Thomas P. Dousa

Pathogenesis of nephrogenic diabetes insipidus due to chronic administration of lithium in rats.

A polyuric syndrome with nephrogenic diabetes insipidus (NDI) is a frequent consequence of prolonged administration of lithium (Li) salts. Studies in the past, mainly the acute and in vitro experiments, indicated that Li ions can inhibit hydroosmotic effect of [8-arginine]vasopressin (AVP) at the step of cAMP generation in vitro. However, the pathogenesis of the NDI due to chronic oral administration of low therapeutic doses of Li salts is not yet clarified. We conducted a comprehensive study to clarify the mechanism by which Li administered orally for several weeks induces polyuria and NDI in rats. Albino rats consuming a diet which contained Li (60 mmol/kg) for 4 wk developed marked polyuria and polydipsia; at the end of 4 wk the plasma Li was 0.7 +/- 0.09 mM (mean +/- SEM; n = 36). Li-treated rats had a significantly decreased (-33%) tissue osmolality in papilla and greatly reduced cortico-papillary gradient of urea (cortex--43%; medulla--64%; papilla--74%). Plasma urea was significantly (P less than 0.001) lower in Li-treated rats (5.4 +/- 0.2 mM) compared with controls (6.8 +/- 0.3 mM). Medullary collecting tubules (MCT) and papillary collecting ducts (PCD) microdissected from Li-treated animals had higher content of protein than MCT and PCD from the control rats. The cAMP accumulation in response to AVP added in vitro was significantly (delta = -60%) reduced. Also, the cAMP accumulation in MCT and PCD after incubation with forskolin was markedly lower in Li-treated rats. Addition of 0.5 mM 1-methyl,3-isobutyl-xanthine did not restore the cAMP accumulation in response to AVP and forskolin in MCT from Li-treated animals. In collecting tubule segments from polyuric rats with hypothalamic diabetes insipidus (Brattleboro homozygotes) the AVP-dependent cAMP accumulation was not diminished. The activity of adenylate cyclase (AdC) in MCT of Li-treated rats, both the basal and the activity stimulated by AVP, forskolin, or fluoride, was significantly (delta approximately equal to -30%) reduced, while the activity of cAMP phosphodiesterase (cAMP-PDIE) in the same segment showed no significant difference from the controls. Also, the content of ATP in MCT microdissected from Li-treated rats and incubated in vitro did not differ from controls. The rate of [14C]succinate oxidation to 14CO2 in MAL was inhibited (-77%) by 1 mM furosemide, which indicates that this metabolic process is coupled with NaCl cotransport in MAL. The rate of (14)CO(2) production from [14C]succinate in MAL was not significantly different between control and Li-treated rats. In MCT of control rats, the rate of [14C]succinate oxidation was approximately 3 times lower than in MAL. The rate of (14)CO(2) production from [(14)C]succinate in MCT of Li-treated rats was significantly (delta +33%) higher than in MCT dissected from control rats. Based on these results, we conclude that at least two factors play an important role in the pathogenesis of NDI consequent to chronic oral administration of Li: (a) decreased ability of MCT and PCD to generate and accumulate cAMP in response to stimulation by AVP; this defect is primarily due to diminished activity of AdC in these tubular segments caused by prolonged exposure to Li; and (b) lower osmolality of renal papillary tissue, due to primarily to depletion of urea, which decreases osmotic driving force for water reabsorption in collecting tubules. On the other hand, NaCI reabsorption in MAL is apparently not affected by chronic Li treatment.

Possible role of nicotinamide adenine dinucleotide as an intracellular regulator of renal transport of phosphate in the rat.

