Hemodynamic and inotropic effects of endothelin-1 in vivo

Endothelin-1 (ET-1) is known to have strong vasoactive properties. Contradictory results have been reported with regard to its inotropic effects. This study examined the dose-dependent (500, 1000, 2500, 5000 and 10,000 ng ET-1/kg vs. NaCl controls) hemodynamic and inotropic effects of ET-1 in 53 open-chest rats during and after a 7-min infusion. Besides measurements in the intact circulation the myocardial function was examined by isovolumic registrations independent of peripheral vascular effects. A transient ET-1 induced (500, 1000, 2500, 5000 ng ET-1/kg) decrease of the left ventricular systolic pressure (LVSP) and the mean aortic pressure (AoPmean) was followed by a dose-related rise of these pressures (LVSP: -1%, -1%, +8%, +16% vs. preinfusion values; AoPmean: -11%, +9%, +39%, +52%). Heart rate (HR) was not influenced by ET-1. Due to the dose-dependent decrease of the stroke volume (SV) the cardiac output (CO) was reduced (CO: -8%, -23%, -40%, -50%). After an initial vasodilatation ET-1 elevates the total peripheral resistance (TPR: -1%, +49%, +139%, +215%) dose-dependently. 10,000 ng ET-1/kg was a lethal dose resulting in cardiac failure within minutes (low output). Since the maximum of the isovolumic LVSP (peak LVSP) and the corresponding dP/dtmax (peak dP/dtmax) were unchanged under ET-1, the isovolumic measurements do not indicate a positive inotropic effect of ET-1 in vivo in contrast to published results of in vitro experiments. It may be possible that a direct positive inotropic effect of ET-1 observed in in vitro studies is counterbalanced in vivo by an indirect negative inotropic effect due to the coronary-constrictive effect of ET-1

Enzyme-linked immunosorbent assay for urinary albumin at low concentrations

We describe an enzyme-linked immunosorbent assay (ELISA) for urinary albumin. It requires only commercially available reagents, can detect as little as 16 micrograms of albumin per liter, and analytical recovery ranges from 92 to 116%. The assay is simple, rapid, and inexpensive. Albumin excretion was 6.2 (SD 4.1) mg/24 h in healthy subjects (n = 40), 14.7 (SD 7.2) mg/24 h in albumin-test-strip-negative Type I diabetics (n = 11), and 19.7 (SD 16.2) mg/24 h in patients with essential hypertension (n = 12)

Cyclosporine A attenuates the natriuretic action of loop diuretics by inhibition of renal COX-2 expression

Cyclosporine A attenuates the natriuretic action of loop diuretics by inhibition of renal COX-2 expression.BackgroundIt is known that inhibition of cyclooxygenase (COX) impairs the renal actions of loop diuretics. Recently, we found that cyclosporine A (CsA) inhibits renal COX-2 expression. Therefore, we examined the interferences of CsA with the renal actions of loop diuretics.MethodWe investigated the renal effects of furosemide administration (12mg/day subcutaneously) in male Sprague-Dawley rats receiving in addition vehicle, CsA (15mg/kg × day), rofecoxib (10mg/kg × day), or a combination of both.ResultsCsA, rofecoxib, and their combination lowered the furosemide-induced increase of prostaglandin E2 (PGE2) and of 6-keto prostaglandin F1α (6-keto PGF1α) excretion by 55% and by 70%. They also lowered furosemide stimulated renal excretion of sodium and water by about 65% and 60%. Basal as well as furosemide-induced stimulation of plasma renin activity (PRA) and of renal renin mRNA was further enhanced by CsA. In contrast, rofecoxib attenuated the furosemide-induced rise of PRA and of renin mRNA, both in the absence and in the presence of CsA. In addition, the increase in plasma 6-keto PGF1α levels by furosemide was further enhanced by CsA and was attenuated by rofecoxib.ConclusionTaken together, our data suggest that CsA acts as an antinatriuretic, likely by the inhibition of COX-2–mediated renal prostanoid formation. Since the furosemide-induced stimulation of the renin system is not attenuated by CsA but by COX-2 inhibition, we speculate that extrarenal COX-2–derived prostanoids may be involved in the stimulation of the renin system by CsA and by loop diuretics

