Insulin markedly potentiates the capacity of parathyroid hormone to increase expression of 25-hydroxyvitamin D3-24-hydroxylase in rat osteoblastic cells in the presence of 1,25-dihydroxyvitamin D3.

We have previously shown that insulin alters the renal metabolism of 25-hydroxyvitamin D. To examine the effect of insulin on vitamin D metabolism in bone, we have used UMR-106 osteoblast-like cells to study the regulation of 25(OH)D3-24-hydroxylase (24-hydroxylase) expression by insulin. The 24-hydroxylase is an important enzyme in degrading 1,25-dihydroxyvitamin D3 (1,25(OH)2D) in target tissues. Insulin alone had no effect on mRNA levels of the cytochrome P450 component (CYP24) of the 24-hydroxylase or on 24-hydroxylase activity itself in UMR cells. However, insulin increased the capacity of parathyroid hormone (PTH) to elevate CYP24 mRNA levels by 3-4-fold and to increase 24-hydroxylase activity by 2-fold in the presence of 1,25(OH)2D. Insulin increased the maximal responsiveness of UMR cells to PTH without altering their sensitivity. The action of insulin required the presence of 1,25(OH)2D and was partly dependent on new protein synthesis. Insulin-like growth factor 1 also potentiated the effects of PTH. This marked stimulation of the 24-hydroxylase by PTH and insulin may serve to regulate 1,25(OH)2D action and/or to produce 24,25-dihydroxyvitamin D in bone cells.

Expression of plasma membrane calcium pump mRNA in rat intestine: effect of age and 1,25-dihydroxyvitamin D.

The capacity of the small intestine to actively transport Ca declines markedly with increasing age in the rat. The basal-lateral plasma membrane Ca pump is thought to be an important component of the active transport mechanism. Therefore, the purpose of this study was to determine if there are changes in the expression of the intestinal Ca pump with age, mRNA levels were quantitated by Northern and dot blot analysis using a cDNA probe based on the sequence of the plasma membrane Ca pump expressed in the rat intestine (PMCA1). In the duodenum, Ca pump mRNA levels were 3-4 times higher in young (2 months) rats compared to adult (12 months) and old (27 months) rats. In the ileum, Ca pump mRNA levels were one third those of the duodenum, and ileal levels were higher in young rats compared to adult rats. These changes in mRNA levels with age and segment were significantly correlated with Ca pump activity as measured in basal-lateral membrane vesicles in vitro. To determine intestinal responsiveness to 1,25(OH)2D, rats were fed a strontium diet to induce vitamin D deficiency. In young animals, 1,25(OH)2D significantly increased Ca pump mRNA levels 4-fold in the duodenum. 1,25(OH)2D had a similar effect in the adult duodenum. These studies demonstrate that there are changes in Ca pump mRNA levels with age and intestinal segment. Since there was no change in the capacity of 1,25(OH)2D to increase Ca pump mRNA levels, the decline in Ca pump expression may be due to the age-related decrease in serum 1,25(OH)2D rather than to decrease responsiveness to 1,25(OH)2D.

Measurement of protein phosphatase activity in biological samples using synthetic phosphopeptides.

A method has been developed for measuring specific protein phosphatase activity in biological samples using synthetic, phospho-Kemptide and phospho-GS-peptide. This method uses ion-exchange chromatography to determine phosphatase activity by quantifying the release of [32P]phosphate directly. The method was used to measure phosphatase activity of rat kidney, adrenals, heart, and liver cytosol and the activity of purified alkaline phosphatases, protein phosphatase 1, and protein phosphatase 2A. Ion-exchange chromatography was also used for the preparation of the radiolabeled phosphopeptide substrates. This method results in high recovery and specific activity of the labeled peptides. These techniques should be useful in isolating and characterizing specific protein phosphatases found in cells.

Effect of parathyroid hormone on rat renal calcium/calmodulin-dependent protein kinase II.

