Renal angiotensin I-converting enzyme-deficient mice are protected against aristolochic acid nephropathy.

The renal renin-angiotensin system (RAS) is involved in the development of chronic kidney disease. Here, we investigated whether mice with reduced renal angiotensin I-converting enzyme (ACE-/-) are protected against aristolochic acid nephropathy (AAN). To further elucidate potential molecular mechanisms, we assessed the renal abundances of several major RAS components. AAN was induced using aristolochic acid I (AAI). Glomerular filtration rate (GFR) was determined using inulin clearance and renal protein abundances of renin, angiotensinogen, angiotensin I-converting enzyme (ACE) 2, and Mas receptor (Mas) were determined in ACE-/- and C57BL/6J control mice by Western blot analyses. Renal ACE activity was determined using a colorimetric assay and renal angiotensin (Ang) (1-7) concentration was determined by ELISA. GFR was similar in vehicle-treated mice of both strains. AAI decreased GFR in controls but not in ACE-/- mice. Furthermore, AAI decreased renal ACE activity in controls but not in ACE-/- mice. Vehicle-treated ACE-/- mice had significantly higher renal ACE2 and Mas protein abundances than controls. AAI decreased renal ACE2 protein abundance in both strains. Furthermore, AAI increased renal Mas protein abundance, although the latter effect did not reach statistical significance in the ACE-/- mice. Renal Ang(1-7) concentration was similar in vehicle-treated mice of both strains. AAI increased renal Ang(1-7) concentration in the ACE-/- mice but not in the controls. Mice with reduced renal ACE are protected against AAN. Our data suggest that in the face of renal ACE deficiency, AAI may activate the ACE2/Ang(1-7)/Mas axis, which in turn may deploy its reno-protective effects.

High-Mobility Group Box Protein 1 Is an Independent Prognostic Marker for All-Cause Mortality in Patients With Dilated Cardiomyopathy.

High-mobility group box protein 1 (HMGB1) is released during tissue damage and activates the innate immune system through toll-like receptor 4. Because mortality in dilated cardiomyopathy (DCM) is associated with activation of the innate immune system, we hypothesized that HMGB1 possesses a prognostic value in estimating mortality in patients with DCM. We determined HMGB1 and N-terminal B-type natriuretic peptide (NT-proBNP) levels in 67 patients with DCM (12 women, mean age 53.6 ± 1.5 years). Kaplan-Meier analyzes revealed that higher levels of HMGB1 and NT-proBNP are related to increased all-cause mortality. Multivariable Cox regression confirmed HMGB1 as a risk factor for mortality in patients with DCM, independent of NT-proBNP, age, and gender (hazard ratio per 1 SD 1.920, 95% confidence interval 1.401 to 2.631, p <0.001). HMGB1 is a promising candidate to estimate the prognosis of patients with DCM.

Renal Soluble Guanylate Cyclase Is Downregulated in Sunitinib-Induced Hypertension.

Background The tyrosine kinase inhibitor sunitinib causes hypertension associated with reduced nitric oxide (NO) availability, elevated renal vascular resistance, and decreased fractional sodium excretion. We tested whether (1) nitrate supplementation mitigates sunitinib-induced hypertension and NO contributes less to renal vascular resistance as well as fractional sodium excretion regulation in sunitinib-treated rats than in controls; and (2) renal soluble guanylate cyclase (sGC) is downregulated and sGC activation lowers arterial pressure in rats with sunitinib-induced hypertension. Methods and Results Arterial pressure responses to nitrate supplementation and the effects of systemic and intrarenal NO synthase (NOS) inhibition on renal hemodynamics and fractional sodium excretion were assessed in sunitinib-treated rats and controls. Renal NOS and sGC mRNA as well as protein abundances were determined by quantitative polymerase chain reaction and Western blot. The effect of the sGC activator cinaciguat on arterial pressure was investigated in sunitinib-treated rats. Nitrate supplementation did not mitigate sunitinib-induced hypertension. Endothelium-dependent reductions in renal vascular resistance were similar in control and sunitinib-treated animals without and with systemic NOS inhibition. Selective intrarenal NOS inhibition lowered renal medullary blood flow in control but not in sunitinib-treated rats without significant effects on fractional sodium excretion. Renal cortical sGC mRNA and sGC α1-subunit protein abundance were less in sunitinib-treated rats than in controls, and cinaciguat effectively lowered arterial pressure by 15-20 mm Hg in sunitinib-treated rats. Conclusions Renal cortical sGC is downregulated in the presence of intact endothelium-dependent renal vascular resistance regulation in developing sunitinib-induced hypertension. This suggests that sGC downregulation occurs outside the renal vasculature, increases renal sodium retention, and contributes to nitrate resistance of sunitinib-induced hypertension.

