EGF inhibits muscarinic receptor-mediated calcium signaling in a human salivary cell line.

The effects of epidermal growth factor (EGF) on intracellular calcium ([Ca(2+)](i)) responses to the muscarinic agonist carbachol were studied in a human salivary cell line (HSY). Carbachol (10(-4) M)-stimulated [Ca(2+)](i) mobilization was inhibited by 40% after 48-h treatment with 5 x 10(-10) M EGF. EGF also reduced carbachol-induced [Ca(2+)](i) in Ca(2+)-free medium and Ca(2+) influx following repletion of extracellular Ca(2+). Under Ca(2+)-free conditions, thapsigargin, an inhibitor of Ca(2+) uptake to internal stores, induced similar [Ca(2+)](i) signals in control and EGF-treated cells, indicating that internal Ca(2+) stores were unaffected by EGF; however, in cells exposed to thapsigargin, Ca(2+) influx following Ca(2+) repletion was reduced by EGF. Muscarinic receptor density, assessed by binding of the muscarinic receptor antagonist L-[benzilic-4,4'-(3)HCN]quinuclidinyl benzilate ([(3)H]QNB), was decreased by 20% after EGF treatment. Inhibition of the carbachol response by EGF was not altered by phorbol ester-induced downregulation of protein kinase C (PKC) but was enhanced upon PKC activation by a diacylglycerol analog. Phosphorylation of mitogen-activated protein kinase (MAP kinase) and inhibition of the carbachol response by EGF were both blocked by the MAP kinase pathway inhibitor PD-98059. The results suggest that EGF decreases carbachol-induced Ca(2+) release from internal stores and also exerts a direct inhibitory action on Ca(2+) influx. A decline in muscarinic receptor density may contribute to EGF inhibition of carbachol responsiveness. The inhibitory effect of EGF is mediated by the MAP kinase pathway and is potentiated by a distinct modulatory cascade involving activation of PKC. EGF may play a physiological role in regulating muscarinic receptor-stimulated salivary secretion.

Modulation by food restriction of intracellular calcium signaling in parotid acinar cells of aging Fischer 344 rats.

Previous studies suggest that alpha 1-adrenergic (alpha 1-AR)-induced intracellular calcium ([Ca2+]i) mobilization in rat parotid acinar cells declines with age. In this study, we examined the effects of food restriction on alpha 1-AR and muscarinic-stimulated [Ca2+]i mobilization in parotid acinar cells during aging. [Ca2+]i levels in response to the alpha 1-AR agonist epinephrine and the muscarinic agonist carbachol were evaluated in Fura-2-loaded parotid acinar cells from ad libitum-fed (AL) and food-restricted (FR) Fischer 344 male rats at 4, 6, 14, and 24 months of age. [Ca2+]i responses to epinephrine and carbachol (10 microM) were significantly reduced (48% and 35%, respectively; p < .05) in cells from 24-month-old AL rats as compared to younger AL rats. In contrast, no significant reduction of epinephrine and carbachol responses was observed in 24-month-old FR animals. An age-related increase in basal [Ca2+]i (peak around 14 months; p < .02) was observed in both AL and FR rats. In addition, basal [Ca2+]i was higher in FR than in AL rats at 14 and 24 months of age (p < .02). These studies suggest that FR partially attenuates or delays age-related impairments in alpha 1-AR- and muscarinic-cholinergic signal transduction systems of parotid acinar cells. Basal [Ca2+]i also appears to be altered during aging and by FR.

Protein kinase C differentially modulates PTH- and PGE2-sensitive adenylate cyclase in osteoblast-like cells.