Gene expression of adenosine receptors along the nephron

Gene expression of adenosine receptors along the nephron.BackgroundIn view of the multiple effects of adenosine on kidney function, this study aimed to determine the expression of adenosine receptors (AR) along the rat and mouse nephron.MethodsFor this purpose, we semiquantified mRNA abundance for adenosine A1-, A2A-, A2B-, and A3 receptors by RNAse protection and by reverse transcription-polymerase chain reaction (RT-PCR) in the kidney zones and in the different nephron segments of mice and rats.ResultsWe found very similar expression patterns for rat and mice. For the kidney zones A1-AR mRNA and A2A-AR mRNA abundance displayed a marked difference, with an increase from cortex to the inner medulla. This was not seen for A2B receptors, which showed in general a rather weak expression. Along the nephron, A1-AR was strongly expressed in the thin limbs of Henle and in the collecting duct system and to a lesser extent in the medullary thick ascending limb. A2A-AR mRNA was clearly detected in glomeruli but not in other nephron segments. A2B-AR mRNA was strongly expressed in the cortical thick ascending limb of Henle and in the distal convoluted tubule. A3-AR mRNA was not found in any nephron segment.ConclusionOur data demonstrate a distinct mutual expression of the AR subtypes along the nephron. A1 receptors are expressed in medullary tubular structures, while A2B receptors are predominant in cortical tubular structures. A2A receptor expression in the kidney appears to be restricted to vascular cells

Toll-like receptor 4 in experimental kidney transplantation: early mediator of endogenous danger signals

The role of toll-like receptors (TLRs) has been described in the pathogenesis of renal ischemia/reperfusion injury, but data on the expression and function of TLR4 during renal allograft damage are still scarce. We analyzed the expression of TLR4 in an experimental rat model 6 and 28 days after allogeneic kidney transplantation in comparison to control rats and rats after syngeneic transplantation. On day 6, a significant induction in TLR4 expression - restricted to the glomerular compartment - was found in acute rejecting allografts only. TLR4 expression strongly correlated with renal function, and TLR4 induction was accompanied by a significant increase in CC chemokine expression within the graft as well as in urinary CC chemokine excretion. TLR4 induction may be caused by an influx of macrophages as well as TLR4-expressing intrinsic renal cells. Fibrinogen deposition in renal allografts correlated with renal TLR4 expression and may act as a potent stimulator of chemokine release via TLR4 activation. This study provides, for the first time, data about the precise intrarenal localization and TLR4 induction after experimental kidney transplantation. It supports the hypothesis that local TLR4 activation by endogenous ligands may be one pathological link from unspecific primary allograft damage to subsequent chemokine release, infiltration and activation of immune cells leading to deterioration of renal function and induction of renal fibrosis. Copyright (c) 2012 S. Karger AG, Base

Biological Activity of Different Forms of Oxidized Parathyroid Hormone

Preclinical studies have shown that parathyroid hormone (PTH) loses its biological effects through oxidation. PTH can be oxidized at methionines 8 and 18. Three possible variations of oxidized PTH (oxPTH) exist: Met8(ox)PTH, Met18(ox)PTH, and Met8, Met18(di-ox)PTH. A recent study showed that Met18(ox)PTH retained biological activity and was able to upregulate Fgf23 gene expression, whereas Met8(ox)PTH and Met8, Met18(di-ox)PTH showed less or no biological activity. An earlier study likewise showed that the oxidation of Met18 has minor effects on the secondary structure of PTH, whereas the oxidation of Met8 causes substantial structural changes, consistent with another study showing that oxidization just at Met8 blocks the generation of the second messenger cAMP, whereas the effect of the oxidation of Met18 is much less potent in inhibiting cAMP formation. A considerable percentage of circulating PTH in chronic kidney disease (CKD) patients is oxidized. However, we do not know the relative amounts of the different forms of oxPTH with agonistic, partial agonistic, or even antagonistic biological actions in different CKD populations. This might explain different clinical findings in the different CKD populations analyzed so far. The currently available method that was used in these clinical studies just distinguishes between oxPTH and noxPTH without being able to differentiate between different forms of oxPTH. Only methods of PTH measurement that are able to differentiate between PTH forms (noxPTH, Met8(ox)PTH, Met18(ox)PTH, and Met8, Met18(di-ox)PTH) have the potential to improve patient care, because only these methods will definitively separate bioactive from non-bioactive PTH forms. Such methods need to be developed, validated, and used in prospective randomized clinical trials to define the potential value of bioactive PTH forms as a predictor of cardiovascular events, mortality, and bone turnover

Short- and long-term effects of hemodialysis on platelet and monocyte activity markers of atherosclerosis in patients with end-stage renal disease

Background: In hemodialysis (HD) patients cardiovascular events represent the predominant cause of mortality. Since platelet and monocyte activity markers play an important role in cardiovascular mortality, this study assessed the influence of HD on these markers. Methods: Forty one HD patients (25 male, 16 female) were included. Blood samples were obtained before and after a single HD session at baseline and again after an elapsed period of 114 ± 21 days (91–175 days) on maintenance hemodialysis. Surface expression of CD40L and CD62P on platelets, tissue factor binding on monocytes and platelet-monocyte aggregates were measured by flow cytometry. Plasma levels of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFa) and soluble CD40L were analyzed by enzyme linked immunosorbent assay. Results: Tissue factor on monocytes was significantly increased after a single HD session at baseline (p = 0.041), whereas platelet-monocyte aggregates, the expression of CD40L and CD62P on platelets did not change significantly. After a mean of 114 ± 21 days of HD therapy, tissue factor on monocytes (p < 0.0001), platelet-monocytes aggregates (p < 0.0001), plasma levels of MCP-1 (p = 0.012) and TNFa (p = 0.046) were significantly decreased compared to baseline values. In contrast, platelet surface expression of CD40L and CD62P as well as plasma levels of sCD40L and IL-6 were not attenuated significantly. There was no significant correlation detected between the markers examined and the cumulative time on hemodialysis. Conclusions: Platelet and monocyte activity markers assessed in this study do not appear to be significantly increased by HD therapy. Therefore, these markers probably cannot be accountable for increased cardiovascular mortality in chronic HD patients

