Horst Schweer

Localization of the succinate receptor in the distal nephron and its signaling in polarized MDCK cells

When the succinate receptor (SUCNR1) is activated in the afferent arterioles of the glomerulus it increases renin release and induces hypertension. To study its location in other nephron segments and its role in kidney function, we performed immunohistochemical analysis and found that SUCNR1 is located in the luminal membrane of macula densa cells of the juxtaglomerular apparatus in close proximity to renin-producing granular cells, the cortical thick ascending limb, and cortical and inner medullary collecting duct cells. In order to study its signaling, SUCNR1 was stably expressed in Madin-Darby Canine Kidney (MDCK) cells, where it localized to the apical membrane. Activation of the cells by succinate caused Gq and Gi-mediated intracellular calcium mobilization, transient phosphorylation of extracellular regulated kinase (ERK)1/2 and the release of arachidonic acid along with prostaglandins E2 and I2. Signaling was desensitized without receptor internalization but rapidly resensitized upon succinate removal. Immunohistochemical evidence of phosphorylated ERK1/2 was found in cortical collecting duct cells of wild type but not SUCNR1 knockout streptozotocin-induced diabetic mice, indicating in vivo relevance. Since urinary succinate concentrations in health and disease are in the activation range of the SUCNR1, this receptor can sense succinate in the luminal fluid. Our study suggests that changes in the luminal succinate concentration may regulate several aspects of renal function.

Tandem mass spectrometric determination of 11-dehydrothromboxane B2, an index metabolite of thromboxane B2 in plasma and urine☆

11-Dehydrothromboxane B2 is one of the major enzymatic metabolites of thromboxane B2 (TXB2), a biologically inactive product of thromboxane A2. The short half-life of thromboxane A2 and ex vivo production of thromboxane B2 by platelet activation make these prostanoid metabolites inappropriate as indices of systemic thromboxane biosynthesis, whereas 11-dehydro-TXB2 has been shown to reflect the release of thromboxane A2 in the human blood circulation. Analysis of 11-dehydro-TXB2 in plasma and urine was performed by gas chromatography-mass spectrometry-mass spectrometry using the chemically synthesized tetradeuterated compound as an internal standard. The high selectivity of triple-stage quadrupole mass spectrometry (tandem mass spectrometry) considerably facilitates sample purification as compared to single quadrupole mass spectrometric determination. Plasma concentrations in five healthy male volunteers were in the range 0.8-2.5 pg/ml. Urinary excretion of 11-dehydro-TXB2 was higher than that of 2,3-dinor-TXB2: 1.2 +/- 0.36 micrograms/24 h vs 0.53 +/- 0.33 micrograms/24 h (n = 5). Thus 11-dehydro-TXB2 appears at present to be the best index metabolite of systemic TXA2 activity in plasma as well as in urine.

Delayed In Vivo Catabolism of Intermediate-Density Lipoprotein and Low-Density Lipoprotein in Hemodialysis Patients as Potential Cause of Premature Atherosclerosis

Objective—Premature cardiovascular disease is the leading cause of death in patients with end-stage renal disease treated by hemodialysis (HD). Low-density lipoprotein (LDL) levels are not generally increased in HD patients, but their LDL metabolism is still poorly understood. We therefore investigated the in vivo metabolism of apoB-containing lipoproteins in two different ethnic populations of HD patients and controls. Methods and Results—We performed stable isotope kinetic studies using a primed constant infusion of deuterated leucine in 12 HD patients and 13 healthy controls. Tracer/tracee ratio of apoB was determined by means of gas chromatography/mass spectrometry, and the modeling program SAAMII was used to estimate the fractional catabolic rate (FCR) of apoB. Mean LDL-apoB plasma concentrations were almost identical in both groups (HD: 95±30 mg/dL, controls: 91±40 mg/dL), whereas LDL-apoB FCR was 50% lower in HD patients as compared with controls (0.22±0.12 days−1 versus 0.46±0.20 days−1, P=0.001) with concomitantly decreased production rates of LDL. Compared with controls, intermediate-density lipoprotein (IDL)-apoB FCR was 65% lower (2.87±1.02 days−1 versus 8.89±4.94 days−1, P=0.014), accompanied by 1.5-fold higher IDL-apoB levels in HD. Very low-density lipoprotein metabolism was similar in both study groups. Conclusions—In vivo catabolism of LDL and IDL is severely impaired in HD patients but misleadingly masked by normal plasma cholesterol levels. The resulting markedly prolonged residence times of both IDL and LDL particles might thus significantly contribute to the well-documented high risk for premature cardiovascular disease in HD patients.