The effects of phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, on receptor-mediated stimulation of adenylate cyclase were evaluated in a rat osteosarcoma cell line (UMR-106) with the osteoblast phenotype. Pretreatment of UMR-106 cells with PMA increased parathyroid hormone (PTH)-stimulated adenylate cyclase activity and inhibited prostaglandin E2 (PGE2)-responsive enzyme activity. In addition, PMA enhanced enzyme activation by forskolin, which is thought to exert a direct stimulatory action on the catalytic subunit of adenylate cyclase. The regulatory effects of PMA were concentration dependent and of rapid onset (less than or equal to 1 min). Treatment with PMA also resulted in translocation of protein kinase C activity from the cytosol to the particulate cell fraction. Pertussis toxin, which attenuates inhibition of adenylate cyclase mediated by the inhibitory guanine nucleotide-binding regulatory protein (Gi), augmented PTH-sensitive adenylate cyclase activity and reduced the incremental increase in PTH response produced by PMA. The results suggest that activation of protein kinase C increases PTH-stimulated adenylate cyclase activity by actions on Gi and/or the catalytic subunit and decreases PGE2 responsiveness by a mechanism involving the PGE2 receptor.

B2-microglobulin and associated amyloidosis presenting as bilateral popliteal tumors.

Chronic hemodialysis has led to the prolongation of life in many patients with end-stage renal disease, but has also allowed for the development of new diseases that are a consequence of this clinical setting. B2-microglobulin accumulation leading to systemic amyloidosis may be the most recent disease in this category. This case report documents the development of bilateral popliteal tumors in a patient undergoing chronic hemodialysis for 9 years. Removal of one of the tumors and pathological examination demonstrated amyloid that was positive for B2-microglobulin by specific antibody testing. This case adds further support to the suggestion that B2-microglobulin amyloidosis in chronic hemodialysis patients is truly a systemic disorder. The development of popliteal tumors, particularly in proximity to joints, in a chronic hemodialysis patient, must include amyloidosis in the differential diagnosis.

Prostaglandins and the urinary concentrating defect in potassium-depleted rabbits.

Urinary prostaglandin E (UPGE) excretion increased significantly after 1 and 2 wk of potassium depletion (KD) in female New Zealand White rabbits on ad libitum water intake [UPGE control, 21.3 +/- 4.6 ng PGE/mg creatinine; 1 wk KD, 40.4 +/- 6.1 ng PGE/mg creatinine (P less than 0.01); 2 wk KD, 31.9 +/- 14.9 ng PGE/mg creatinine (P less than 0.05)]. In vivo prostaglandin inhibition with indomethacin or meclofenamate significantly increased urinary osmolality after 12 h of dehydration and exogenous vasopressin (1.25 U) from 794 +/- 59 to 1,163 +/- 113 mosmol/kgH2O (P less than 0.01). In vitro prostaglandin inhibition with indomethacin or meclofenamate corrected the antidiuretic hormone (ADH) unresponsiveness of isolated perfused cortical collecting tubules (CCTs) from KD rabbits. Furthermore, preincubation with pertussis toxin, an agent that inactivates the guanine nucleotide inhibitory (Ni) subunit of adenylate cyclase, normalized the ADH response of KD CCTs, suggesting that prostaglandins may attenuate ADH action on the CCT through activation of Ni and contribute to the urinary concentrating defect associated with KD.

Contribution of AV3V region in anephric NaCl-induced hypertension in the rat.

The role of the anteroventral third ventricle (AV3V) region in mediating hypertonic sodium chloride-induced pressor responses was investigated in conscious rats. Sham- and AV3V-lesioned rats were prepared with femoral artery and vein catheters and subjected to bilateral nephrectomy under gaseous anesthesia. After recovery, animals were infused intravenously with isotonic (0.5 meq/kg) or hypertonic (10 meq/kg) saline at a rate of 0.0103 ml/min over 2 h. Isotonic saline infusion did not affect arterial pressure or heart rate in either group of rats. Hypertonic saline increased arterial pressure 35 +/- 3 mmHg in sham-lesioned rats and only 10 +/- 4 mmHg in AV3V-lesioned animals (P less than 0.0005). Sham-lesioned rats infused with hypertonic saline had a greater vasopressin-dependent component maintaining arterial pressure than the other groups of rats. Conversely, the sympathetic nervous system-dependent component of blood pressure was suppressed in the hypertonic saline-infused sham-lesioned animals compared with the other animals. However, when the vasopressin receptors were blocked, the neurally mediated portion of blood pressure was similar in all four groups of rats. These results emphasize that circulating vasopressin is important for the rise in arterial pressure accompanying the osmotic stimulus. Furthermore, this study demonstrates that the AV3V region is necessary for vasopressin-dependent pressor responses caused by an osmotic stimulus.

