Influence of dietary sodium on the renal isoforms of 11 beta-hydroxysteroid dehydrogenase.

Endogenous glucocorticoids are converted to their biologically inert 11-dehydroderivatives by isoforms of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). The low-K(m), NAD(+)-dependent renal isoform (Type 2) identified in the distal nephron protects mineralocorticoid receptors from activation by endogenous glucocorticoids. The function of high-K(m), NADP(+)-dependent renal isoform (Type 1) is less well understood. Since glucocorticoids may modulate sodium transport in renal proximal tubules (PT), we hypothesized that Type 1 activity in this segment may be regulated by dietary Na(+)-11 beta-HSD activity was assessed in homogenates of canine PT by the conversion of cortisol to cortisone in the presence of NADP+ 200 microM. A high-Na+ diet for 4 days increased the Vmax 4-fold, with no change in the Type 1 K(m) (40 mEq/day Na+ diet: K(m) 0.959 microM, Vmax 3.40 pmoles/min/mg protein versus 150 mEq/day Na+ diet: K(m) 0.962 microM, Vmax 14.8 pmoles/min/mg protein). Type 1 mRNA also rose in the salt repleted animals. The high-Na+ diet produced no detectable change in the Type 2 isoform enzyme kinetics and mRNA level. No reverse oxo-reductase activity was noted with either renal isoform. Thus, renal Type 1 11 beta-HSD can be regulated by dietary Na+ independent of changes in the renal Type 2 isoform.

Interactions between D-glucose and phosphate in renal proximal tubule cells.

The addition of D-glucose to renal proximal tubule cells reduces phosphate transport and in conditions of limited phosphate availability reduces mitochondrial respiration, oxidative phosphorylation and ATP content, a phenomenon called the Crabtree effect. The present study examined the effects of D-glucose on cellular content of inorganic phosphate, phosphomonoesters, and ATP. These studies were performed in suspensions of canine proximal tubules using 31P NMR spectroscopy. When the extracellular medium contained 0.4 mM phosphate, addition of 8.3 mM D-glucose to the perifusion fluid significantly lowered cell content of inorganic phosphate (by 31%), phosphomonoesters (by 23%) and ATP (by 25%). Addition of a similar concentration of mannitol had no significant effect on these measured parameters. Removal of inorganic phosphate from the extracellular medium in the absence of glucose reduced the cellular content of inorganic phosphate by only 9%. These data indicate that a reduction in cellular phosphate occurs during the Crabtree effect. The glucose effect on cellular phosphates appears to be an inhibition of phosphate uptake and an enhancement of phosphate efflux. The data do not support the hypothesis that glucose induces an increase in sugar monophosphates.

An NMR study of cellular phosphates and membrane transport in renal proximal tubules.

Technical limitations in the measurement of cellular phosphates have hindered studies of interrelationships between cellular Pi, its transport, and its metabolism in renal proximal tubule (PT) cells. We have developed a noninvasive 31P-nuclear magnetic resonance (NMR) probe-perifusion system to measure cellular Pi and have utilized this system to investigate relationships in canine PT cells between the membrane transport and the cellular content of Pi. With 1.2 mM Pi in the extracellular medium, the cellular Pi content of PT averaged 4.94 +/- 0.55 nmol/mg protein. Inhibition of Pi uptake by removal of extracellular Pi rapidly decreased all cellular phosphate compounds to values that were between 55 and 85% of control. Partial replacement of extracellular Pi (0.4 mM) increased cellular phosphates up to 84-100% of control values. Inhibition of Na(+)-K(+)-adenosinetriphosphatase uptake by the addition of ouabain failed to change either cellular Pi or organic phosphates. Reducing the basolateral membrane potential with the addition of barium chloride increased cellular Pi content by nearly 30%. Maximal contents of cellular Pi and ATP were achieved at 0.4 mM Pi in the presence of an inwardly directed Na+ gradient and at 0.8 mM Pi in its absence. These data indicate that cellular Pi content in canine PT is regulated by Na(+)-dependent and -independent transport mechanisms and by the membrane potential across the basolateral membrane. Lastly, cellular ATP content was found to be directly proportional to the cellular Pi content over a physiological range.

A perifusion loop-gap resonator NMR probe for aerobic cell suspensions.

NMR studies of aerobic cell metabolism require that spectral data of high sensitivity and resolution be acquired from samples maintained under suitable conditions. The loop-gap resonator 31P NMR probe described here was designed for investigations of highly aerobic-dependent renal proximal tubules in an environment similar to those of conventional incubation flasks. The inherently higher sensitivity of the loop-gap resonator design and associated circuitry made it possible to obtain adequate spectra in short periods of time with small amounts of sample. Cellular physiological properties of perifused aerobic renal proximal tubule cell suspensions in the loop-gap resonator probe were found to be similar to equivalent tissue samples incubated in conventional flasks. Furthermore, the loop-gap perifusion probe was found to provide useful 31P NMR spectra of the kidney tubule system after acquisition times as short as 3 min.