Effects of celecoxib and diclofenac on blood pressure, renal function, and vasoactive prostanoids in young and elderly subjects

Cyclooxygenase (COX) inhibitors are among the most widely used drugs, especially in the elderly. It has been claimed that the new COX‐2 inhibitors offer advantages in terms of drug safety. To test this hypothesis, the authors compared in a double‐blind, randomized trial the effects of celecoxib (200 mg bid) and diclofenac (75 mg bid) on blood pressure and renal function in two groups (each n =12) of young (mean age = 32 years) and elderly (mean age = 68 years) normotensive subjects. Changes from baseline in the 24‐hour blood pressure profiles, parameters of the renin‐angiotensin‐aldosterone system, inulin clearance, urinary marker proteins, and eicosanoid excretion were monitored during the treatment period of 2 weeks. Comparison between celecoxib and diclofenac showed no significant difference in minor alterations of blood pressure. During daytime, there was a trend to elevation of mean arterial blood pressure (mmHg) by celecoxib in the elderly of 2.8 (95% confidence interval [Cl] = −2.5 to 8.2) in comparison with the young subjects of −1.3 (95% Cl = −3.7 to 1.0); there was also a trend to elevation of mean arterial blood pressure by diclofenac in the elderly of 4.1 (95% Cl = −1.2 to 9.4) in comparison with the young subjects of 0.4 (95% Cl =−2.4 to 3.2). In both populations, the authors found no significant drug effects on the parameters of the renin‐angiotensin‐aldosterone system, inulin clearance, and urinaiy marker proteins. As expected, diclofenac reduced excretion of all prostanoids, whereas celecoxib did not affect production of TxB2 and its metabolites. Neither in young nor in elderly normotensive subjects were blood pressure and renal function significantly affected by a short‐terrn treatment with standard doses of celecoxib and diclofenac. Therefore, normal aging appears not to represent a special risk factor in therapy with these two agents.

In vivo stable-isotope kinetic study suggests intracellular assembly of lipoprotein(a)

OBJECTIVE Lipoprotein(a) [Lp(a)] consists of apolipoprotein B-100 (apoB-100) as part of an LDL-like particle and the covalently linked glycoprotein apolipoprotein(a) [apo(a)]. Detailed mechanisms of its biosynthesis, assembly, secretion and catabolism are still poorly understood. To address the Lp(a) assembly mechanism, we studied the in vivo kinetics of apo(a) and apoB-100 from Lp(a) and LDL apoB-100 in nine healthy probands using stable-isotope methodology. METHODS The level of isotope enrichment was used to calculate the fractional synthesis rate (FSR), production rate (PR) and retention time (RT) using SAAMII software and multicompartmental modeling. RESULTS We observed a similar mean PR for apo(a) (1.15 nmol/kg/d) and apoB-100 (1.31 nmol/kg/d) from Lp(a), which differed significantly from the PR for apoB-100 from LDL (32.6 nmol/kg/d). Accordingly, mean FSR and RT values for Lp(a)-apo(a) were similar to those of Lp(a)-apoB and different from those for LDL-apoB. CONCLUSION Two different kinetic apoB pools within Lp(a) and LDL suggest intracellular Lp(a) assembly from apo(a) and newly synthesized LDL.

Lithium reduces aquaporin-2 transcription independent of prostaglandins.

Vasopressin (AVP)-stimulated translocation and transcription of aquaporin-2 (AQP2) water channels in renal principal cells is essential for urine concentration. Twenty percent of patients treated with lithium develop nephrogenic diabetes insipidus (NDI), a disorder in which the kidney is unable to concentrate urine. In vivo and in mouse collecting duct (mpkCCD) cells, lithium treatment coincides with decreased AQP2 abundance and inactivation of glycogen synthase kinase (Gsk) 3β. This is paralleled in vivo by an increased renal cyclooxygenase 2 (COX-2) expression and urinary prostaglandin PGE(2) excretion. PGE(2) reduces AVP-stimulated water reabsorption, but its precise role in lithium-induced downregulation of AQP2 is unclear. Using mpkCCD cells, we here investigated whether prostaglandins contribute to lithium-induced downregulation of AQP2. In these cells, lithium application reduced AQP2 abundance, which coincided with Gsk3β inactivation and increased COX-2 expression. Inhibition of COX by indomethacin, leading to reduced PGE(2) and PGF(2α) levels, or dexamethasone-induced downregulation of COX-2 both increased AQP2 abundance, while PGE(2) addition reduced AQP2 abundance. However, lithium did not change the prostaglandin levels, and indomethacin and dexamethasone did not prevent lithium-induced AQP2 downregulation. Further analysis revealed that lithium decreased AQP2 protein abundance, mRNA levels and transcription, while PGE(2) reduced AQP2 abundance by increasing its lysosomal degradation, but not by reducing AQP2 gene transcription. In conclusion, our data reveal that in mpkCCD cells, prostaglandins decrease AQP2 protein stability by increasing its lysosomal degradation, indicating that in vivo paracrine-produced prostaglandins might have a role in lithium-induced NDI via this mechanism. However, lithium affects also AQP2 gene transcription, which is prostaglandin independent.

