Miroslawa Szczepanska-Konkel

Insulin induces expression of adenosine kinase gene in rat lymphocytes by signaling through the mitogen-activated protein kinase pathway.

The activity of adenosine kinase (AK) was significantly impaired in splenocytes isolated from diabetic rats. Administration of insulin to diabetic animals restored AK activity, protein, and mRNA levels in diabetic splenocytes. Experiments performed on cultured rat lymphocytes demonstrated that insulin did not change the stability of AK mRNA. Insulin induced AK gene expression in a dose- and time-dependent manner. Maximal increases in AK mRNA (3.9-fold) and activity level (3.7-fold) were observed at the fourth and fifth hours of cell incubation with 10 nM insulin, respectively. The insulin effect on AK expression was not influenced by dibutyryl cAMP (dcAMP). On the other hand dcAMP weakly increased (1.7-fold) basal expression of AK. Exposure of rat lymphocytes to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), or rapamycin, an inhibitor of mTOR, did not affect the ability of insulin to stimulate expression of AK. Prior treatment of the cells with 10 microM PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK) completely blocked insulin-stimulated expression of AK gene. Insulin produced a significant transient increase in the tyrosine phosphorylation of ERK1/2, and PD98059 inhibited this phosphorylation. Furthermore exposure of cells to insulin has resulted in transient phosphorylation of Elk-1 on Ser-383 and sustained elevation of c-Jun and c-Fos protein. The maximal phosphorylation of Elk-1 was observed at 15 min, and was blocked by PD98059. We concluded that insulin stimulates AK gene expression through a series of events occurring sequentially. This includes activation of the MAPK cascade and subsequent phosphorylation of Elk-1 followed by increased expression of c-fos and c-jun genes.

The effects of P2X receptor agonists on renal sodium and water excretion in anaesthetized rats.

Aim:  To investigate in vivo effects of P2X receptor activation on sodium and water excretion in urine.

INHIBITION OF CGMP : PHOSPHODIESTERASE RESTORES THE GLOMERULAR EFFECTS OF ATRIAL NATRIURETIC FACTOR IN LOW SODIUM DIET RATS

It was shown that atrial natriuretic factor (ANF)-induced increase in glomerular filtration rate (GFR) is blunted in low sodium diet. We investigated whether saralasin, as an angiotensin II receptor antagonist, or verapamil, as a calcium entry blocker, or theophylline and zaprinast, as inhibitors of cGMP-phosphodiesterase activity, could restore the effect of ANF on GFR increase in low sodium diet rats. ANF alone (5 micrograms/kg bolus then 0.5 micrograms/min/kg BW maintenance) increased diuresis and natriuresis to the same extend in low and normal sodium diet rats but had no GFR-increasing effect in low sodium diet rats. Infusion of ANF in the presence of verapamil, saralasin, theophylline or zaprinast induced a significant increase in GFR and filtration fraction (FF). Administration of verapamil or saralasin alone did not alter GFR and FF whereas theophylline or zaprinast alone resulted in moderate but significant increases in both parameters. The increase of nephrogenous cGMP excretion in response to ANF infusion in low sodium diet rats was markedly lower as compared to normal sodium diet rats (243.4 +/- 43.3 vs. 444.0 +/- 35.6 pmol/min, respectively, p < 0.01). Administration of a selective inhibitor of cGMP-phosphodiesterase activity (zaprinast) abolished the differences in ANF-stimulated nephrogenous cGMP excretion in low and normal sodium diet rats. Basal and peak ANF (0.5 microM)-stimulated cGMP formation by isolated glomeruli was significantly lower in low than normal sodium diet rats (1.4 +/- 0.1 vs. 2.9 +/- 0.2 and 7.1 +/- 0.5 vs. 12.5 +/- 1.1 pmol/mg protein, for basal and ANF-stimulated cGMP formation, respectively; both p < 0.05). Zaprinast both alone and in combination with ANF, potentiated cGMP formation by glomeruli isolated from both groups of rats. In the presence of zaprinast, there were no differences in both basal and peak ANF-stimulated cGMP formation by glomeruli isolated from low and normal sodium diet rats. cGMP-phosphodiesterase activity was the same in the medulla of both groups of rats but markedly higher in the renal cortex of low sodium diet rats as compared to normal sodium diet rats (82.6 +/- 6.0 vs. 59.8 +/- 4.3, respectively; p < 0.05). These data suggest that the lack of GFR-increasing response to ANF in low sodium diet rats is primarily due to the increase of cGMP hydrolysis in glomeruli.

P1,P4-diadenosine tetraphosphate (Ap4A) inhibits proximal tubular reabsorption of sodium in rats.

Background/Aims: P¹,P⁴-diadenosine tetraphosphate (Ap₄A) is a vasoactive dinucleotide possessing natriuretic activity. It is unclear, however, which part of the nephron is the target site of action for Ap₄A. Methods: We evaluated the tubular sites of Ap₄A action using the lithium clearance technique. Results: Ap₄A at a priming dose of 2 µmol/kg with subsequent infusion at 20 nmol/kg/min increased fractional water and sodium excretion 2.5- and 5.6-fold, respectively. Moreover, Ap₄A increased lithium clearance 1.9-fold and fractional lithium excretion 2.8-fold. Fractional water and sodium excretion from distal nephron segments was not significantly affected by Ap₄A. Conclusion: These results suggest that Ap₄A induces natriuresis mainly through inhibition of proximal tubular reabsorption of sodium.

