High glucose inhibits glucose uptake in renal proximal tubule cells by oxidative stress and protein kinase C

BACKGROUND: High glucose has been considered to play an important role in alteration of renal proximal tubule transporter's activity. This study examined the mechanism by which high glucose modulates alpha-methyl-D-glucopyranoside (alpha-MG) uptake in primary cultured rabbit renal proximal tubule cells (PTCs). METHODS: PTCs were incubated with 25 mmol/L glucose alone or combined with taurine, ascorbic acid, catalase, staurosporine, and bisindolylmaleimide I. Then alpha-MG uptake and lipid peroxide (LPO) formation were examined. RESULTS: Twenty-five mmol/L glucose from four hours, but not 25 mmol/L mannitol, inhibited alpha-MG uptake by 23% compared with 5 mmol/L glucose (control). In the study to examine the relationship of oxidative stress in the high-glucose-induced inhibition of alpha-MG uptake, 25 mmol/L glucose significantly increased LPO by 27% compared with control. However, 10 mmol/L glucose did not affect alpha-MG uptake and LPO formation. Taurine (2 mmol/L), ascorbic acid (1 mmol/L), endogenous antioxidants, or catalase (600 U/mL) significantly blocked 25 mmol/L glucose-induced increase of LPO formation and inhibition of alpha-MG uptake. In the experiment to examine the effects of protein kinase C on LPO formation, 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 ng/mL) increased LPO formation, and staurosporine (10(-7) mol/L) and bisindolylmaleimide I (10(-6) mol/L) totally blocked 25 mmol/L glucose-induced increase of LPO formation and inhibition of alpha-MG uptake. In addition, taurine reduced TPA-induced increase of LPO formation and inhibition of alpha-MG uptake. CONCLUSION: High glucose induces, in part, the inhibition of alpha-MG uptake through LPO formation, and activation of protein kinase C may play a role in high-glucose-induced LPO formation in the primary cultured rabbit renal PTCs.     

Effect of bee venom and its melittin on apical transporters of renal proximal tubule cells

Renal failure by bee venom may be related to a malfunction of renal transporters. However, the effects of bee venom on apical membrane transporters of renal proximal tubular cells are not yet known. The aim of this study was to examine the effects of dried bee venom of Apis mellifera and its melittin on apical transporter activity of primary cultured rabbit kidney proximal tubule cells. Bee venom (1 microg/ml) decreased the cell viability and increased lactate dehydrogenase activity over 30-min treatments. Its effect was blocked by mepacrine or AACOCF(3) (10(-6) M; phospholipase A(2) inhibitors). However, there was no effect on cell viability at a concentration of 0.01 microg/ml of bee venom. Thus, we investigated the effect of bee venom (1 microg/ml) on the activity of renal transporters at 30 min. Bee venom inhibited alpha-methyl-D-glucopyranoside, Pi, and Na(+) uptakes, but increased Ca(2+) uptake. These effects of bee venom were blocked by mepacrine or AACOCF(3) (10(-6) M), and bee venom-induced stimulation of Ca(2+) uptake was also blocked by methoxyverapamil and nifedipine (L-type calcium channel blockers). In addition, bee venom increased [(3)H]-arachidonic acid release by 216 % of that of control. In all experiments, bee venom melittin (0.5 microg/ml) had an identical effect to that of bee venom itself. In conclusion, bee venom inhibited, in part, alpha-MG, Pi, and Na(+) uptakes through its melittin which increased Ca(2+) uptake and arachidonic acid release in primary cultured rabbit renal proximal tubule cells.     

Mechanism of regulation of Na+ transport by angiotensin II in primary renal cells