Determination of 11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid and 9α,11α-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry

Abstract 11α-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGE-M) and 9α,11α-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGF-M) in urine were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of the 2H7-labeled internal standard, O-methylhydroxylamine hydrochloride in acetate buffer was added either directly (PGE-M) or after standing overnight at pH 10 (PGF-M) to form the methoxime. The sample was acidified to pH 2.5 and PGE-M and PGF-M were extracted with ethyl acetate/hexane. Then the prostanoids were derivatized to the pentafluorobenzyl ester and purified by thin-layer chromatography and the trimethylsilyl ether was formed. The products were quantified by gas chromatography/triple-stage quadrupole mass spectrometry. For PGE-M, the fragment ions m z 349 and m z 356 (2H7 standard) (daughter ions of m z 637 and m z 644 (2H7 standard)) were used. The results of the PGE-M assay were compared with those of an assay using the [2H3]methoxime as the internal standard. For determination of PGF-M, the daughter ions m z 484 and m z 491 (2H7 standard) with the parent ions m z 682 and m z 689 (2H7 standard) were chosen.

Pharmacokinetics of prostaglandin E1 and its main metabolites after intracavernous injection and short-term infusion of prostaglandin E1 in patients with erectile dysfunction.

PURPOSE Alprostadil (prostaglandin E1) is the preferred monotherapy for intracavernous injection in the diagnosis and treatment of erectile dysfunction. Our study was designed to evaluate whether there is a difference in the pharmacokinetics of prostaglandin E1 and its main metabolites after intracavernous injection or short-term intravenous infusion. In addition, we also investigated the influence of the erectile response on prostaglandin E1 kinetics after intracavernous injection. MATERIALS AND METHODS A total of 24 patients with erectile dysfunction received, in a randomized order at an interval of 5 hours, an intracavernous injection or a 30-minute intravenous infusion of 20 microg. of alprostadil alfadex (prostaglandin E1). Venous blood samples were obtained 5 minutes before and at various times after the applications. We used highly sensitive gas chromatography/double-mass spectrometry method to measure prostaglandin E1 and its metabolites in plasma. RESULTS We demonstrated the presence of relevant systemic blood levels of prostaglandin E1 and its metabolites immediately after intracavernous injection. We found significantly lower systemic prostaglandin E1 concentrations between 7 and 20 minutes after intracavernous injection in patients with an erectile response compared with those without. CONCLUSIONS We found significant systemic concentrations of prostaglandin E1 and its metabolites after intracavernous injection. The systemic presence did not lead to significant changes in vital signs.

Increased Renal Biosynthesis of Prostaglandin E2 and Thromboxane B2 in Human Congenital Obstructive Uropathy

ABSTRACT: Animal experiments have shown that after ureter obstruction hydronephrotic kidneys release increased amounts of prostaglandin E2(PGE2) and thromboxane A2 (TxA2), suggesting that these prostanoids modify renal blood flow and excretory function in this model. To test the hypothesis that these mechanisms are also operative in congenital obstructive uropathy, we measured prostanoid excretion rates in 12 neonates and infants with congenital unilateral or bilateral hydronephrosis. Prostanoid determinations were performed by gas chromatography mass spectrometry. PGE2 and thromboxane B2 (TxB2) (non-enzymatic metabolite of TxA2) excretion exceeded the normal range in eight and 11 of 12 patients, respectively. Median PGE2 excretion was 22, range 4-572 ng/h/1.73 m2 (normal 3-16). Median TxB2 excretion was 22, range 3-188 ng/h/1.73 m2 (normal 3-7). No other renal prostanoids (prostaglandin F2α, 6-keto-prostaglandin F1α) or systemic prostanoid metabolites (PGE-M, 2,3-dinorthromboxane B2, 11-dehydro-thromboxane B2, 2,3-dinor- 6-keto-prostaglandin F1α) were consistently elevated. A second group of 12 neonates with congenital obstructive uropathy was followed sequentially. PGE2 and thromboxane B2 excretion rates increased even further after surgical decompression and gradually normalized during follow-up. There was a significant relationship between elevated FeNa and enhanced PGE2 and TxB2 excretion. These data suggest that endogenous renal formation of PGE2 and TxA2 is selectively stimulated in hydronephrotic kidneys in neonates and infants. PGE2 and TxA2 may be involved in modulating renal function in these infants.

Prostanoid formation during feeding of a preterm formula with long-chain polyunsaturated fatty acids in healthy preterm infants during the first weeks of life.

The objective of this study was to evaluate the effect of conventional and long-chain polyunsaturated fatty acids (LCP)-enriched preterm formula on prostanoid formation in preterm infants during their first weeks of life. In a prospective, randomized, double-blind study, healthy infants received either formula enriched with LCP (n = 10), standard preterm formula(n = 10), or (expressed) breast milk (n = 10). Urine was sampled, and anthropometric measurements were taken at study entry and after the study period of 3 wk. In vivo formation of prostaglandin E2, thromboxane A2, and prostacyclin was evaluated by measuring the urinary excretion of the respective index metabolites by gas chromatography-mass spectrometry. There were no significant differences in urinary prostanoid excretion and anthropometric data between the groups at the end of the study period. We conclude that neither conventional formula nor supplementation of a preterm formula with LCP for a period of 3 wk substantially influence prostanoid formation in healthy preterm infants.