Gerd Luippold

Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography.

A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N6-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25-1.0 nmol/g wet weight for renal tissue and 0.25-0.5 microM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1 +/- 2.6% and 102.5 +/- 4.0% for renal tissue and urine, respectively (mean +/- S.D., n = 3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64 +/- 2.2, 0.67 +/- 0.18 and 46.2 +/- 1.9 nmol/g wet weight, respectively (mean +/- S.D., n = 6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low microM range and are negatively correlated with urine flow-rate.

Dopamine D3 receptor activation modulates renal function in anesthetized rats

The renal effects of the D3 receptor agonist R(+)-7-hydroxy-dipropyl-aminotetraline (7-OH-DPAT) were studied in anesthetized Sprague-Dawley rats using standard clearance experiments. 7-OH-DPAT infusion (0.01, 0.1, and 1.0 µg kg–1 min–1) dose-dependently increased glomerular filtration rate (GFR) compared to baseline by a maximum of 20±2% while arterial blood pressure was not affected. Heart rate was not altered during the two lower doses of 7-OH-DPAT whereas a slight reduction occurred due to infusion of 1.0 µg kg–1 min–1. In contrast, higher doses of 7-OH-DPAT, starting from 3 µg kg–1 min–1, markedly influenced systemic hemodynamics. In addition to the hyperfiltration, 7-OH-DPAT (1.0 µg kg–1 min–1) also induced a significant diuresis (27.7±4.3 µl min–1 100 g–1 vs 16.2±5.4 µl min–1 100 g–1) and increased both absolute (3.30±0.58 µmol min–1 100 g–1 vs 0.95±0.26 µmol min–1 100 g–1) and fractional sodium excretion (2.48±0.32% vs 0.79±0.19%). These changes in renal function were not modulated by pretreatment with the D2 receptor antagonist S(–)-sulpiride but abolished by the D3 antagonist 5,6-dimethoxy-2-(di-n-propylamino)indane (U-99194A). In coincidence with the action of 7-OH-DPAT on both glomerular and tubular function, reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of D3 receptors in both glomerular and tubular fractions of kidneys taken from Sprague-Dawley rats. These data indicate that D3 receptors in the kidney are involved in the regulation of renal hemodynamics and tubular function.

Subapical Localization of the Dopamine D3 Receptor in Proximal Tubules of the Rat Kidney

The dopamine D3 receptor (D3R), intensively studied in neuroscience, also plays an important role in the regulation of renal and cardiovascular function. In contrast to functional findings, less information is available on its localization in the kidney. Neither RT-PCR studies nor radioligand binding assays are suitable to selectively determine the distribution of renal D3R at the level of cellular or even subcellular structures. We studied the renal D3R distribution in Sprague-Dawley rats by a polyclonal antiserum directed against an epitope in the third intracytoplasmic loop. D3R immunoreactivity was detected by indirect immunofluorescence and confocal laser scanning microscopy. D3R staining was confined to the renal cortex and occurred in proximal convoluted tubules near or in direct connection with the urinary pole of the glomeruli. The fluorescent spots were restricted to the subapical portion of the proximal tubular cells. Double staining with the F-actin marker phalloidin revealed a localization of the D3R below the brush border region. However, staining by anti-P1/p2-adaptins, recognizing clathrin-coated compartments, did not correspond to the distribution of the D3R signal. This is the first description of a D3R accumulation in a cytoplasmic pool in the kidney, probably corresponding to a recycling mechanism or storage compartment.

Arterial blood pressure and renal sodium excretion in dopamine D3 receptor knockout mice.