Rat parathyroid hormone (PTH) stimulates cAMP-dependent protein kinase and protein kinase C activity in the kidney. However, PTH increases intracellular Calcium in primary cultures of proximal tubular cells. We have investigated the possibility that PTH also stimulates Calcium/calmodulin-dependent protein kinase II (CaM kinase II). We have employed the tandem chromatographic column method, using synthetic peptide as a substrate, to measure the renal CaM kinase II activity. PTH (250 nM) stimulated CaM kinase II activity by about 50% after 15 sec., and activity returned to baseline by 2 min. Calmodulin antagonists significantly impaired the stimulatory action of PTH whereas basal levels of CaM kinase II activity were relatively unaffected. This study demonstrates that PTH does activate CaM kinase II in renal tissue, and suggests another pathway for the actions of PTH in the kidney.

Characterization and regulation of the vitamin D hydroxylases.

The metabolism of vitamin D is regulated by three major cytochrome P450-containing h hydroxylases-the hepatic 25-hydroxylase, the renal 1α-hydroxylase, and the renal and intestinal 24-hydroxylase. In the liver, the 25-hydroxylation reaction is catalyzed by microsomal and mitochondrial cytochrome P450cc25. The microsomal P450 accepts electrons from the NADPH-cytochrome P450 reductase, and the mitochondrial P450 accepts electrons from NADPH-ferredoxin reductase and ferredoxin. In the kidney, the 1α- and 24-hydroxylation reactions are catalyzed by mitochondrial cytochromes P450cc1α and P450cc24, respectively. The 24-hydroxylase is also found in vitamin D target tissues such as the intestine. The rat hepatic mitochondrial P450cc25 and the rat renal mitochondrial P450cc24 have been purified, and their cDNAs have been cloned and sequenced. 1,25-Dihydroxyvitamin D, the active metabolite of vitamin D, markedly stimulates renal P450cc24 mRNA and 24-hydroxylase activity in the intact animal and in renal cell lines. This stimulation occurs via a receptor-mediated mechanism requiring new protein synthesis. Despite the availability of a clone, no studies have yet been reported of the regulation of hepatic P450cc25 at the mRNA level. The study of one of the most important enzymes in vitamin D metabolism, the renal 1α-hydroxylase which produces the active metabolite, awaits the definitive cloning of the cDNA for the P450cc1α.

Effects of diabetes mellitus on parathyroid hormone-stimulated protein kinase activity, ferredoxin phosphorylation, and renal 1,25-dihydroxyvitamin D production.

Impairment in the stimulation of renal production of 1,25-dihydroxyvitamin D[1,25 (OH)2D] by parathyroid hormone (PTH) occurs in diabetes. Renal response to PTH in terms of 25-hydroxyvitamin D-1-hydroxylase (1-OHase) stimulation involves increased cyclic adenosine monophosphate (cAMP) production, increased cAMP-dependent protein kinase activity, and dephosphorylation of renal ferredoxin (renoredoxin). To identify the step where diabetes might impair PTH stimulation of 1-OHase, we studied the effects of PTH on 1,25(OH)2D production, cAMP content, cAMP-dependent protein kinase activity, and the phosphorylation state of renoredoxin by using renal slices from diabetic and nondiabetic rats. PTH and forskolin significantly stimulated 1,25(OH)2D production in renal slices from nondiabetic animals but not from diabetic animals. PTH-stimulated cAMP production and cAMP-dependent protein kinase activity in renal slices were not altered by diabetes. However, diabetes significantly impaired the capacity of PTH to dephosphorylate renoredoxin and to increase the activity of the 1-OHase enzyme complex. These results suggest that the decreased capacity of PTH to stimulate 1-OHase activity in diabetic animals may reflect the decreased capacity of PTH to alter the phosphorylation state of renoredoxin in these animals.

Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates.

The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.

Calcitonin stimulates 1,25-dihydroxyvitamin D production in diabetic rat kidney.