Suprofen-related nephrotoxicity. A distinct clinical syndrome.

We describe a unique clinical syndrome of flank pain and acute renal failure that is associated with suprofen, a nonsteroidal anti-inflammatory drug that has recently been made available in the United States. In the initial 6 months of the drug's distribution in this country, at least 16 patients developed this syndrome. All 16 had acute flank pain and 13 developed mild reversible renal failure within 12 hours of ingestion of one to three suprofen capsules. This syndrome is unlike other nephrotoxic syndromes related to nonsteroidal anti-inflammatory drugs. The mechanism is not known.

Renal physiology of the prostaglandins and the effects of nonsteroidal anti-inflammatory agents on the kidney.

The prostaglandins are a series of fatty acid products derived from the cellular metabolism of arachidonic acid. The kidney makes prostaglandins and the endogenous renal prostaglandins appear to play a role in the regulation of renal hemodynamics, renal salt and water excretion, and control of the level of activity of the renin-angiotensin system. The administration of nonsteroidal anti-inflammatory drugs blocks cyclooxygenase activity, an early step in the synthesis of prostaglandins. This class of drugs, under certain circumstances, leads to sodium retention, hyperkalemia and several different forms of acute and chronic renal failure. The potential role of altered prostaglandin synthesis in leading to these clinical syndromes is reviewed.

The role of high-energy phosphate in norepinephrine-induced acute renal failure in the dog.

Previous studies have demonstrated that pretreatment with mannitol, furosemide, or bradykinin can attenuate the severity of norepinephrine-induced renal functional impairment. The present studies were designed to evaluate the possibility that these agents are protective, in part, by preserving cellular metabolic integrity. The renal cortex was repetitively biopsied during the course of this study, and high-pressure liquid chromatography was used to analyze the tissue content of adenine nucleotides (expressed in nanomoles per gram of wet tissue). The adenine nucleotide charge ratio (CR) and total adenine nucleotide (TAN) content were calculated as indices of cellular metabolic integrity. In addition to the above-established protective agents, phenoxybenzamine was used to evaluate a direct toxic effect of norepinephrine on renal tissue. Inulin clearance at 3 hours post infusion (expressed as a percent of control) was 7% with norepinephrine alone and, in the protected groups, 36% with bradykinin, 61% with furosemide, 51% with mannitol, and 100% with phenoxybenzamine. There was no change in CR or TAN with phenoxybenzamine. In contrast, during norepinephrine administration CR fell significantly in all other groups. Three hours after stopping norepinephrine, CR had returned toward control values and the level of CR was significantly better in all protected groups when compared with norepinephrine alone. Similarly, the levels of TAN were significantly diminished in the norepinephrine-alone group when compared to all protected groups, and there was significantly more tubular necrosis as well. The maintenance of higher levels of TAN and the preserved ability to regenerate adenosine triphosphate in the protected groups, when compared to the norepinephrine-alone group, support the contention that these agents offer protection, at least in part, by preserving cellular metabolic integrity.

Prostaglandins may mediate chloride concentration gradient across domes formed by MDCK1 cells.