Activated neutrophils inhibit Na(+)-K(+)-ATPase in canine renal basolateral membrane.

To examine the effects of activated neutrophils (PMNs) on Na(+)-K(+)-ATPase, phorbol 12-myristate 13-acetate (PMA)-stimulated PMNs were incubated with canine renal cortical basolateral membrane (BLM), and BLM ouabain-sensitive Na(+)-K(+)-ATPase activity was subsequently quantified. Na(+)-K(+)-ATPase activity decreased to 40.0 +/- 8.7% (SE) of control in the presence of activated PMNs, from 0.89 +/- 0.12 to 0.34 +/- 0.05 mumol Pi.mg protein-1.min-1. This inhibition coincided with a decrease in the apparent Michaelis constant (Km) for ATP from 0.18 +/- 0.02 to 0.05 +/- 0.01 mM. Inclusion of catalase (CAT) and superoxide dismutase (SOD) in the BLM/PMN/PMA incubation mixture resulted in partial preservation of enzyme activity, with an increase to 57.0 +/- 4.6% of control with CAT alone and to 70.0 +/- 5.3% with both CAT and SOD. SOD alone had no protective effect. Neither the myeloperoxidase inhibitor azide nor the hypochlorous acid scavenger L-methionine preserved enzyme activity. Hydroxyl radical scavengers and iron chelators were also ineffective in attenuating Na(+)-K(+)-ATPase inhibition by activated PMNs. These results indicate that activated PMNs mediate a decrease in BLM Na(+)-K(+)-ATPase activity characterized by a reduction in maximum velocity and Km for ATP that appears to be mediated in part by reactive oxygen metabolites.

Growth hormone regulates ammoniagenesis in canine renal proximal tubule segments.

To determine whether growth hormone (GH) directly affects ammoniagenesis in the renal proximal tubule, ammonia production was measured in suspensions of isolated canine renal proximal tubule segments (IPTs) incubated with 2.5 mM L-glutamine and varying concentrations of human growth hormone (hGH). Ammonia production from IPTs significantly increased by nearly threefold in the presence of hGH (10(-6) M) at 60 min. This increase was dose dependent, with as little as 10(-9) M hGH significantly stimulating ammonia production. In addition, hGH enhanced glucose production when lactate, alanine, and succinate replaced L-glutamine as substrate. hGH significantly stimulated ammonia production when IPTs were incubated at alkalotic and neutral pH. The effect of hGH was lost at acidic pH. When hGH was added to IPTs incubated under Na(+)-equilibrated conditions, ammonia production was not different from control. hGH stimulated ouabain-sensitive Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity by 8.1 +/- 1.1% in basolateral membranes isolated from IPTs. hGH stimulation of proximal tubule ammonia production from L-glutamine occurs at physiological concentrations of hGH and when the extracellular-to-intracellular Na+ gradient favors L-glutamine transport. This effect is associated with an increase in basolateral Na(+)-K(+)-ATPase activity. The data suggest a role for hGH in the regulation of renal acid-base metabolism under physiological conditions in which increased net acid excretion is important.

Angiotensin II stimulates ammoniagenesis in canine renal proximal tubule segments.

To determine whether angiotensin II (ANG II) affects ammoniagenesis in renal proximal tubule, ammonia production was measured in suspensions of canine renal proximal tubule segments (PCT) incubated with L-glutamine and varying concentrations of ANG II. Ammonia production from PCT was significantly increased by 15.5 +/- 1.1% in the presence of ANG II (10(-6) M) at 2 h. Similarly, glucose production significantly increased by 10.0 +/- 0.9%. Half-maximal stimulation occurred at approximately 10(-9) M ANG II. Stimulation of ammonia production by ANG II was blocked in the presence of the ANG II antagonist, [Sar1-Ile8]ANG II (10(-6) M). Enhancement of ammonia production in PCT by ANG II occurred in acidotic and neutral media but not in alkalotic medium. When extracellular [Na+] = intracellular [Na+] ANG II significantly increased ammonia production in PCT. Absence of extracellular Ca2+ or addition of trifluoperazine or N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) (Ca2(+)-calmodulin-dependent pathway inhibitors) blocked the action of ANG II to enhance ammonia production. We conclude that ANG II stimulates ammonia and glucose production in canine renal PCT via a receptor-mediated signal. The action of ANG II on ammoniagenesis may be mediated by a calcium-calmodulin-dependent pathway. Stimulation of ammoniagenesis in vitro under normal and acidotic conditions may reflect a role in vivo for ANG II in the regulation of renal acid-base metabolism.

Hormonal control of renal ammonium metabolism: a review.

Hormonal regulation of renal ammonium production and handling has undergone recent intensive investigation. The relationship between hormone action and ammonium metabolism has been known since the beginning of this century. Hormones may directly affect production or excretion of ammonium. Second messengers have also been shown to effect changes in renal ammonium metabolism. Hormonal control of renal ammonium production and handling may play a role in regulation of acute acid-base metabolism, but its role in chronic acid-base pathophysiological states has yet to be determined.