In these experiments we investigated whether NAD could serve as an intracellular modulator of the brush border membrane (BBM) transport of inorganic phosphate (Pi). NAD, both oxidized (NAD+) and reduced (NADH) form, inhibited the Na+-dependent uptake of 32Pi in the concentration range of 10-300 microM NAD when added in vitro to BBM vesicles isolated from rat kidney cortex, but did not inhibit BBM uptake of D-[3H]glucose or BBM uptake of 22Na+. Neither nicotinamide (NiAm) nor adenosine alone influenced BBM uptake of 32Pi. NAD had a similar relative effect (percent inhibition) in BBM from rats stabilized on low Pi diet (0.07% Pi), high Pi diet (1.2% Pi), or normal Pi diet (0.7% Pi). Subsequently, we examined the renal effects of changing the tissue NAD level in vivo. Rats stabilized on low Pi diet were injected intraperitoneally with NiAm (0.25-1.0 g/kg body wt); urinary excretions of Pi (UPiV), of fluid, and of other solutes were measured before and after NiAm injection, then renal cortical tissue nucleotide content was determined, and a BBM fraction was isolated for transport measurements. In BBM from NiAm-treated rats, the Na+-dependent uptake of 32Pi was decreased, but BBM uptake of D-[3H]glucose and BBM uptake of 22Na+ were not changed. NiAm injection elicited an increase in NAD+ (maximum change, 290%), a lesser increase in NADH (maximum change, +45%), but no change in the content of ATP or cyclic AMP in the renal cortex. Na+-dependent BBM uptake of 32Pi ws inversely correlated with NAD+ content in renal cortex (r = -0.77 +/- 0.1; P less than 0.001) and with UPiV (r = -0.67 +/- 0.13; P less than 0.01). NAD+ in renal cortex was positively correlated with UPiV (r = 0.88 +/- 0.05; P less than 0.001). Injection of NiAm elicited a marked increase in UPiV, but no change in excretions of creatinine or K+, or in urine flow; excretion of Na+ and Ca declined. NiAm injection caused similar renal responses, in normal and in thyroparathyroidectomized rats, as well as in rats on normal Pi diet and low Pi diet. We conclude that NAD can serve as an intracellular modulator (inhibitor) of Na+-dependent transport of Pi across the renal luminal BBM and across the proximal tubular wall by its direct interaction with BBM. We propose that at least some hormonal and/or metabolic stimuli elicit phosphaturia by increasing NAD+ in cytoplasm of proximal tubular cells.

Administration of atrial natriuretic factor inhibits sodium-coupled transport in proximal tubules.

The newly discovered peptides extracted from cardiac atria, atrial natriuretic factors (ANFs), when administered parenterally cause renal hemodynamic changes and natriuresis. The nephron sites and cellular mechanism accounting for profound increase in Na+ excretion in response to ANFs are not yet clarified. In the present study we investigated whether synthetic ANF peptide alters the reabsorption of Na+ and reabsorption of solutes cotransported with Na+ in the proximal tubules of rats. Synthetic ANF peptide consisting of 26 amino acids, 4 micrograms/kg body wt/h, or vehicle in controls, was infused to surgically thyroparathyroidectomized anesthetized rats. After determination of the fractional excretion (FE) of electrolytes (Na+, K+, Pi, Ca2+, Mg2+, HCO3), the kidneys were removed and luminal brush border membrane vesicles (BBMVs) were prepared from renal cortex. Solute transport was measured in BBMVs by rapid filtration techniques. Infusion of ANF peptide increased FENa, FEPi, and FEHCO3; but FECa, FEK, and FEMg were not changed. The increase in FENa was significantly correlated, on the one hand, with increase of FEPi (r = 0.9, n = 7; P less than 0.01) and with increase of FEHCO3 (r = 0.89, n = 7; P less than 0.01). On the other hand, FENa did not correlate with FEK, FECa, or with FEMg. The Na+ gradient-dependent uptake of Pi by BBMVs prepared from renal cortex of rats receiving ANF infusion was significantly (P less than 0.05) decreased (-25%), whereas the Na+ gradient-dependent uptake of L-[3H]proline and of D-[3H]glucose or the diffusional uptake of 22Na+ were not changed. ANF-elicited change in FEPi showed a close inverse correlation with decrease of Na+-dependent Pi uptake by BBMVs isolated from infused rats (r = 0.99, n = 7; P less than 0.001). Direct addition of ANF to BBMVs in vitro did not change the Na+ gradient-dependent Pi uptake. In rats infused with ANF, the rate of amiloride-sensitive Na+-H+ exchange across the brush border membrane (BBM) was significantly (P less than 0.05) decreased (-40%), whereas the diffusional 22Na+ uptake (0.5 min) and the equilibrium (120 min) uptake of 22Na+ were not changed. The inhibition of Na+-H+ exchange after ANF was likely due to alteration of the BBM antiporter itself, in that the H+ conductance of BBMVs was not increased. We conclude that synthetic ANF (a) decreases tubular Na+ reabsorption linked to reabsorption of HCO3 in proximal tubules, and (b) inhibits proximal tubular reabsorption of Pi coupled to Na+ reabsorption, independent of secretion and/or action of parathyroid hormone or calcitonin. These ANF effects are associated with inhibition of Na+-Pi synport and of Na+-H+ antiport in luminal BBMs. Our findings document that inhibition of Na+-coupled transport processes in proximal tubules is an integral part of the renal response to ANF.