BACKGROUND: Angiotensin II (Ang II) has a dose-dependent, biphasic effect on the activity of the Na+/H+ antiport system in the renal proximal tubule (RPT). The aim of the present study was to further delineate the signaling pathways involved in Ang II action. METHODS: To examine Ang II signaling, 22Na+ uptake studies were conducted with a primary rabbit RPT cell culture system. The activation of phospholipase A2 (PLA2) was assessed by measuring the release of [3H]-arachidonic acid (AA), and changes in intracellular calcium levels were determined by means of confocal microscopy. RESULTS: Low dosages of Ang II (<10-10 mol/L) stimulated Na+ uptake, whereas high dosages of Ang II (>10-10 mol/L) inhibited Na+ uptake. Ang II (>10-10 mol/L) also caused an increase in AA release associated with an increase in intracellular calcium. Not only did exogenous AA inhibit Na+ uptake, but two PLA2 inhibitors (mepacrine and AACOCF3) blocked the Ang II-mediated inhibition of Na+ uptake. However, the cytochrome P450-dependent epoxygenase inhibitor econazole also blocked the Ang II-induced inhibition of Na+ uptake. Inhibition of Na+ uptake was obtained by the metabolic product of the epoxygenase 5,6-EET. In turn, the inhibitory effect of 5,6-EET was blocked by indomethacin. CONCLUSIONS: The results indicate the involvement of a calcium-dependent PLA2 in mediating the inhibitory effect of Ang II on Na+ uptake. The AA, which is released following PLA2 activation, acts indirectly, through its own metabolism, via a cytochrome P450 epoxygenase pathway and ultimately cyclooxygenase itself.     

The water-soluble fraction (<10 kD) of bee venom (Apis mellifera) produces inhibitory effect on apical transporters in renal proximal tubule cells

Human envenomation caused by bee stings has been reported to cause acute renal failure and the pathogenetic mechanisms of these renal functional changes are still unclear. Bee venom is also a complex mixture of enzymes and proteins. Thus, this study was conducted to examine the effects of bee venom (BV, Apis mellifera) fractions on apical transporters' activity and its related signal pathways in primary cultured renal proximal tubule cells. Whole BV was extracted into three fractions according to solubility [a water-soluble fraction (BVA), an ethylacetate-soluble fraction (BVE), and a hexane-soluble fraction (BVH)]. BVA fraction was further separated to three portions according to molecular weights: BF1 (>20 kD), BF2 (10-20 kD), and BF3 (<10 kD). Each fraction was treated to the PTCs to the ratio of BV (1 microg/ml). BVA (930 ng/ml) significantly decreased cell viability, but BVH (27 ng/ml) and BVE (43 ng/ml) did not. BF3 (710 ng/ml) among BVA fractions predominantly decreased cell viability and inhibited alpha-methyl-D-glucopyranoside (alpha-MG), phosphate (Pi), and Na(+) uptake. In addition, BF3 increased [(3)H] arachidonic acid release, lipid peroxide formation, and Ca(2+) uptake. These effects of BF3 were blocked by mepacrine and AACOCF(3) (phospholipase A(2) inhibitors) or N-acetylcysteine, vitamin C, and vitamin E (antioxidants). In conclusion, BF3 (<10 kD) among BV fractions is the most effective portion in BV-induced inhibition of alpha-MG, P(i), and Na(+) uptake and these effects of BF3 are associated with phospholipase A(2)-oxidative stress-Ca(2+) signal cascade in the primary cultured rabbit renal proximal tubule cells.     

Modulation of rat prostatic inhibinlike peptide (PIP) by steroids and protein hormones

Prostatic inhibinlike peptide (PIP) was detected in the ventral but not in the dorsal lobe of rat prostate. On orchiectomy, PIP concentration in the ventral prostate increased significantly, whereas it decreased on testosterone administration and attained value comparable with that in intact control. Estradiol-17 beta treated animals exhibited high levels of PIP in absence of significant alterations in the protein content. The effect of prolactin and human chorionic gonadotropin on PIP concentration was not so prominent at the dose levels studied. The present study thus demonstrates negative regulation of PIP by testosterone and stimulatory effect of estradiol-17 beta on PIP in rat ventral prostrate.     

Dopamine inhibits renal Na+:HCO3- cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats

BACKGROUND: Dopamine (DA) is thought to regulate renal proximal transport through the inhibition of the Na+,K+-ATPase and/or Na+/H+ exchanger. Defects in this dopaminergic system are proposed to be a pathogenic factor of genetic hypertension. However, microperfusion studies have not consistently confirmed direct tubular effects of DA. METHODS: Isolated proximal straight tubules were perfused peritubularly with Dulbecco's modified Eagle's tissue culture medium (DMEM) containing norepinephrine (NE) to improve incubation conditions. Intracellular Na+ concentrations ([Na+]i) and cell pH (pHi) were measured with fluorescence probes. RESULTS: When incubated in DMEM plus NE, DA increased [Na+]i in rabbit tubules. Inhibition of Na+,K+-ATPase could not explain this response, as it was not suppressed by ouabain. An analysis of pHi responses to bath HCO3- reduction revealed that DA, SKF 38393 (a DA1 agonist), and adenosine 3',5'-cyclic monophosphate (cAMP) inhibited the basolateral Na+:HCO3- cotransporter in rabbit and Wistar-Kyoto rat (WKY), if its transport stoichiometry was converted to 3 HCO3-:1 Na+ by DMEM plus NE incubation. The inhibitory effect of DA was abolished by SCH 23390, a DA1 antagonist, but not by (-)-sulpiride, a DA2 antagonist. In spontaneously hypertensive rats (SHRs), however, DA and SKF 38393 failed to inhibit the cotransporter, although the inhibitory effects of cAMP and parathyroid hormone were comparable to those in WKY. CONCLUSION: These results indicate that DA inhibits the Na+:HCO3- cotransporter in renal proximal tubules and also suggest that dysregulation of the cotransporter, possibly through the defect in DA1 receptor signaling, could play an important role in development of hypertension in SHRs.     

In vitro cyclosporine toxicity. The effect of verapamil

The epithelial cell line LLC-PK1, which expresses many proximal tubular characteristics, was used to investigate the relationship between calcium, the calcium channel blocker verapamil, and cyclosporine toxicity. The LLC-PK1 cells took up cyclosporine when this was added in a concentration of 2 micrograms/ml, and this uptake was maximal at 30 min (112 +/- 3 ng cyclosporine/mg cell protein). At 12 micrograms/ml it inhibited the sodium glucose cotransporter, as assessed by phlorizin-inhibitable 14C-alpha-methyl glucopyranoside (alpha-MG) uptake (control 37.2 +/- 6.3, 12 micrograms/ml 21.2 +/- 1.1 mumol/hr/mg protein). Cyclosporine at 2 micrograms/ml did not affect cell growth after 5 days (control 945 +/- 60 micrograms cell protein per 25 cm2 flask, 2 micrograms/ml cyclosporine/ml 1046 +/- 32 micrograms protein/flask), even in the presence of 7.6 mM ionized calcium (862 +/- 37 micrograms protein/flask). Cyclosporine at 12 micrograms/ml inhibited cell growth (286 +/- 27 micrograms protein/flask), and raising the ambient ionized calcium concentration to 7.6 mM reduced cell growth further (91 +/- 6 micrograms protein/flask). Cyclosporine at concentrations of 2 and 12 micrograms/ml produced increasing cell vacuolation, as seen in vivo. Short-term uptake of 2 micrograms/ml cyclosporine could be inhibited by 1.0 mM and 0.5 mM verapamil (49 +/- 9.5 and 71 +/- 6.4 ng cyclosporine/mg cell protein, respectively, at 30 min). However, in the presence of 2 micrograms/ml cyclosporine 0.1 mM verapamil was toxic to the cells grown over five days (44 +/- 5 micrograms protein/flask). At 0.01 mM verapamil was not toxic to cell growth (921 +/- 29 micrograms protein/flask), but raising the medium calcium to 7.6 mM reduced cell growth (652 +/- 96 micrograms/ml). Inhibition of cyclosporine uptake did not occur with 0.01 mm verapamil (control 145.6 +/- 12.3 vs. 0.01 mM verapamil 150.4 +/- 3.8 ng cyclosporine/mg cell protein). The LLC-PK1 cell line represents a good in vitro model for cyclosporine renal tubular toxicity, as the in vivo observation of glycosuria and proximal tubular cell vacuolation in cyclosporine nephrotoxicity can be reproduced. In vitro this is shown to be associated with inhibition of sodium-dependent glucose cotransport. Verapamil inhibited cyclosporine uptake, but only at concentrations that were toxic to the cells. Verapamil potentiated rather than reduced the increased cyclosporine toxicity produced by increasing the medium calcium concentration. The suggested protective effect of verapamil against cyclosporine nephrotoxicity is therefore unlikely to be due to inhibition of cyclosporine uptake or of calcium entry into proximal tubular cells.     