Alterations in the dopaminergic system may contribute to the development of hypertension. Recently, it has been reported that pentobarbital-anesthetized mice with deficient dopamine D3 receptors showed renin-dependent elevation in blood pressure. In a series of experiments, we evaluated the contribution of the dopamine D3 receptor to the renal sodium excretion and arterial blood pressure behavior in conscious as well as anesthetized dopamine D3 receptor knockout (−/−) mice. The blood pressure measuring study was designed as a cross-over trial to investigate the influence of different sodium loads. The animals were fed a normal salt diet (0.6% NaCl, NS) for 1 week and afterwards a low (0.2% NaCl, LS) or a high salt diet (4.6% NaCl, HS) for 2 weeks. After the third week, the animals were switched to the corresponding protocol. Systolic blood pressure in conscious (−/−) mice measured by tail-cuff plethysmography was not different from that of wild-type (+/+) animals, irrespective of the time course or the salt diet. In another experiment, challenge of an acute sodium loading per gavage in conscious D3 receptor (−/−) and (+/+) animals on HS or NS diet did not show significant differences in renal sodium excretion between the two genotypes. Additionally, animals were fed an NS diet for 1 week and an HS diet for another week. As expected, sodium excretion significantly increased after the change from the NS to the HS diet. A slightly lower urinary sodium excretion was observed when comparing D3 receptor (−/−) mice to their corresponding (+/+) mice, both on an HS diet. Clearance experiments with anesthetized D3 receptor (−/−) and (+/+) mice were performed to investigate the renal sodium excretion capacity, when exposed to a moderate volume expansion (VE). Urinary sodium excretion increased in response to the VE; however, no difference were observed between the two genotypes. Taking these results together, we conclude that in the present animal model renal dopamine D3 receptors are not significantly involved in the regulation of blood pressure associated with a deficiency in renal sodium elimination.

Renal response to infusion of dopamine precursors in anaesthetized rats.

In the present study the renal response to intravenous infusion of the catecholamine precursors l-dihydroxyphenylalanine (l-DOPA) or l-tyrosine was investigated in thiopentone sodium-anaesthetized Sprague-Dawley rats. Glomerular filtration rate (GFR) was assessed by renal clearance of inulin, urinary concentration of dopamine (UDAV) by HPLC and sodium excretion (UNaV) by flame photometry. We found that basal UDAV was 6.5 ± 0.5 pmol/min per 100 g body weight (mean ± SEM). Intravenous infusion of l-tyrosine at 0.1–3.0 μmol/min dose dependently enhanced UDAV (17 ± 3 to 144 ± 14 pmol/ min respectively) with higher doses of l-tyrosine resulting in no further increase in UDAV. Compared with l-tyrosine administration significantly lower doses of l-DOPA (0.07 to 35 nmol/min) caused increases in UDAV which were orders of magnitude higher (18 ± 1 to 7800 ± 470 pmol/min, respectively) and did not show saturation characteristics. GFR did not change in response to l-tyrosine or l-DOPA infusion. No variations in urinary flow rate or in UNaV could be observed which were significantly correlated to changes in UDAV. In contrast, intravenous infusion of dopamine at a dose of 6 nmol/min significantly increased GFR by 35 ± 6.2% and urinary flow rate by over 2-fold. Immunohistochemistry with light microscopy revealed no tyrosine hydroxylase in the kidney. Therefore, dopamine synthesis in the tubular cells mainly depends on the renal supply of l-DOPA. The unchanged GFR and UNaV in spite of large variations of UDAV argue against the hypothesis that intratubular dopamine plays a functional role in the regulation of hemodynamics or sodium transport in the kidney. Renal dopamine excretion may rather represent an effective pathway for the elimination of catecholamine precursors from the plasma.

The Salt Paradox of the Early Diabetic Kidney Is Independent of Renal Innervation