Effects of hypoxia on renin secretion and renal renin gene expression

Effects of hypoxia on renin secretion and renal renin gene expression. Plasma renin activity (PRA) and renal renin mRNA levels were measured in male rats exposed to hypoxia (8% O2) or to carbon monoxide (CO; 0.1%) for six hours. PRA increased fourfold and 3.3-fold, and renin mRNA levels increased to 220% and 200% of control, respectively. In primary cultures of renal juxtaglomerular (JG) cells, hypoxia (lowering medium O2 from 20% to 3% or 1%) for 6 or 20hours did not affect renin secretion or gene expression. Renal denervation did not prevent stimulation of the renin system by hypoxia. Because norepinephrine increased 1.7-fold and 3.2-fold and plasma epinephrine increased 3.9-fold and 7.8-fold during hypoxia and CO inhalation, respectively, circulating catecholamines might mediate the stimulatory effects of hypoxia on renin secretion and renin gene expression. Stimulation of β-adrenergic receptors by continuous infusion of 160 μg/kg/hr isoproterenol increased PRA 17-fold and 20-fold after three and six hours, respectively, and renin mRNA by 130% after six hours. In rats with a stimulated renin system (low-sodium diet), isoproterenol did not stimulate PRA or renal renin mRNA further. In summary, both arterial and venous hypoxia can stimulate renin secretion and renin gene expression powerfully in vivo but not in vitro. These effects seem not to be mediated by renal nerves or by a direct effect on JG cells but might be mediated by circulating catecholamines

Inverse correlation of intact PTH, oxidized PTH as well as non-oxidized PTH with 25-hydroxyvitamin D3 in kidney transplant recipients

Background 25-hydroxyvitamin D (25(OH)D) and potentially also 1,25-dihydroxyvitamin D (1,25(OH)2D) inhibits the synthesis of parathyroid hormone (PTH) in the chief cells of the parathyroid gland. Clinical studies showing a negative correlation between (25(OH)D and PTH are in good agreement with these findings in basic science studies. However, PTH was measured in these studies with the currently clinically used 2nd or 3rd generation intact PTH (iPTH) assay systems. iPTH assays cannot distinguish between oxidized forms of PTH and non-oxidized PTH. Oxidized forms of PTH are the by far most abundant form of PTH in the circulation of patients with impaired kidney function. Oxidation of PTH causes a loss of function of PTH. Given that the clinical studies done so far were performed with an PTH assay systems that mainly detect oxidized forms of PTH, the real relationship between bioactive non-oxidized PTH and 25(OH)D as well as 1,25(OH)2D is still unknown. Methods To address this topic, we compared for the first time the relationship between 25(OH)D as well as 1,25(OH)2D and iPTH, oxPTH as well as fully bioactive n-oxPTH in 531 stable kidney transplant recipients in the central clinical laboratories of the Charité. Samples were assessed either directly (iPTH) or after oxPTH (n-oxPTH) was removed using a column that used anti-human oxPTH monoclonal antibodies, a monoclonal rat/mouse parathyroid hormone antibody (MAB) was immobilized onto a column with 500 liters of plasma samples. Spearman correlation analysis and Multivariate linear regression were used to evaluate the correlations between the variables. Results There was an inverse correlation between 25(OH)D and all forms of PTH, including oxPTH (iPTH: r=-0.197, p<0.0001; oxPTH: r=-0.203, p<0.0001; n-oxPTH: r=-0.146, p=0.001). No significant correlation was observed between 1,25(OH)2D and all forms of PTH. Multiple linear regression analysis considering age, PTH (iPTH, oxPTH and n-oxPTH), serum calcium, serum phosphor, serum creatinine, fibroblast growth factor 23 (FGF23), osteoprotegerin (OPG), albumin, and sclerostin as confounding factors confirmed these findings. Subgroup analysis showed that our results are not affected by sex and age. Conclusion In our study, all forms of PTH are inversely correlated with 25-hydroxyvitamin D (25(OH)D). This finding would be in line with an inhibition of the synthesis of all forms of PTH (bioactive n-oxPTH and oxidized forms of PTH with minor or no bioactivity) in the chief cells of the parathyroid glad