Effects of gonadal hormones on thymus gland after bilateral ovariectomy and orchidectomy in rats

This study examined the histological changes that occurred in the thymus gland after gonadectomy and the administration of various sex steroids following gonadectomy. Male and female Wistar albino rats that were 6 weeks of age were used. The rats were subjected to bilaterally gonadectomy and then gonadal steroid hormones (testosterone, estrogen, and progesterone, 2.5 mg/kg) were given. Effects of gonadal steroid hormones on the thymus gland were microscopically examined. Thymic weight increased in all the groups after gonadectomy. Testosterone, estrogen, and estrogen + progesterone treatment decreased thymic weight after gonadectomy. Progesterone treatment also decreased weight, but there was no statistical significance. In the light microscopy, testosterone and estrogen treatment induced a loss of lymphoid elements from the thymic cortex, increased the number of phagocytic macrophages and mast cells, and enlarged blood vessels and connective tissue were observed in the thymic medulla. In the electron microscopic study it was observed that rough endoplasmic reticulum enlarged in the thymic lymphocytes. The same results were also found after estrogen + progesterone treatment. No histologically identifiable changes were observed in the thymus gland after progesterone treatment. This study demonstrated that the thymus gland undergoes involution after testosterone and estrogen treatment, but not progesterone, following gonadectomy.     

Hormonal effects of high dose medroxyprogesterone acetate treatment in males with renal or prostatic adenocarcinoma

In 18 patients, 12 with renal and 6 with prostatic carcinoma, the gonadal, pituitary and adrenal functions were studied by measurements of steroid hormones and gonadotrophins, before and after six weeks treatment with medroxyprogesterone acetate (MPA), injected intramuscularly 500 mg per day for 5 days each week. The testosterone-oestradiol-binding globulin (TeBg) was measured and the amount of albumin and TeBg bound and unbound testosterone was calculated. Treatment with high doses of MPA caused a profound decrease in serum concentrations of testosterone, dehydroepiandrosterone sulphate (DHEAS), cortisol and TeBg. There were significant decreases in serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH) and oestradiol-17 beta. The serum concentration of prolactin was significantly elevated. The protein unbound testosterone fraction was lowered by MPA treatment but less than total testosterone. In conclusion, MPA therapy in high dose alters the gonadal, pituitary and adrenal functions suppressing serum concentrations of androgens, gonadotrophins, cortisol and TeBg but elevating prolactin concentration.     

The role of the functional interrelation of the adrenals and testes in the pathogenesis of sterility in patients with left-sided varicocele

Selective renal phlebography, phlebotonometry, selective analysis of the blood from the renal veins and vena cava inferior for the activity of plasma renin, catecholamines, prostaglandins E2, pO2 and pCO2, peripheral blood analysis for levels of progesterone, androstendione, testosterone, ACTH, ACTH-tolerance test, orchidometry, ejaculate microscopy, evaluation of seminal plasma testosterone, trochanter index and parameters of sexual maturation were performed during the treatment of 70 patients with left varicocele. Based on the results the authors concluded that not only the left testicle, but the left adrenal was involved in the course of organic renal venous hypertension. A significant feedback correlation was revealed between the peripheral blood progesterone and the left kidney venous pressure (r = -0.50, p less than 0.01) and between the peripheral blood progesterone and the number of motile spermatozoa in the ejaculate (r = -0.31, p less than 0.05). Pathogenesis of organic renal venous hypertension and spermatogenesis failure were supplemented by the conclusion that the left adrenal central vein was the first to involve into the compensation of venous hypertension. Retrograde alterations in the direction of the blood flow in the left adrenal central vein resulted in the abnormal stimulation of steroidogenesis in the cortical layer. The excessive production of antiandrogenic steroid hormones by the left adrenal gland was a cause of spermatogenesis damage in both testicles. The blockage of the left adrenal androgenic hormones of the hypothalamo-hypophyseal system can deteriorate the process.(ABSTRACT TRUNCATED AT 250 WORDS)     

gamma-L-glutamyl-L-DOPA inhibits Na(+)-phosphate cotransport across renal brush border membranes and increases renal excretion of phosphate