Glomerular filtration rate (GFR) is inversely and thus paradoxically related to dietary NaCl intake in rats and patients with early type 1 diabetes mellitus (DM). Enhanced sensitivity of proximal reabsorption to NaCl diet inducing secondary adaptations in GFR through actions of tubuloglomerular feedback causes this salt paradox. We studied the role of renal nerves for the salt paradox in rats with streptozotocin (STZ)-induced DM since a regulatory influence of renal nerves on proximal reabsorption is well established. The left kidney (LK) was denervated before induction of STZ-DM. Subsequently, the normal diet was continued or a low NaCl diet was initiated and 1 week later animals were prepared for clearance experiments under anesthesia including ureter catheterization to measure GFR for each kidney. In diabetic rats, the right innervated as well as the left denervated kidney showed higher values for GFR and kidney weight in animals on a low versus a normal NaCl diet indicating that the salt paradox occurs independent of renal innervation. In addition, evidence is provided that the renal nerves of non-diabetic rats do not contribute to renal Na+ retention during dietary NaCl restriction but modulate renal hemodynamics and kidney weight under these conditions.

Renal effects of exogenous dopamine: modulation by renal nerves and dopamine receptor antagonists

The modulation of the renal response to exogenous dopamine by renal denervation (DNX) and dopamine receptor antagonists was investigated in thiopental-anesthetized Sprague-Dawley rats. Experiments were performed after reaching stable systemic hemodynamics and urinary flow rate. These conditions were obtained with an infusion rate of approximately 1.2% of body weight per hour. In the vehicle group (VHC) i.v. infusion of dopamine (1, 3 and 9 µg kg–1 min–1) significantly increased glomerular filtration rate (GFR), assessed by renal clearance of [3H]inulin, by 14±1.5, 16±1.6 and 31±2.6%, respectively. Infusion of 1 and 3 µg kg–1 min–1 dopamine did not change systemic hemodynamics while the highest dose elevated heart rate, potentially contributing to the GFR increase. The specific D1 receptor antagonist SCH 23390 (10 µg kg–1 min–1 i.v.) did not affect the GFR response to dopamine infusion. In contrast, domperidone (DOM; 8 µg kg–1 min–1 i.v.), a specific, peripherally acting D2 antagonist, attenuated the glomerular hyperfiltration induced by the three doses of dopamine to 11±1.7, 13±2.2 and 16±2.6%, respectively. DNX diminished the GFR response to dopamine infusion to almost the same extent (11±2.8, 10±2.2 and 17±2.6%, respectively) as did DOM. When DNX animals were treated with DOM, the GFR responses to dopamine were further attenuated to non-significant increases. These additive effects of DOM and DNX suggest that two different mechanisms are involved. Both DNX and SCH 23390 decreased sodium excretion at baseline whereas DOM enhanced it. Under the present experimental condition, neither D1 nor D2 receptor blockade affected the natriuretic and diuretic response to dopamine. Whereas D1 receptors do not appear to be involved, both D2 receptors and renal nerves play a role in the renal hemodynamic response to dopamine, indicating involvement of both pre- and postsynaptical dopamine receptors.

Norepinephrine and cerebral blood flow regulation in patients with arteriovenous malformations

OBJECTIVE To test the hypothesis that the sympathetic nervous system plays a role in cerebral blood flow regulation in patients with arteriovenous malformations (AVM). METHODS Cortical interstitial norepinephrine was measured by means of microdialysis, regional cerebral blood flow was measured by a thermal diffusion technique, and regional oxygen saturation (SO2) was measured by microspectrophotometry in 12 patients harboring cerebral AVMs (AVM group) and in 15 patients with deep-seated nonvascular lesions (control group) before and after resection. Measurements were compared according to groups and times of measurements. All values are given as means +/- standard deviation. RESULTS Cortical regional SO2 increased significantly (P < 0.05) in both groups after surgery (AVM group: presurgery 52.4 +/- 12.5% SO2, postsurgery 71.4 +/- 7.4% SO2; control group: presurgery 57.1 +/- 8.4% SO2, postsurgery 69.9 +/- 8.7% SO2), whereas regional cerebral blood flow increased only in the AVM group (AVM group: presurgery 18.9 +/- 6.6 ml/100 g/min, postsurgery 26.2 +/- 6.9 ml/100 g/min; control group: presurgery 20.1 +/- 7.6 ml/100 g/min, postsurgery 19.4 +/- 7.8 ml/100 g/min). Norepinephrine concentrations were significantly lower in the AVM group as compared with the control group before surgery. Although there was no significant difference between pre- and postsurgery conditions in the AVM group, the norepinephrine level of the control group was significantly lower after surgery (AVM group: presurgery 3.3 +/- 1.2 nmol/L, postsurgery 2.9 +/- 1.7 nmol/L; control group: presurgery 5.4 +/- 1.4 nmol/L, postsurgery 4.2 +/- 1.1 nmol/L). CONCLUSION Chronically lowered perfusion pressure seems to induce the hypothesized adaptive down-regulation of sympathetic nervous system activity, yet protective up-regulation after a sudden elevation of cerebral perfusion pressure after AVM excision could not be shown in this study.