Inhibitors of cyclic nucleotide phosphodiesterase isozymes type-III and type-IV suppress mitogenesis of rat mesangial cells.

We studied interactions between the mitogen-activated protein kinase (MAPK) signalling pathway and cAMP-protein kinase (PKA) signaling pathway in regulation of mitogenesis of mesangial cells (MC) determined by [3H]thymidine incorporation, with or without added EGF. Forskolin or dibutyryl cAMP strongly (by 60-70%) inhibited [3H]thymidine incorporation into MC. Cilostamide, lixazinone or cilostazol selective inhibitors of cAMP-phosphodiesterase (PDE) isozyme PDE-III, inhibited mitogenesis to similar extent as forskolin and DBcAMP and activated in situ PKA, but without detectable increase in cAMP levels. Cilostamide and cilostazol were more than three times more effective at inhibiting mesangial mitogenesis than rolipram and denbufylline, inhibitors of isozyme PDE-IV, even though PDE-IV was two times more abundant in MC than was PDE-III. On the other hand, when incubated with forskolin, rolipram-enhanced cAMP accumulation was far greater (10-100x) than with cilostamide. EGF increased MAPK activity (+300%); PDE isozyme inhibitors which suppressed mitogenesis also inhibited MAPK. PDE isozyme inhibitors also suppressed PDGF-stimulated MC proliferation. We conclude that cAMP inhibits the mitogen-dependent MAPK-signaling pathway probably by decreasing the activity of Raf-1 due to PKA-catalyzed phosphorylation. Further, we surmise that minor increase in the cAMP pool metabolized by PDE-III is intimately related to regulation of mesangial proliferation. Thus, PDE isozyme inhibitors have the potential to suppress MC proliferation by a focused effect upon signaling pathways.

Phosphate transport across renal cortical brush border membrane vesicles from rats stabilized on a normal, high or low phosphate diet

Abstract Phosphate uptake measured at 0.25, 0.5 and 1.0 min time intervals was 2-fold higher in renal brush border membrane vesicles from rats fed a low phosphate diet compared to control rats given a normal phosphate diet. In rats on a high phosphate diet the uptake of phosphate at the same time intervals was 2-fold lower compared to controls. D-glucose uptake by the same vesicle preparations was not significantly different between the three groups at 0.25, 0.5 and 1.0 min. At each of these time points there was a positive correlation between phosphate uptake and the activity of alkaline phosphatase which was measured in the vesicle preparations from each group of rats. There was no correlation between phosphate uptake and the activities of three other brush border enzymes.

Renal Adaptation to a Low Phosphate Diet in Rats: BLOCKADE BY ACTINOMYCIN D

The major renal adaptive changes in response to selective dietary phosphate restriction are a marked reduction in urinary excretion of phosphate and an increased urinary excretion of calcium; at the cellular level, there is selective increase in renal cortical brush border membrane phosphate uptake and increase in specific activity of alkaline phosphatase. In the present study we examined whether these functional and biochemical adaptive changes could be blocked by drugs known to inhibit protein synthesis. Administration of actinomycin D or cycloheximide to rats switched from a diet with normal phosphate content (0.7%) to a diet with low (0.07%) phosphate content either completely (actinomycin D) or partially (cycloheximide) prevented the expected decrease in urinary excretion of phosphate and increase in the urinary excretion of calcium. The specific activity of alkaline phosphatase measured in crude membrane fraction (washed 100,000 g pellet) from renal cortical homogenate in animals fed a low phosphate diet and treated with actinomycin D or with cycloheximide was significantly lower than in control animals also on a low phosphate diet receiving placebo; but there were no differences between treated and untreated animals in the activities of two other brush border enzymes, gamma-glutamyltransferase and leucine aminopeptidase. Actinomycin D administered to rats maintained on a normal phosphate diet throughout the course of the experiment caused an increase in the urinary excretion of phosphate on the last (6th) day of the experiment but did not change urinary excretion of calcium. In acute clearance experiments, infusion of actinomycin D to rats adapted to a low phosphate diet did not increase fractional excretion of phosphate. In separate experiments, using the same dietary protocol as above, brush border membrane fraction (vesicles) was prepared from renal cortex of rats sacrificed at the end of the experiment. In this preparation Na(+)-dependent (32)Pi and d-[(3)H]glucose uptake and activities of brush border enzymes membrane were determined. Brush border membrane vesicles prepared from rats fed a low phosphate diet showed significantly higher Na(+)-dependent (32)Pi uptake compared with rats fed a normal phosphate diet. This increase in (32)Pi uptake was completely prevented when rats on a low phosphate diet were simultaneously treated with actinomycin D. These differences were specific for (32)Pi transport as no differences were observed in d-[(3)H]glucose uptake among the three groups. There was a positive correlation (r = 0.82, P < 0.01) between (32)Pi uptake and specific activity of alkaline phosphatase measured in aliquots of the same brush border membranes, whereas no such correlation was observed with two other brush border membrane enzymes gamma-glutamyltransferase and leucine aminopeptidase. These observations show that actinomycin D prevents both the functional and cellular renal adaptive changes induced by a low phosphate diet. Taken together, these observations suggest that renal adaptation to a low phosphate diet could be prevented by inhibition of de novo protein synthesis.