BACKGROUND: For treatment of phosphate (Pi) overload in various pathophysiological states, an agent that selectively increases renal Pi excretion would be of major value. Previously, we have shown that dopamine (DA) inhibits Na(+)-Pi cotransport in renal epithelia. However, the administration of DA or its immediate precursor L-DOPA increases DA in multiple tissues. Synthetic dipeptide gamma-L-glutamyl-L-DOPA (gludopa) can serve as an inactive precursor (pro-pro-drug) of DA. This study tested the hypothesis that, because of the unique colocalization of gamma-glutamyltransferase (gamma-GT), aromatic amino acid decarboxylase, Na(+)-Pi cotransporter, and Na(+)-L-DOPA cotransporter in brush border membrane (BBM) of proximal tubular cells, gludopa may elicit phosphaturia by action of DA generated within the kidney. METHODS: Thyroparathyrectomized rats were given placebo, or gludopa, or gludopa + gamma-GT inhibitor acivicin. Urinary excretion of Pi, Ca2+, Na+, K+, DA, cAMP, and cGMP was determined, and Na(+)-Pi cotransport was measured in BBM prepared from kidneys of rats at the end of the experiment. RESULTS: The administration of gludopa resulted in: (a) an inhibition of Na(+)-Pi cotransport, but not cotransport of Na(+)-proline and Na(+)-alanine in BBM; (b) an increase (+300%) of fractional excretion (FE) of Pi and a drop (-35%) of plasma Pi, whereas the plasma levels and FEs of Ca2+, Na+, and K+ were unchanged; (c) an increase in urinary excretion of cAMP. but not cGMP; (d) a 1000-fold increase of urinary excretion of DA, without a change in excretion of norepinephrine; and (e) an incubation of gludopa with BBM in vitro, which caused a release of L-DOPA, and the in vivo administration of acivicin, which blocked actions of gludopa to inhibit Na(+)-Pi cotransport and to increase urinary excretions of Pi and DA. CONCLUSIONS: We conclude that colocalization of enzymes of biotransformation, BBM transporters, and the autocrine/paracrine DA system in cells of proximal tubules constitutes a cellular basis for the potent and specific phosphaturic action of gludopa.     

Molecular aspects of renal tubular handling and regulation of inorganic sulfate

The renal proximal tubular reabsorption of sulfate plays an important role in the maintenance of sulfate homeostasis. Two different renal sulfate transport systems have been identified and characterized at the molecular level in the past few years: NaSi-1 and Sat-1. NaSi-1 belongs to a Na(+)-coupled transporter family comprising the Na(+)-dicarboxylate transporters and the recently characterized SUT1 sulfate transporter. NaSi-1 is a Na(+)-sulfate cotransporter located exclusively in the brush border membrane of renal proximal tubular and ileal cells. Recently, NaSi-1 was shown to be regulated at the protein and mRNA level by a number of factors, such as vitamin D, dietary sulfate, glucocorticoids and thyroid hormones, which are known to modulate sulfate reabsorption in vivo. The second member of renal sulfate transporters, denoted Sat-1, belongs to a family of Na+-independent sulfate transporter family comprising the DTDST, DRA and PDS genes. Sat-1 is a sulfate/bicarbonate-oxalate exchanger located at the basolateral membrane of proximal tubular epithelial cells and canalicular surface of hepatic cells. Contrary to NaSi-1, no physiological factor has been found to date to regulate Sat-1 gene expression. Both NaSi-1 and Sat-1 transporter activities are implicated in pathophysiological states such as heavy metal intoxication and chronic renal failure. This review focuses on recent developments in the molecular characterization of NaSi-1 and Sat-1 and the mechanisms involved in their regulation.     

PTH-Induced downregulation of the type IIa Na/P(i)-cotransporter is independent of known endocytic motifs

Parathyroid hormone (PTH)-induced inhibition of renal proximal tubular Na/P(i) cotransport involves two consecutive steps: endocytosis followed by lysosomal degradation of the type IIa Na/P(i) cotransporter. Tyrosine-, dileucine-, and diacidic-based motifs are suggested to be involved in endocytosis and/or lysosomal targeting of different plasma membrane proteins. The rat type IIa cotransporter (NaPi2) contains two cytoplasmic tyrosine residues (Y) within sequences highly homologous to tyrosine-based motifs (GY(402)FAM and Y(509)RWF), three cytoplasmic dileucine (LL(101), LL(374), and LI(591)) and two cytoplasmic diacidic motifs (EE(81) and EE(616)). We studied the role of these motifs on the PTH-induced retrieval and lysosomal degradation of the NaPi2 cotransporter. To follow its trafficking in vivo, the NaPi2 protein was fused to the carboxyl-terminal end of the enhanced green fluorescence protein. This fusion did not impair the apical targeting or the PTH-induced endocytosis of the wild-type cotransporter when transfected in opossum kidney cells. Single and multiple Y and LL mutants retained the apical targeting and the PTH-induced degradation. Mutations of the diacidic motifs were also without effect. These data suggest that the above three motifs are not required for the PTH-induced internalization and/or degradation of the cotransporter.     