Disprocynium24 induces a dopamine-independent, eukaliuric diuresis and natriuresis in the anaesthetized rat

In the anaesthetized rat, intravenous administration of the isocyanine 1,1′-diisopropyl-2,4′-cyanine (disprocynium24) at doses up to 600 μg/kg resulted in marked diuresis and natriuresis without affecting urinary potassium excretion. Fractional sodium excretion was increased over 10-fold indicating a high ceiling-diuretic action. The effects of disprocynium24 on renal function were accompanied by a dose-dependent reduction in heart rate (HR) and mean arterial blood pressure (MAP). Acute administration of 600 μg/kg disprocynium24 decreased MAP by 25% and, in addition, caused a fall in glomerular filtration rate (GFR). Since i) disprocynium24 has been shown to interfere with urinary dopamine excretion (UDAV) and ii) dopamine has been implicated with the regulation of renal sodium excretion, we hypothesized that the effects of disprocynium24 might be mediated by its effects on renal dopamine handling. The following findings, however, argue against this hypothesis. First, administration of disprocynium24 in single doses up to 600 μg/kg caused a diuresis and natriuresis, but did not significantly affect UDAV. Second, neither the systemic nor the renal response to disprocynium24 were markedly altered by pretreatment with the dopamine D1- or D2-receptor blockers SCH23390 (10 μg · kg–1· min–1) or S(-)sulpiride (15 μg · kg–1· min–1), respectively.

Effect of L-Dopa Decarboxylase Inhibitor Benserazide on Renal Function in Streptozotocin-Diabetic Rats

Background/Aims: Benserazide (BZD), an inhibitor of the dopamine synthesis, abolished the increase in glomerular filtration rate (GFR) following the infusion of a mixed amino acid solution. These results reveal endogenous dopamine as a mediator in the renal response to amino acids. The aim of the present study was to evaluate whether dopamine is also involved in the regulation of glomerular hyperfiltration during the early state of diabetes mellitus (DM). Methods: Male Sprague-Dawley rats were injected with a single dose of streptozotocin (60 mg/kg i.p.) for induction of experimental DM (n = 7–8/group). Age-matched non-diabetic animals, injected with citrate buffer, served as controls (CON, n = 8/group). Clearance experiments were performed 2 weeks after induction of DM in thiopental-anesthetized rats (80 mg/kg i.p.), which were continuously infused either with BZD (30 µg/min/kg) or vehicle (VHC). Results: Mean arterial blood pressure was around 110 mm Hg and did not significantly differ among the groups. GFR was 0.95 ± 0.02 ml/min/100 g b.w. in VHC-treated CON. BZD treatment did not significantly change GFR in the CON group (0.92 ± 0.06 ml/min/100 g b.w.). As expected, glomerular hyperfiltration was observed in diabetic rats infused with VHC (1.24 ± 0.08 ml/min/100 g b.w.). Treatment with BZD significantly reduced the diabetes-induced increase in GFR to control levels (0.95 ± 0.05 ml/min/100 g b.w.). Conclusion: Our results show that the inhibition of dopamine synthesis prevented the increase in GFR due to diabetic conditions, indicating that endogenous dopamine is involved in the regulation of DM-induced changes in renal hemodynamics.