Parathyroid hormone stimulates ammoniagenesis in canine renal proximal tubular segments.

To determine whether parathyroid hormone (PTH) affects ammoniagenesis in renal proximal tubule, we measured ammonia production in suspensions of canine renal proximal tubular segments incubated with 10 mM L-glutamine in the absence or presence of 10(-7) M PTH. Productions of ammonia were linear functions of time for 120 min and averaged 231 +/- 55 and 311 +/- 67 mumol ammonia/g protein in the absence and presence of PTH, respectively. When measured over the range of 10(-11)-10(-7) M PTH, half-maximal stimulation of ammonia production occurred between 10(-10) and 10(-9) M PTH. Maximal production of ammonia was observed at 10(-8) M PTH. Incubation of proximal tubular segments with PTH increased levels of adenosine 3',5'-cyclic monophosphate (cAMP) in vitro. Ammonia production was significantly enhanced by incubation of segments with the cAMP analogue, 8-bromoadenosine 3',5'-cyclic monophosphate. PTH also increased ammonia production in segments incubated with 1 mM L-glutamine. We conclude that PTH stimulates ammonia production in canine renal proximal tubular segments. This effect appears to be mediated, at least in part, through cAMP in vitro. Such stimulation could reflect a direct action of PTH on the proximal tubule to enhance ammoniagenesis in vivo.

Insulin stimulates ammoniagenesis in canine renal proximal tubular segments.

To characterize the effect of insulin on ammoniagenesis in renal proximal tubule, ammonia productions were measured in suspensions of canine renal proximal tubular segments incubated with 10 mM L-glutamine and varying concentrations of insulin. Productions of ammonia were linear functions of time for 120 min. Insulin (10(-6) M) increased ammonia production at 2 h by 34 +/- 5%. At the same time, gluconeogenesis, as measured by glucose production, was decreased by 16 +/- 2%. Significant enhancement of ammoniagenesis occurred in suspensions of segments incubated with as little as 10(-9) M insulin. Half-maximal stimulation occurred at between 10(-9) and 10(-8) M insulin. Oxidation of L-glutamine in cells within segments was also increased by insulin in a concentration-dependent manner. Insulin increased ammonia productions in segments incubated at pH 7.5 but not at 7.0. Under the former conditions, insulin enhanced ammoniagenesis in proximal tubular segments under conditions such that extracellular [Na+] was greater than intracellular [Na+], but not when extracellular [Na+] equaled intracellular [Na+]. We conclude that insulin stimulates ammonia production in suspensions of canine renal proximal tubular segments. Stimulation of ammonia production in vitro could reflect an action of insulin to enhance proximal tubular ammoniagenesis in vivo.

Phorbol esters inhibit ammoniagenesis and gluconeogenesis in proximal tubular segments.

To characterize the regulation of ammoniagenesis and gluconeogenesis in renal proximal tubule, ammonia and glucose productions were measured in suspensions of canine proximal tubular segments incubated with 10 mM L-glutamine. Productions were linear functions of time for 120 min and were decreased as extracellular pH was increased from 7.0 to 7.5 To ascertain whether activation of protein kinase c affects either process, we incubated segments with tumor-promoting phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA), or phorbol 12,13-dibutyrate, or with the inactive phorbol ester 4 alpha-phorbol. Ammoniagenesis and gluconeogenesis were inhibited by incubation with 10(-6) M of the two former compounds but not the latter compound. Inhibition of ammoniagenesis and gluconeogenesis occurred after incubation with as little as 10(-9) M phorbol 12,13-dibutyrate. Phorbol ester-induced inhibition was observed under conditions such that extracellular [Na+] was greater than intracellular [Na+], but not when extracellular [Na+] equaled intracellular [Na+], and was not observed in the presence of amiloride. Our findings are consistent with a role for protein kinase c in the control of ammoniagenesis and gluconeogenesis in proximal tubule. Such control could be mediated via stimulation of Na+-H+ exchange.

Early onset of clinical diabetic nephropathy in children--a new subgroup?

The onset of fixed proteinuria and hypertension in insulin-dependent diabetic is generally associated with eventual renal insufficiency due to diabetic nephropathy or with a superimposed glomerulopathy. We report three adolescents with normal renal function who developed fixed proteinuria and hypertension after only 7 to 11 years of insulin-dependent diabetes mellitus. Blood pressure ranged from 130/95 to 165/104 mmHg, urinary protein excretion was 1.31 to 1.37 g/24 hours, and creatinine clearance ranged from 98-133 ml/min/1.73 m2. Renal biopsy revealed changes consistent only with diabetic glomerulosclerosis. Follow-up evaluation for 11 months to 3 1/2 years revealed blood pressure reductions to 125/78-140/85 mmHg as a result of antihypertensive medications. Creatinine clearance increased by 12-20% and urinary protein excretion remained unchanged. We conclude that these patients may represent an unusual subgroup of insulin-dependent diabetics with early development of clinical and pathological diabetic nephropathy in the face of normal renal function.