In diabetic animals, there is a decrease in serum 1,25-dihydroxyvitamin D [1,25(OH)2D] and in renal production of 1,25(OH)2D. In nondiabetic animals, renal 1,25(OH)2D production is markedly stimulated by parathyroid hormone (PTH) and calcitonin (CT). There is evidence that diabetes impairs the responsiveness of the kidney to PTH. The effect of diabetes on responsiveness to CT is unknown. The studies reported here determined the effect of streptozotocin-induced diabetes on renal responsiveness to PTH and CT. Experiments were performed in 7- to 8-week-old rats that were fed a diet sufficient in calcium and vitamin D and were thyroparathyroidectomized (TPTX) 5 days before hormone treatment. PTH (0.33 U/g body weight at 24, 12, and 2 hours before death) significantly increased renal 1,25(OH)2D production by threefold in nondiabetic rats. This effect was markedly attenuated by diabetes. On the other hand, CT (20 U/100 g body weight at 12 and 2 hours before death) produced a maximal response in both groups of animals. In diabetic rats, CT stimulated renal 1,25(OH)2D production fivefold, whereas PTH stimulated production only 1.5-fold. Diabetes did not affect the capacity of PTH to increase serum calcium or decrease renal tubular reabsorption of phosphorus (TRP). These findings suggest that the decrease in renal 1,25(OH)2D production seen in experimental diabetes may be due to decreased renal responsiveness to PTH, but not to decreased responsiveness to CT.

Phosphorylation of ferredoxin and regulation of renal mitochondrial 25-hydroxyvitamin D-1 alpha-hydroxylase activity in vitro.

The kidney is the principal physiologic site of production of biologically active 1,25-dihydroxyvitamin D. The 25-hydroxyvitamin D-1 alpha-hydroxylase (1-OHase) activity found in renal mitochondria is under tight hormonal control. Parathyroid hormone stimulates the renal conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D in young animals, which is accompanied by dephosphorylation of ferredoxin (Fx), a component of the mitochondrial 1-OHase enzyme complex (Siegel, N., Wongsurawat, N., and Armbrecht, H. J. (1986) J. Biol. Chem. 261, 16998-17003). The present study investigates the capacity of Fx to be phosphorylated in vitro and to modulate the 1-OHase activity of a reconstituted system. Fx was phosphorylated by renal mitochondrial type II protein kinase. Phosphorylation did not alter Fx mobility on sodium dodecyl sulfate gels but did decrease the pI as measured by isoelectric focusing. Amino acid analysis demonstrated that 1 mol of serine and 1 mol of threonine were phosphorylated per mol of Fx. Peptide mapping of phosphorylated Fx was consistent with phosphorylation of serine 88 and threonine 85 or 97. Fx was selectively dephosphorylated by rabbit skeletal muscle protein phosphatase C2 but not C1. Phosphorylation of Fx significantly inhibited the 1-OHase activity of a reconstituted system consisting of Fx reductase, Fx, and renal mitochondrial cytochrome P-450. These findings suggest that phosphorylation/dephosphorylation of Fx may play a role in modulating renal 1,25-dihydroxyvitamin D production.

Effect of age on renal responsiveness to parathyroid hormone and calcitonin in rats.

The purpose of these studies was to determine whether the responsiveness of the kidney to parathyroid hormone (PTH) and calcitonin changed with age. Experiments were performed in young (3 months old), adult (12-14 months old) and old (22-24 months old) male Fischer 344 rats fed normal diets and thyroparathyroidectomized. Parathyroid hormone was administered i.p. at 24, 12 and 2 h before death and calcitonin was given i.p. at 12 and 2 h before death. Parathyroid hormone significantly increased the conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by renal slices from young but not adult or old animals. A similar age-related decline in the capacity of PTH to raise serum 1,25-dihydroxyvitamin D (1,25-(OH)2D) levels was also seen. Parathyroid hormone significantly decreased tubular reabsorption of phosphorus, increased concentrations of urinary cyclic AMP (cAMP) and increased serum concentrations of calcium in all age groups. In contrast, calcitonin significantly increased 1,25-(OH)2D3 production by renal slices from both young and adult animals. Calcitonin decreased serum concentrations of calcium in young but not in adult rats. These results suggest that there are maturational changes in the PTH- and cAMP-dependent pathways in the kidney but not in the calcitonin- and cAMP-independent pathways. The changes in the PTH- and cAMP-dependent pathways affect the stimulation of 1,25-(OH)2D production but not the inhibition of phosphate transport.

Comparison of calcium effect on in vitro calcitonin and parathyroid hormone release by young and aged thyroparathyroid glands.