Interaction of prostaglandins and histamine with enzymes of cyclic AMP metabolism from guinea pig gastric mucosa.

Prostaglandins (PGE1, PGE2, PGA1) and histamine have opposing effects on gastric HCl secretion, but we found that both stimulate adenylate cyclase activity in cell-free membrane preparations of guinea pig gastric fundic mucosa. The stimulatory effect of prostaglandins was found in this study to be specific and dose-dependent over a concentration range from 10(-7) to 10(-4) M. In similar preparations from antral regions of guinea pig gastric mucosa, the adenylate cyclase was stimulated only by PGE1, PGE2, and PGA1 and not by histamine. Maximum stimulating doses of PGE1, PGE2, or PGA1, and of histamine had an additive effect on the adenylate cyclase activity from fundic gastric mucosa. Metiamide, a histamine H2-receptor antagonist, inhibited the stimulation of fundic mucosa adenylate cyclase by histamine but did not interfere with the stimulation by prostaglandins. Cyclic AMP phosphodiesterase activity of guinea pig gastric mucosa was unaffected by PGE1 and PGE2 or by histamine, and was slightly depressed by PGA1. These results indicate that histamine and prostaglandins stimulate two different adenylate cyclase systems both present in guinea pig gastric mucosa tissue. Therefore, the known inhibitory effect of prostaglandins on gastric acid secretion is not related to the interference with the stimulation of the histamine H2-receptor-sensitive adenylate cyclase complex by histamine nor do prostaglandins accelerate cyclic AMP breakdown by cyclic AMP phosphodiesterase to reduce cyclic AMP levels.

Cellular Action of Vasopressin in Medullary Tubules of Mice with Hereditary Nephrogenic Diabetes Insipidus

Our previous studies (1974. J. Clin. Invest.54: 753-762.) suggested that impaired metabolism of cyclic AMP (cAMP) may be involved in the renal unresponsiveness to vasopressin (VP) in mice with hereditary nephrogenic diabetes insipidus (NDI). To localize such a defect to specific segments of the nephron, we studied the activities of VP-sensitive adenylate cyclase, cAMP phosphodiesterase (cAMP-PDIE), as well as accumulation of cAMP in medullary collecting tubules (MCT) and in medullary thick ascending limbs of Henles loop (MAL) microdissected from control mice with normal concentrating ability and from mice with hereditary NDI. Adenylate cyclase activity stimulated by VP or by NaF was only slightly lower (-24%) in MCT from NDI mice, compared with controls. In MAL of NDI mice, basal, VP-sensitive, and NaF-sensitive adenylate cyclase was markedly (> -60%) lower compared with MAL of controls. The specific activity of cAMP-PDIE was markedly higher in MCT of NDI mice compared with controls, but was not different between MAL of control and NDI mice. Under present in vitro conditions, incubation of intact MCT from control mice with VP caused a striking increase in cAMP levels (>10), but VP failed to elicit a change in cAMP levels in MCT from NDI mice. When the cAMP-PDIE inhibitor 1-methyl-3-isobutyl xanthine (MIX) was added to the above incubation, VP caused a significant increase in cAMP levels in MCT from both NDI mice and control mice. Under all tested conditions, cAMP levels in MCT of NDI mice were lower than corresponding values in control MCT. Under the present experimental setting, VP and other stimulating factors (MIX, cholera toxin) did not change cAMP levels in MAL from either control mice or from NDI mice. The results of the present in vitro experiments suggest that the functional unresponsiveness of NDI mice to VP is perhaps mainly the result of the inability of collecting tubules to increase intracellular cAMP levels in response to VP. In turn, this inability to increase cAMP in response to VP is at least partly the result of abnormally high activity of cAMP-PDIE, a somewhat lower activity of VP-sensitive adenylate cyclase in MCT of NDI mice, and perhaps to a deficiency of some other as yet unidentified factors. The possible contribution of low VP-sensitive adenylate cyclase activity in MAL of NDI mice to the renal resistance to VP remains to be defined.