The MDCK1 and LLC-PK1 cell lines have been used to characterize transepithelial transport in isolated culture. Both can be maintained in continuous culture and form domes that represent fluid actively transported from luminal to contraluminal surface. Fluid in domes from LLC-PK1 cells was isotonic with the culture medium and no Cl gradient between domes and medium was present. Fluid from domes formed by MDCK1 cells was also isotonic with respect to Na and K but there was a persistent Cl concentration gradient. To evaluate prostaglandin (PG) production by these cell lines, the culture medium was analyzed for PGE. LLC-PK1 cells released no measurable PGE; MDCK1 cells regularly released PGE both during basal conditions and after augmentation of PG synthesis by a high Ca concentration in the medium. Qualitatively similar results had previously been found with these cell lines when PG synthesis was augmented with addition of arachidonic acid or Ca ionophore A23187, and PGE or 6-keto-prostaglandin F1 alpha was measured in the medium. To evaluate whether PGs might be responsible for the Cl gradient across MDCK1 cells, indomethacin or meclofenamate was added. There was a marked decrease in the Cl gradient with either agent at 5 h. To evaluate the possibility that PGE2 or PGI2 plays a role in maintaining this Cl gradient, further studies were done in which indomethacin was added to the culture medium to block endogenous PG synthesis, and exogenous PGE2 or PGI2 was added. Although addition of PGE2 was without effect, addition of PGI2 to indomethacin-treated cells maintained the Cl gradient for at least 5 h.(ABSTRACT TRUNCATED AT 250 WORDS)

The Spitzer-Weinstein syndrome: one form of type IV renal tubular acidosis and its response to hydrochlorothiazide.

A twelve-year-old girl with persistent hyperkalemia, metabolic acidosis, normal blood pressure and glomerular filtration rate, and short stature (first percentile for height) was studied using metabolic balance techniques. Prior to therapy with hydrochlorothiazide (HCTZ), urinary potassium and acid excretion were low and urine pH was inappropriately high at 5.8. HCTZ (25 mg orally per day) (1 mg/kg) was then started and rapidly corrected her serum electrolytes. The therapy with HCTZ was associated with a diuresis, a decrease in urine pH to 4.8, and concomitant increases in potassium, titratable acid (TA) and ammonium excretion. The increase in TA excretion was explicable, in part, to the decrease in urine pH and, in part, to the considerable increase in phosphate excretion (from 56 to 81 mmol/d). Plasma renin activity and plasma aldosterone increased markedly following HCTZ but urinary prostaglandin E (PGE) excretion was unchanged. These observations suggest that administration of HCTZ in this setting increases hydrogen ion secretion. It is unclear whether this effect is a direct consequence of HCTZ at the level of the tubule or is secondary to some other action of HCTZ. However, it is clear that this effect is not related to an alteration in PGE excretion.

Effect of prostaglandins on renal salt and water excretion.

There are several important mechanisms by which renal prostaglandins modulate renal salt and water excretion. The role of endogenous renal prostaglandins in facilitating urinary sodium excretion and the individual nephron segments that are affected by renal prostaglandins are reviewed. The role of the administration of nonsteroidal anti-inflammatory agents on the kidney's ability to excrete salt and water both physiologically and clinically is summarized. The potential role for endogenous prostaglandins to antagonize the effect of antidiuretic hormone and to alter renal water excretion is also described. The clinical consequences of taking nonsteroidal anti-inflammatory drugs in terms of hyperkalemia, sodium retention with associated edema, and possible hyponatremia are all discussed. Although these clinical consequences are quite uncommon statistically, there are certain subsets of patients for whom additional concern is important.

Relationship between urine flow rate and prostaglandin E excretion in human beings.