Serum immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) levels are higher in young than aged rats. However, serum calcium concentration does not change with age suggesting that the calcium regulation of PTH and CT secretion may be affected by aging. We compared iPTH and iCT secretion in vitro at low and high calcium concentrations using thyroparathyroid glands removed from young (2-3 months), adult (12-13 months), and old (24-27 months) F-344 male rats fed regular rat chow. Glands from each animal were incubated for 3 h in serum-free culture media containing 1.0 mM calcium and then transferred to media containing 2.5 mM calcium for another 3 h. Immunoreactive PTH and iCT concentrations of the media after each incubation period were determined by radioimmunoassay. Immunoreactive PTH and iCT secretion per pair of glands was significantly higher in glands from older animals regardless of calcium concentration. The decrease in iPTH, and increment in iCT, secretion in response to 2.5 mM calcium by glands from old rats was smaller than that observed for glands from young animals. These age-related changes in the regulation of secretion by calcium may contribute to the increased iPTH and iCT secretion and serum levels seen in older animals.

Parathyroid hormone stimulates dephosphorylation of the renoredoxin component of the 25-hydroxyvitamin D3-1 alpha-hydroxylase from rat renal cortex.

Parathyroid hormone (PTH) stimulates the renal conversion of 25-OH-vitamin D3 to 1,25-(OH)2-vitamin D3 in young animals. There is evidence that PTH acts via cAMP and cAMP-dependent protein kinase, but the identity of the phosphorylated protein(s) is unknown. The present study investigates the possibility that phosphorylation modification of specific components of the renal mitochondrial, cytochrome P-450-linked 25-OH-vitamin D3-1 alpha-hydroxylase is involved in the regulation of 1,25-(OH)2-vitamin D3 production. Mitochondria were isolated from [32P]phosphate-labeled renal cortical slices which had been divided into control and agonist-treated groups. The hydroxylase protein components from the solubilized mitochondria were partially purified using p-chloroamphetamine-Sepharose affinity chromatography and polyacrylamide gel electrophoresis. Phosphorylation was observed only in a protein with an Mr = 12,000 and a pI of 4.2 by autoradiography of the gels. This radiolabeled protein was immunoprecipitated with adrenodoxin antibody. Additionally, the protein in the same Mr region of the polyacrylamide gel reacted with adrenodoxin antibody and co-migrated with bovine adrenodoxin. PTH and forskolin treatment resulted in decreased phosphate incorporation into the protein, whereas A23187 treatment increased the phosphorylation. In parallel experiments, affinity-isolated hydroxylase from control and PTH-treated slices was used to assess in vitro hydroxylase activity using [3H]25-hydroxyvitamin D3 as substrate. The hydroxylase activity derived from PTH-treated tissue was significantly higher than that of control. From these data, it is proposed that renal response to PTH in terms of 25-hydroxyvitamin D3 hydroxylase stimulation involves dephosphorylation of renoredoxin, the ferrodoxin component of this hydroxylase complex.

Aging and the kidney.

Numerous anatomic and physiologic alterations occur in the kidney with aging. These changes affect the ability of elderly patient(s) to maintain homeostasis and alter response to medications, stress, illness, or changes in diet, mobility, or environment. Drug-induced illness and drug interactions are major problems in the elderly. Bone disease and fractures are associated with negative calcium balance and decreased production of 1,25-dihydroxycholecalciferol seen with aging. The geriatric patient is not immune to the primary glomerular diseases that occur in younger patients, although the relative incidence of pathologic diagnoses may differ. The high incidence of membranous glomerulonephritis in the elderly, and the well-known association between malignancy and membranous nephropathy strongly favor aggressive evaluation of the nephrotic syndrome in the geriatric age group. Attention must be given to consideration of appropriate end-stage renal disease treatment alternatives for the geriatric population, which now comprises the fastest-growing segment of the end-stage renal disease population.

Insulin modulates the stimulation of renal 1,25-dihydroxyvitamin D3 production by parathyroid hormone.