Effects of diadenosine polyphosphates on glomerular volume

1 Diadenosine polyphosphates (P1,P3‐diadenosine triphosphate, Ap3A; P1,P4‐diadenosine tetraphosphate, Ap4A; and P1,P5‐diadenosine pentaphosphate, Ap5A) are vasoactive molecules. The experimental model of isolated rat renal glomeruli was used to investigate their effects on glomerular vasculature. We measured the changes of glomerular inulin space (GIS) as a marker of glomeruli contractility. 2 Ap4A and Ap5A induced concentration‐ and time‐dependent reduction of GIS whereas Ap3A had no effect. The effects of Ap4A and Ap5A (both at 1 μM) were prevented by a nonselective P2 receptor antagonist, that is, suramin (10 μM) and P2Y receptor antagonist – reactive blue 2 (50 μM). However, the antagonist of P1 receptor, that is, theophylline (1 μM) and A1 receptor 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 10 μM) did not affect the responses of glomeruli to Ap4A or Ap5A. 3 Ap3A, in contrast to Ap4A and Ap5A, prevented angiotensin II‐induced reduction of GIS in a concentration‐ and time‐dependent manner. This effect was partially prevented by suramin and markedly reduced by reactive blue 2 and the specific antagonist of P2Y1 receptor – MRS 2179 (10 μM). However, theophylline and the specific antagonist of A2 receptor – 3,7‐dimethyl‐1‐propargylxanthine (DMPX; 10 μM) – did not affect Ap3A action. 4 We indicate that diadenosine polyphosphates changed the glomerular volume via activation of P2 receptors. We suggest that extracellular Ap4A and Ap5A via P2X and P2Y receptors may decrease and Ap3A via, at least in part, P2Y1 receptors may increase filtration surface, which in turn may modify glomerular filtration rate.

Modulation by low sodium intake of glomerular response to cicletanine and atrial natriuretic factor

The aim of the study was to investigate whether cicletanine (CIC), as a potential inhibitor of cyclic GMP phosphodiesterase, is able to restore glomerular response to atrial natriuretic factor (ANF) in rats under conditions of diet deprived of sodium. We examined the effects of CIC on glomerular filtration rate (GFR), natriuresis and nephrogenous cyclic GMP excretion in response to ANF and the effects of both agents on intracapillary volume and cyclic GMP accumulation in isolated glomeruli of rats on normal and low sodium diets. CIC (0.25 mg min−1 kg−1 BW) of ANF (0.5 μg min−1 BW) alone, given in pharmacological doses, increased Cin significantly in normal sodium rats, whereas the effect of each agent was blunted in low sodium diet rats. Pretreatment with CIC restored the increase in Cin in response to ANF infusion in low sodium diet rats. In rats on either diet, there were no differences in the extent of diuresis and natriuresis induced by CIC or ANF alone. In contrast to FENa, combined effects of both agents on V and UNaV in rats on normal and low sodium diets were observed. In normal sodium diet rats, CIC 10−4 M or ANF 10−6 M alone inhibited angiotensin II 10−6 M (AII)‐induced decrease in intracapillary volume reflected by the glomerular [3H]‐inulin space (GIS). In contrast, CIC or ANF alone did not inhibit AII‐induced decrease in GIS in low sodium diet rats. Both agents given together inhibited AII‐induced decrease in GIS in low sodium diet rats. CIC both alone and in combination with ANF increased nephrogenous cyclic GMP excretion and cyclic GMP accumulation in isolated glomeruli of rats on normal and low sodium diets. In rats on either diet, CIC abolished the difference in ANF‐stimulated increase in nephrogenous cyclic GMP excretion and cyclic GMP accumulation in glomeruli. These results suggest that CIC and ANF alone induce relaxation of glomeruli and a resultant increase in glomerular filtration rate in normal sodium diet rats; in contrast, these effects are blunted in the low sodium diet rats. CIC restores glomerular response to ANF in low sodium diet rats, apparently involving inhibition of the cyclic GMP phosphodiesterase.

Extracellular purines' action on glomerular albumin permeability in isolated rat glomeruli: insights into the pathogenesis of albuminuria

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes.

Cicletanine New insights into its pharmacological actions

Cicletanine ((+/-)3-(4-chlorophenyl)-1,3-dihydro-7-hydroxy-6-methylfuro-[3,4-c ] pyridine) 3-(4-chlorophenyl)-1,3-dihydro-7-hydroxy-6-methylfuro-[3,4-c] pyridine) is a novel antihypertensive agent that has been shown to possess vasorelaxant, natriuretic, and diuretic properties in preclinical and clinical studies. The mechanism(s) by which cicletanine induces these biological effects has not been definitely established, although it appears to differ from that of other classes of antihypertensive drugs. The salidiuretic activity appears to be the result of an action of the sulfoconjugated metabolite of cicletanine, which inhibits the apical Na+-dependent Cl-/HCO3- anion exchanger in the distal convoluted tubule. The mechanism of the vasodilating effect of cicletanine seems to be complex; it may include stimulation of vascular prostaglandin synthesis, inhibition of the low Km cyclic GMP phosphodiesterases, and blockade of Ca2+ channels either directly or indirectly through a K+-channel opening effect. The drug has also been shown to interact with alpha-adrenergic, vascular histamine, and muscarinic receptors. We have also reviewed the other vascular effects of the drug, such as stimulation of nitric oxide synthesis and inhibition of both myosin light chain kinase and protein kinase C. Cicletanine protects cardiovascular and renal systems against the injuries induced by hypertension, in addition to its lowering of arterial pressure. Similarly to the vasorelaxant action of cicletanine, the various properties of the drug likely contribute to its protective effect against injury in hypertension.