MD-2 is a new predictive biomarker in dilated cardiomyopathy and exerts direct effects in isolated cardiomyocytes.

BACKGROUND: Myeloid differentiation factor-2 (MD-2) has been shown to be an important modulator of the innate immune system, but its role in cardiac diseases is unknown. We investigated whether MD-2 plays a role as risk predictor and contributor in dilated cardiomyopathy (DCM). METHODS AND RESULTS: We included 174 patients with reduced left ventricular (LV) ejection fraction (LVEF <45%) due to DCM. Coronary artery disease and severe valvular diseases were excluded in all patients by angiography or echocardiography. Cardiac inflammation, viral infection and MD-2 expression were analyzed from right ventricular endomyocardial biopsies. MD-2 was quantified by ELISA in serum upon first hospital admission. Myocyte contractility and inflammatory response after stimulation with recombinant MD-2 protein were analyzed in isolated rat cardiomyocytes. Median follow-up of the patients was 3.51 years (2.73; 4.48) with 34 deaths. Absolute mortality risk increases in patients displaying a MD-2 serum concentration greater than the median (302 ng/ml) was 23% (P < 0.0001). Age- and sex-adjusted Cox regression analyses demonstrated that mortality risk was highly related to MD-2 concentrations (P < 0.001), but not to age or sex. An increase of 100 ng/ml in the MD-2 level was associated with an absolute mortality risk increase of 50.4%. Receiver operating characteristic (ROC) analyses showed no difference between MD-2 and nterminal-pro brain natriuretic peptide (NT-pro-BNP), while the combination of both MD-2 and NT-pro-BNP resulted in a significantly increased capability of risk prediction when compared to NT-pro-BNP alone (P = 0.014). In-vitro, recombinant MD-2 decreases cell shortening and modulates cytokine activation in isolated cardiomyocytes. CONCLUSION: MD-2 predicts long-term outcome in DCM patients and improves mortality risk prediction capability compared to NT-pro-BNP alone. In addition, MD-2 exerts direct negative inotropic effects on isolated cardiomyocytes in-vitro. Further randomized trials should confirm MD-2 as a diagnostic and therapeutic target.

The role of distal tubule and collecting duct sodium reabsorption in sunitinib-induced hypertension.