Although an increase in urine flow rate has been shown to augment urinary prostaglandin E (PGE) excretion, the relationship between these two variables has not been quantitated. Because we have previously shown that water immersion to the neck induces diuresis and augmentation of PGE excretion, we utilized water immersion to the neck to assess kinetically the relationship between changes in flow rate and PGE excretion. Fourteen normal male subjects were studied twice during 4 hours of water immersion to the neck, once after 11 hours of fluid deprivation and again during moderate hydration. PGE excretion as determined by radioreceptor assay was measured each hour. When subjects deprived of fluids underwent immersion to the neck, flow rate increased from 0.5 ml/min (control) to 1.5 ml/min, and PGE excretion rose from 1.4 to 2.6 ng/min (both p less than 0.01). In contrast, when subjects were studied during hydration, flow rate increased from 4.1 to 7.2 ml/min and PGE excretion increased from 8.1 to 13.0 ng/min at the same time intervals (both p less than 0.01). There was a significant positive correlation between flow rate and PGE excretion during both fluid deprivation and hydration. Were there an effect, independent of flow rate, of hydration on PGE, the slope of these two regression lines would differ. When the regression line slopes of these relationships were analyzed by the F test, there was a statistically significant (p less than 0.01) difference between the two regression coefficients. Our data suggest that, in addition to the previously characterized relationship between flow rate and PGE excretion, there is also a direct effect of hydration on PGE excretion.

Ammonia production and amino acid metabolism by rat renal papillary epithelial cells in culture.

A significant percentage of excreted ammonium is added to tubular fluid along the medullary collecting duct. However, it is not clear whether this ammonia is produced in the cortex and delivered into the medulla or is produced directly by medullary cells. To address this issue, rat epithelial cells derived from the renal papilla were grown in continuous culture and their ability to generate ammonia was examined. When grown in Dulbecco's modified Eagle's medium with 4 mM glutamine, these cells produced ammonia at a rate of approximately 27 nmol/10(6) cells/h. When these cells were grown in minimum essential medium without glutamine, ammonia production fell to 7 nmol/10(6) cells/ h. Increasing the glutamine concentrations of minimum essential medium to 4 mM increased ammonia production to slightly greater than 30 nmol/10(6) cells/ h. Increasing the media concentration of glutamate, glycine, or asparagine resulted in no significant increase in ammoniagenesis. Analysis of media amino acid concentration revealed that glutamine was the main amino acid consumed while alanine was the predominant amino acid produced. The glutaminase activity of these cells appears to be primarily phosphate-dependent, similar to that observed in vitro in papillary tubules. Alterations of K+ or H+ ion concentration did not alter ammoniagenesis, but addition of 2.5 mM ammonium chloride significantly reduced net ammonia production. It is concluded that rat papillary epithelial cells have the intrinsic ability to utilize glutamine to generate ammonia and alanine. In vivo ammonia produced locally in the medulla may contribute to final urinary ammonium excretion.

Prostaglandin I2 attenuates ischemic acute renal failure in the rat.

We gave prostaglandin I2 (PGI2) (8 ng X kg-1 X min-1 i.v.) for 20 min before, during, and 20 min after clamping of the rat left renal artery for 40 min to evaluate the effect of PGI2 in this model of acute renal failure. Control animals were given glycine buffer (PGI2 diluent). Glomerular filtration rate was estimated by the clearance of inulin 24 h later from each kidney. In group I rats (studied during hydropenia) inulin clearance in the control (right) kidney averaged 1.4 ml/min. Inulin clearance in kidneys exposed to 40 min of ischemia was 0.05 (glycine treated) versus 0.22 (PGI2 treated) ml/min. Although PGI2 offered significant protection in the group I animals, the differences were small and many of the glycine-treated ischemic kidneys were anuric. In the group II studies the same protocol was employed except that 5% body wt volume expansion was done with Ringer solution prior to measurement of inulin clearance. In the group II rats inulin clearance in control (right) kidneys averaged 1.5 ml/min. Inulin clearance after 40 min of renal ischemia was 0.04 ml/min in glycine-treated rats versus 0.90 ml/min in PGI2-treated animals. Histological examination of the group II ischemic kidneys revealed cellular necrosis and cast formation in the S3 segments of the glycine-treated animals and significantly less necrosis and cast formation in the PGI2-treated animals. The degree of necrosis and casts was inversely related to inulin clearance. Accordingly, PGI2 significantly attenuated the fall in inulin clearance measured 24 h after ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal effects of nonsteroidal anti-inflammatory agents.