G-protein stimulation inhibits amiloride-sensitive Na/H exchange independently of cyclic AMP.

G-proteins are heterotrimeric proteins involved in many transmembrane signaling events. Both the renal basolateral membrane and the renal brush border membrane contain large quantities of these proteins. G-proteins appear related to hormonal signaling in the basolateral membrane and presumably affect ion gating in the brush border. We investigated the influence of G-proteins on the amiloride-sensitive Na/H exchanger, the activity of which is regulated at least in part by cAMP-dependent protein kinase, by measuring the amiloride-sensitive component of [22Na+] uptake in rat renal brush border membrane vesicles (BBMV) in the presence of a pH gradient. Incubation of vesicles with AlF4- (10 microM Al3+, 10 mM F-) resulted in significant inhibition of amiloride-sensitive [22Na+] uptake at both 20 seconds and 5 minutes of incubation. Incorporation of GTP gamma S into BBMV by transient hypotonic lysis also resulted in significantly reduced amiloride-sensitive [22Na+] uptake compared to controls at both time points. This inhibition could be reversed by GDP beta S. Similar lysis in the presence of 10 microM GDP beta S alone had no significant effect. When Na(+)-dependent [14C]-D-glucose uptake into BBMV was studied no significant effect of these G-protein modulating agents was observed. Adenylate cyclase activity could not be stimulated in these BBMV preparations using standard techniques. Furthermore, cAMP-dependent protein kinase activity, strongly stimulated in these BBMV by exogenously added cAMP, was not stimulated by 10 microM GTP gamma S alone. These findings suggest that the amiloride-sensitive Na/H exchanger can be regulated by G-proteins independently of adenylate cyclase and cAMP-dependent protein kinase.     

The binding of sex steroids to human spermatozoa An autoradiographic study

The locations of steroid binding sites with specificities for 17β-oestradiol, progesterone and testosterone have been examined on human spermatozoa by autoradiography. The binding sites were not evenly distributed over the entire surface of human spermatozoa. They were more densely located on the midpiece of the tail, less on the head and neck, and least on the principal- and end pieces of the tail. The binding sites for 17β-oestradiol were specific, with ³H-17β-oestradiol being displayed by unlabelled 17β-oestradiol, but less readily displaced by either progesterone or testosterone. The binding sites for testosterone were less specific than those for 17β-oestradiol or those for progesterone. Both 17β-oestradiol and progesterone were good competitors for testosterone binding sites, while the binding of ³H-progesterone was easily displaced by 17β-oestradiol, but less readily displayed by testosterone. Since there was preferential binding of these steroids to the mid-piece, and strong competition for binding between 17β-oestradiol and progesterone, these studies appear to provide information on the sites of action for the reported effects of these and related steroids on spermatozoal motility, migration and metabolism.     

P-glycoprotein inhibitors stimulate renal phosphate reabsorption in rats

BACKGROUND: Dipyridamole (Dip) was previously shown to increase renal phosphate (Pi) reabsorption in humans. However, the mechanism(s) underlying this renal tubular effect is not fully elucidated. It is known that Dip inhibits the activity of the P-glycoprotein (Pgp) multidrug resistance protein 1 (MDR1) expressed on the apical membrane of renal proximal tubular cells where the Na-Pi cotransporter (NPT2) is also expressed. We hypothesized that Dip could increase renal Pi reabsorption by inhibiting Pgp activity. METHODS: To test this hypothesis, the effects of Dip, verapamil (Ver), and cyclosporine A (CsA), three unrelated Pgp inhibitors, were studied on the renal Pi reabsorption in rats. RESULTS: All three drugs decreased the fractional excretion of Pi (FE(Pi)) in a dose-dependent manner within one hour after beginning the drug infusion, without altering the glomerular filtration rate or serum parathyroid hormone concentration. Sodium-dependent Pi uptake but not Na-glucose transport was increased in brush-border membrane vesicles (BBMVs) when comparing treated with untreated rats. Western blot analysis showed that NPT2 protein was increased in BBMVs from treated rats. Dip and Ver had no effect when applied directly to BBMVs prepared from untreated rats. Pretreatment of rats with colchicine prevented the effects of Dip on the FE(Pi) and NPT2 expression in brush-border membranes. CONCLUSIONS: Our results suggest that inhibition of Pgp in the proximal tubule increases Pi uptake and NPT2 translocation to the apical membrane.     

