cardiotoxic at clinically relevant concentrations (5 microM).

Sphingosine 1-Phosphate Produced by Sphingosine Kinase 2 Intrinsically Controls Platelet Aggregation In Vitro and In Vivo

RATIONALE Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function. OBJECTIVE To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function. METHODS AND RESULTS We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo. CONCLUSIONS We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis.

Combination of Low-Dose Valsartan and Enalapril Improves Endothelial Dysfunction and Coronary Reserve in Nω-Nitro-l-Arginine Methyl Ester–Treated Spontaneously Hypertensive Rats

Combination of nonhypotensive doses of valsartan and enalapril markedly improved survival (+87%) compared with untreated animals (37%) in spontaneously hypertensive rats (SHRs) with endothelial dysfunction. However, the combination had no effect on kidney function, and proteinuria persisted over the 12 weeks of the study. It was hypothesized that the greater survival was due to improvement in endothelial function or coronary vasculature despite blockade of nitric oxide synthase and high blood pressure. Therefore, endothelial function was evaluated in isolated aortic ring and maximal coronary blood flow was studied in isolated perfused SHR hearts (20–24 weeks) treated with Nω-nitro-l-arginine methyl ester (l-NAME) (50 mg/l) for 4 weeks. The animals received vehicle, valsartan 5 mg/kg/d, enalapril 1 mg/kg/d, valsartan 50 mg/kg/d, or the combination valsartan 5 mg/kg/d with enalapril 1 mg/kg/d in drinking water. Normotensive Wistar-Kyoto (WKY) rats were used as control. Blood pressure was measured by telemetry. Histopathology was performed on heart, kidney (hematoxylin-eosin), and aorta (Masson trichrome). l-NAME elevated blood pressure by 50 mm Hg after vehicle (199 ± 5 mm Hg). Valsartan 50 mg/kg/d completely abolished this increase (150 ± 4 mm Hg) whereas the valsartan-enalapril combination synergistically decreased blood pressure (−37 mm Hg at 162 ± 7 mm Hg) compared with monotherapy (valsartan 5 mg/kg/d −10 mm Hg; enalapril 1 mg/kg/d −15 mm Hg). All treatments improved the histopathology, most markedly in those receiving the valsartan-enalapril combination. The severity mean grades for lesions were 2.1, 1.9, 1.7, 1.1, and 0.9 in vehicle-treated SHRs, enalapril 1 mg/kg/d, valsartan 5 mg/kg/d, valsartan 5 or 50 mg/kg/d, and the valsartan-enalapril combination, respectively, compared with 0.02 in WKY rats. Acetylcholine-induced relaxation was significantly greater in treated SHRs than after vehicle (−40% at 0.1 mmol acetylcholine) but the combination induced the maximal relaxation (−85%). The ratio of maximal tension induced by serotonin in rings with and without endothelium was 1.4 and 1.3 in vehicle and valsartan 5 mg/kg/d–treated rats whereas it did not differ from 1 in WKY rats and all other treated groups. The cardiac hypertrophy (+27%) was prevented by valsartan 50 mg/kg/d and the valsartan-enalapril combination. Coronary reserve was significantly increased by valsartan 50 mg/kg/d (+85% at 7.8 ± 0.7 ml/min/g) and the valsartan-enalapril combination (+42% at 6.0 ± 0.4 ml/min/g) compared with 4.2 ± 0.5 (vehicle). This was not different of 8.8 ± 0.5 (WKYs). Despite the maintenance of a high blood pressure, low-dose valsartan-enalapril significantly improved endothelial function and histopathology and increased coronary reserve in SHRs chronically receiving l-NAME.

Preclinical evaluation of potential nilotinib cardiotoxicity

In vitro, concentrations ≥ 10 μM of nilotinib were needed to induce markers of cytotoxicity, apoptosis, and endoplasmic reticulum stress in both neonatal rat ventricular myocytes, a putative target tissue, and non-target heart fibroblasts, indicating a lack of cardiomyocyte-specific nilotinib toxicity in vitro. In rats, oral nilotinib treatment at 80 mg/kg for 4 weeks induced increased heart weight; however, this was not associated with relevant histopathological changes or effects on heart function. Thus, nilotinib at and above clinically relevant concentrations (4.27 μM) did not induce overt cardiovascular pathologies or heart failure in vitro or in vivo under study conditions.

A comparative immunohistochemical study of spontaneous and chemically induced pheochromocytomas in B6C3F1 mice

Spontaneously occurring and chemically induced pheochromocytomas are rare in mice. That the mouse pheochromocytoma is a more appropriate animal model than that of the rat for study of human medullary adrenal tumors has been suggested. The expression of phenylethanolamine-N-methyltransferase (PNMT), the enzyme responsible for production of epinephrine from norepinephrine, is common to both mouse and human pheochromocytomas. This investigation assessed the expression of the immunohistochemical markers PNMT, tyrosine hydroxylase (TH), and chromogranin A (CGA) in spontaneously occurring and chemically induced pheochromocytomas in the B6C3F1 mouse. Spontaneous tumors were derived from control animals from 10 different studies and the pheochromocytomas from treated groups from 4 different studies. All tumors were positive for maximal TH expression. A highly significant difference in PNMT expression (p<0.01) occurred between spontaneously occurring pheochromocytomas classified as benign or “malignant” by the criteria of toxicologic pathology. Chemically induced tumors showed intermediate PNMT staining. A marked reduction in CGA expression occurred in pheochromocytomas induced by technical grade pentachlorophenol, compared to the other three chemicals and the spontaneously occurring tumors. These findings suggest that immunohistochemistry is a reliable tool in investigating the functional capabilities of pheochromocytomas in mice. PNMT expression is a tightly regulated component of the chromaffin cell phenotype and appears to be readily lost in mouse pheochromocytomas, particularly those with aggressive characteristics.

Immunoelectron microscopic localization of cytochrome P-450 isoenzyme CYP4A1 in liver, ileum and kidney of nafenopin treated male rats

A monoclonal antibody raised against and specific for cytochrome P‐450 isoenzyme CYP4A1 was used to investigate the subcellular distribution of this enzyme in the liver, kidney and ileum of nafenopin treated rats by means of immunoelectron microscopy. In the liver and kidney, labelling was restricted to peroxisomes and mitochondria of hepatocytes and proximal tubular epithelial cells whereas in ileum, immunolabelling was exclusively detected in mitochondria of absorptive cells.