OBJECTIVE: Antiangiogenic receptor tyrosine kinase inhibitors (RTKI) induce arterial hypertension which may limit their use. Renal fractional sodium excretion (FENa) is reduced in early RTKI-induced hypertension, whereas fractional lithium excretion is unaltered. Therefore, we tested the hypothesis that activated distal tubule and collecting duct sodium reabsorption contributes to RTKI-induced hypertension. METHODS: Amiloride-sensitive and hydrochlorothiazide (HCTZ)-sensitive fractional sodium reabsorption (FRNa) and renal epithelial sodium channel (ENaC) as well as sodium chloride cotransporter (NCC) abundances were determined in sunitinib-treated and control rats. The antihypertensive effects of amiloride and HCTZ were investigated by radiotelemery. RESULTS: After 4 days of treatment, mean arterial pressure was 20 mmHg higher, FENa was lower (0.32 ±â€Š0.08% vs. 0.65 ±â€Š0.14%; P < 0.05), and renal medullary-ENaC protein abundance was higher in sunitinib-treated rats than in controls. Amiloride-sensitive FRNa was 2.37 ±â€Š0.52% in sunitinib-treated rats vs. 2.66 ±â€Š0.44% in controls (n.s.). HCTZ increased FENa by a similar magnitude without affecting amiloride-sensitive FRNa in both groups. After 14 days of treatment, renal medullary ß-ENaC protein abundance was higher in rats that received sunitinib than in controls, whereas α-ENaC, γ-ENaC, and NCC abundances were similar in both groups. Amiloride and HCTZ reduced the sunitinib-induced mean arterial pressure rise by 8 ±â€Š3 mmHg (P < 0.05) and 12 ±â€Š2 mmHg (P < 0.05), respectively, without additive effects when combined. CONCLUSION: ENaC-dependent and thiazide-sensitive sodium-retaining mechanisms are not overactive in sunitinib-induced hypertension but ENaC blockers and in particular thiazides may be suitable for its treatment.

Stiffness of left ventricular cardiac fibroblasts is associated with ventricular dilation in patients with recent-onset nonischemic and nonvalvular cardiomyopathy.

BACKGROUND: Ventricular dilation is known as a pivotal predictor in recent-onset cardiomyopathy (ROCM), but its pathophysiology is not fully understood. In the present study we investigated whether single-cell stiffness of right and left ventricular-derived fibroblasts has an effect on cardiac phenotype in patients with ROCM. METHODS AND RESULTS: Patients with endomyocardial biopsy-proven ROCM were included (n=10). Primary cardiac fibroblasts (CFBs) were cultured from left and right ventricular endomyocardial biopsies and their single-cell stiffness was analyzed by quantification of Young's modulus using colloidal probe atomic force microscopy. Cardiac fibrosis was analyzed by Masson's trichrome staining. CFBs from the left ventricle showed significantly decreased stiffness when compared with CFBs from the right ventricle, indexed by decreased stiffness (Young's modulus 3,374±389 vs. 4,837±690 Pa; P<0.05). Young's modulus of CFBs derived from the left ventricle correlated negatively with the left ventricular end-diastolic dimension derived from 2-dimensional echocardiography (R(2)=0.77; P<0.01). Neither left nor right ventricular fibrosis correlated with the respective ventricular dimensions. CONCLUSIONS: Our data suggest that a decrease in single-cell stiffness of left ventricular fibroblasts could trigger left ventricular dilation in patients with ROCM. This implies a new potential mechanism for the ventricular dilation with this disease.

Source, topography and excitatory effects of GABAergic innervation in cockroach salivary glands.

Cockroach salivary glands are innervated by dopaminergic and serotonergic neurons. Both transmitters elicit saliva secretion. We studied the distribution pattern of neurons containing gamma-aminobutyric acid (GABA) and their physiological role. Immunofluorescence revealed a GABA-immunoreactive axon that originates within the subesophageal ganglion at the salivary neuron 2 (SN2) and this extends within the salivary duct nerve towards the salivary gland. GABA-positive fibers form a network on most acinar lobules and a dense plexus in the interior of a minor fraction of acinar lobules. Co-staining with anti-synapsin revealed that some putative GABAergic terminals seem to make pre-synaptic contacts with GABA-negative release sites. Many putative GABAergic release sites are at some distance from other synapses and at distance from the acinar tissue. Intracellular recordings from isolated salivary glands have revealed that GABA does not affect the basolateral membrane potential of the acinar cells directly. When applied during salivary duct nerve stimulation, GABA enhances the electrical response of the acinar cells and increases the rates of fluid and protein secretion. The effect on electrical cell responses is mimicked by the GABA(B) receptor agonists baclofen and SKF97541, and blocked by the GABA(B) receptor antagonists CGP52432 and CGP54626. These findings indicate that GABA has a modulatory role in the control of salivation, acting presynaptically on serotonergic and/or dopaminergic neurotransmission.