Effects of Colchicine and Vinblastine on the Cellular Action of Vasopressin in Mammalian Kidney A POSSIBLE ROLE OF MICROTUBULES

To evaluate the possible role of microtubules in the cellular action of vasopressin on the mammalian kidney, the effects of microtubule-disrupting agents were studied in vivo and in vitro. In vivo studies were done in rats in mild to moderate water diuresis induced by drinking 5% glucose. Microtubule-disrupting alkaloids, colchicine (0.1 mg/day) or vinblastine (0.08 mg/day), given intraperitoneally, did not change water and solute excretion itself, but blocked or markedly inhibited the antidiuretic response (increase in urine osmolality and decrease in urine flow) to exogenous vasopressin. Total solute excretion was unaffected by these two alkaloids and there were no substantial changes in excretion of sodium, potassium, or creatinine. Lumicolchicine, a derivative of colchicine that does not interact with microtubules, did not alter the antidiuretic response to exogenous vasopressin. Activities of adenylate cyclase in the renal medullary plasma membrane, and cyclic AMP phosphodiesterase and protein kinase in renal medullary cytosol, were not influenced by 10(-5)-10(-4) M colchicine or vinblastine in vitro. Studies on the subcellular distribution of microtubular protein (assessed as [(3)H]colchicine-binding protein) in renal medulla shows that this protein is contained predominantly in the cytosol. Particulate fractions, including plasma membrane, contain only a minute amount (less than 6%) of the colchicine-binding activity. The results suggest that the integrity of cytoplasmic microtubules in cells of the distal nephron is required for the antidiuretic action of vasopressin, probably in the sites distal to cyclic AMP generation in the mammalian kidney.

Mechanism of resistance to the phosphaturic effect of the parathyroid hormone in the hamster.

The effect of parathyroid hormone and calcitonin on the renal excretion of phosphate, calcium, and cyclic AMP was evaluated in the thyroparathyroidectomized hamster, a mammal apparently reisstant to the phosphaturic effect of parathyroid hormone. Parathyroid hormone did not increase phosphate excretion, although it decreased excretion of calcium and increased urinary excretion of cyclic AMP. This lack of a phosphaturic response to parathyroid hormone was not reversed by administration of 25-OH vitamin D or infusions of calcium or phosphate. Calcitonin, another potentially phosphaturic hormone, also vailed to increase phosphate excretion but markedly elevated urinary excretion of cyclic AMP. In hamsters pretreated with infusion of urinary ammonium chloride, which decreased plasma and urinary pH, both parathyroid hormone and calcitonin increased excretion of phosphate as well as that of cyclic AMP. Acetazolamide had no phosphaturic effect in ammonium chloride-loaded hamsters, and it decreased cyclic AMP and calcium excretion. Alkalinization of urine by acetazolamide did not prevent the phosphaturic effect of parathyroid hormone in ammonium chloride-loaded hamsters, but it blocked the increase in urinary cyclic AMP excretion. Parathyroid hormone and calcitonin both stimulated adenylate cyclase in a cell-free system (600-g pellet) from hamster renal cortex, elevated tissue cyclic AMP levels, and activated protein kinase in tissue slices from hamster renal cortex. In acid medium, the increase in cyclic AMP and activation of protein kinase in response to parathyroid hormone was diminished, but addition of acetazolamide restored responsiveness of both parameters to control values. Acetazolamide, on the other hand, did not influence adenylate cyclase or its response to parathyroid hormone or cyclic AMP phosphodiesterase activity. We conclude that the lack of a phosphaturic effect of parathyroid hormone and calcitonin in the hamster depends on steps in the cellular action of these hormones, steps that are sensitive to pH subsequent to cyclic AMP generation and protein kinase activation. In addition, acetazolamide may potentiate the phosphaturic effect of parathyroid hormone by promoting accumulation of cyclic AMP in tissue. Thus, the hamster is a particularly useful model for studies of syndromes in which there is renal resistance to phosphaturic hormones.