Effects of cardiac glycosides on sodium pump expression and function in LLC-PK1 and MDCK cells

BACKGROUND: The decreases in proximal tubule sodium reabsorption seen with chronic renal failure and volume expansion have been ascribed to circulating digitalis-like substances (DLS). However, the circulating concentrations of DLS do not acutely inhibit the sodium pump to a degree consistent with the observed changes in proximal tubule sodium reabsorption. METHODS: We examined how cell lines that simulated proximal (LLC-PK1) and distal tubule (MDCK) cells responded to acute (30 min) and long-term (up to 12 hours) Na+,K+-ATPase inhibition with DLS. RESULTS: In LLC-PK1, but not MDCK cells, low concentrations of ouabain decreased 86Rb uptake profoundly in a time and dose dependent manner. In LLC-PK1 cells grown to confluence, transcellular 22Na flux was markedly reduced in concert with the decreases in 86Rb uptake. Similar findings were observed with marinobufagenin (MBG) and deproteinated extract of serum derived from patients with chronic renal failure. However, inhibition of the Na+,K+-ATPase with low extracellular potassium concentrations did not produce any of these effects. Western and Northern blots detected no change in alpha1 Na+,K+-ATPase protein and message RNA, respectively, in LLC-PK1 cells treated with ouabain for 12 hours. However, the decrease in enzymatic activity of Na+,K+-ATPase of these cells was comparable to observed decreases in 86Rb uptake. Differential centrifugation as well as biotinylation experiments demonstrated a shift of the Na+,K+-ATPase from the plasmalemma with prolonged ouabain treatment. CONCLUSIONS: The results show that binding of cardiac glycosides by proximal (but not distal) tubular cells results in internalization of Na+,K+-ATPase with the net effect to amplify inhibition of the Na+,K+-ATPase. As the circulating concentrations of DLS increase with chronic renal failure and volume expansion, we suggest that this phenomenon explains some of the decreased sodium reabsorption by the proximal tubule seen in these conditions.     

The stimulatory role of estrogen on sperm motility in the male golden hamster (Mesocricetus auratus)

To clarify the physiological roles of estrogens in the regulation of sperm motility in the golden hamster, two different approaches were used. In the first experiment, silastic tubes containing either low (low E2 group) or high (high E2 group) amount of estradiol-17beta were implanted (Exp 1). In the second experiment, male golden hamsters were actively immunized against estradiol-17beta (Exp 2). In Exp 1, all sperm motility parameters (including motility, straight velocity, curvilinear velocity, beat/cross frequency, and mean amplitude of lateral head displacement) were significantly increased except linear index in the high E2 group as compared with controls at 20 days after the treatment. In the high E2 group, plasma concentrations of luteinizing hormone (LH) significantly increased, whereas levels of circulating testosterone decreased significantly. Plasma concentrations of follicle-stimulating hormone (FSH) and immunoreactive inhibin were not affected by the treatment with estradiol-17beta. In the Exp 2, titer of circulating antibodies to estradiol-17beta consistently increased after the second immunization until the end of experiment (16 weeks). The sperm motility, straight velocity, and curvilinear velocity were significantly decreased after active immunization to estradiol-17beta. Concentrations of circulating LH and FSH were also decreased significantly by the treatment. In conclusion, the current observations indicate that estradiol-17beta affects sperm motility in adult male golden hamsters.     

Effects of oestradiol-17 beta and ethinyl oestradiol on human testicular steroidogenesis in vitro

The inhibition of human testicular testosterone production in vitro by oestrogen was studied by measuring effects of oestradiol-17 beta and ethinyl oestradiol on the 3 beta-hydroxysteroid dehydrogenase/isomerase and the delta 4-pathway of testosterone synthesis. Progesterone, 17 alpha-hydroxyprogesterone, androstenedione and testosterone concentrations of incubation media were all depressed after oestrogen addition. To minimize differences in substrate amounts between incubations, 3H-labelled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone or androstenedione was added to the incubation media. The conversion of pregnenolone, progesterone and 17 alpha-hydroxyprogesterone was inhibited by oestrogen. It is concluded that oestrogen inhibits 3 beta-hydroxysteroid dehydrogenase/isomerase, 17 alpha-hydroxylase and C17-20-lyase enzyme activities of human testicular tissue in vitro while 17 beta-ketosteroid reductase activity is unaffected.