In conclusion, there are a large variety of nonsteroidal anti-inflammatory drugs presently available for clinical use, which on occasion, particularly in human beings with a variety of disease states, can lead to relatively rapid decreases in renal function, particularly glomerular filtration rate. Although it is possible to predict which classes of patients may be more susceptible to this untoward event, even in these groups of patients the incidence of this phenomenon is relatively uncommon and does not preclude the use of these drugs, but rather it suggests that the physician be wary during their administration. Although it is attractive to consider the possibility that these effects are, in part, due to a decrease in endogenous prostaglandins, this issue is still open to active investigation.

Studies on the mechanism of sodium excretion during drug-induced vasodilatation in the dog.

The administration of vasodilating agents such as bradykinin and acetylcholine cause an increase in urinary sodium excretion. Yet the mechanisms involved in this natriuretic effect are not clear. Recent studies with another renal vasodilator, secretin have shown this drug also causes a profound increase in renal blood flow but without major changes in sodium excretion. To attempt to delineate the basis of this difference in sodium excretion with these drugs, the renal functional effects of secretin and bradykinin were compared at an equivalent vasodilating dose. Bradykinin increased renal blood flow from 222 to 342 ml/min, urine volume from 0.2 to 1.2 ml/min, and urine sodium excretion from 28 to 115 mueq/min. Urine osmolality fell from 1,230 to 401 mosmol/kg. Secretin caused a comparable increase in renal blood flow (216 to 325 ml/min) while changes in urine flow, sodium excretion, and urine osmolality were significantly less. In further studies papillary plasma flow was estimated using the albumin accumulation technique. Control papillary plasma flow was 29 ml/min per 100 g. Bradykinin increased urinary sodium excretion 108 mueq/min and decreased urinary osmolality from 1,254 to 516 mosmol/kg in association with a rise in papillary plasma flow to 62 ml/min per 100 g. Urine sodium excretion, urinary osmolality, and urine flow rate, as well as papillary plasma flow rate (32 ml/min per 100 g) were unchanged from control when secretin was administered. Studies with acetylcholine were qualitatively similar to those of bradykinin. Renal blood flow increased from 150 to 248 ml/min, urinary sodium excretion increased from 20 to 243 mueq/min, urinary osmolality decreased from 1,237 to 411 mosmol/kg and papillary plasma flow increased from 39 to 52 ml/min per 100 g. It is suggested that the natriuretic effect of some vasodilators is due, at least in part, to alterations in medullary hemodynamics, as evidenced by the increase in papillary plasma flow seen with bradykinin and acetylcholine, but not secretin.

Gastrointestinal blood loss in patients with chronic renal failure.

In the management of patients with chronic failure one of the persistent medical problems is that of anemia. Since iron deficiency can be an important component of this anemia, this study was designed to evaluate the possible contribution of gastrointestinal blood loss to their anemia. Blood loss was quantitated by 51 chromium labeling red cells in normals and in patients with chronic renal failure both before and during chronic hemodialysis. Four normal volunteers had a gastrointestinal blood loss of 0.83 ml/day, six azotemic patients not yet on hemodialysis had significantly greater gastrointestinal blood loss of 3.15 ml/day (p less than 0.05). Ten patients on chronic regular hemodialysis had a daily gastrointestinal blood loss of 6.27 ml/day which was significantly greater than both the normals (p less than 0.01) and the predialysis azotemic patients (p less than 0.05). Complete gastrointestinal tract evaluation in the chronic dialysis patients revealed several upper gastrointestinal tract mucosal abnormalities although discrete bleeding sites were not identified. In conclusion, azotemic patients both before and after chronic hemodialysis have increased gastrointestinal blood loss. This increased blood loss contributes to the increased iron loss in this patient population.