Previous studies have shown that there is an impairment in renal production of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the major biologically active metabolite of vitamin D3, in diabetes. This impairment is not due to a deficiency in the parathyroid hormone (PTH), a major stimulator of renal 1,25(OH)2D3 production. Therefore, we have investigated the capacity of PTH to stimulate 1,25(OH)2D3 production in insulin deficiency and with insulin replacement. Experiments were performed in rats fed a 0.6% calcium, vitamin D sufficient diet for 2 weeks. Thyroparathyroidectomy was performed on all rats. Rats to be rendered diabetic were injected with streptozotocin immediately after surgery. In non-diabetic rats, PTH administration significantly increased renal 1,25(OH)2D3 production (11 +/- 2 vs 46 +/- 5 pg/min/g; P less than 0.05). In diabetic rats, however, PTH caused only a modest increase in 1,25(OH)2D3 production (11 +/- 1 vs 19 +/- 4 pg/min/g; P less than 0.05). With insulin replacement, PTH stimulation of 1,25(OH)2D3 production was markedly increased over that seen in diabetic rats (48 +/- 12 vs 19 +/- 4 pg/min/g; P less than 0.05). PTH was equally effective in raising serum calcium, depressing serum phosphorus and tubular reabsorption of phosphate in non-diabetic as well as in diabetic rats. These results demonstrate that insulin is necessary for the maximal stimulation of renal 1,25(OH)2D3 production by PTH. However, insulin is not necessary for PTH action in terms of renal handling of phosphate and inducing hypercalcaemia. These results suggest multiple pathways for the action of PTH, only some of which are insulin requiring.

Effects of hypophysectomy and growth hormone treatment on renal hydroxylation of 25-hydroxycholecalciferol in rats.

Growth hormone stimulates intestinal calcium absorption. This action has been linked to vitamin D metabolism. We have investigated the effects of hypophysectomy and GH treatment on renal metabolism of 25-hydroxycholecalciferol (25-OH-D3). Renal hydroxylation of 25-OH-D3 was measured in vitro using the renal slice technique. Experiments were performed in young F344 rats fed a vitamin D-replete, low calcium diet for 4 weeks. In hypophysectomized rats, renal conversion of 25-OH-D3 to 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was markedly reduced compared with sham-operated rats. Renal conversion of 25-OH-D3 to 24,25-(OH)2D3 was markedly increased in hypophysectomized rats compared with sham-operated rats. Treatment of hypophysectomized rats with rat GH (rGH) for 10 days resulted in a significant increase in renal conversion of 25-OH-D3 to 1,25-(OH)2D3 and a significant decrease in conversion to 24,25-(OH)2D3. Rat GH treatment caused no significant changes in serum levels of immunoreactive parathyroid hormone. Serum calcium concentrations were similar in all groups, and serum phosphorus was low in hypophysectomized rats. Treatment of hypophysectomized rats with ovine GH for 6 days caused changes which were much less pronounced than those induced by rGH. Renal conversion of 25-OH-D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3 correlated well with growth rate (weight gain). These results suggest that GH, either directly or indirectly, modulates renal metabolism of 25-OH-D3.

Forskolin increases 1,25-dihydroxyvitamin D3 production by rat renal slices in vitro.

Renal production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] from 25-hydroxyvitamin D3 (25OHD3) is increased by PTH. The complete mechanism by which PTH modulates renal 25OHD3 metabolism is not known, but there is some evidence that the stimulation of renal cAMP production by PTH may be important. Therefore, we have used forskolin, a direct activator of adenylate cyclase in the intact tissue, to further investigate the role of cAMP in regulating renal 25OHD3 metabolism. The effect of forskolin on renal 25OHD3 metabolism and renal adenylate cyclase activity was measured using isolated renal slices from thyroparathyroidectomized rats previously fed a vitamin D-deficient, low calcium diet. Forskolin added to renal slices in vitro for 4 h increased renal 1,25-(OH)2-D3 production in a concentration-dependent manner. In separate experiments, forskolin was found to increase tissue cAMP in a concentration-dependent manner when added for 5 min. The concentration of forskolin necessary for half-maximal stimulation of adenylate cyclase was 10 microM, and that needed for half-maximal stimulation of 1,25-(OH)2-D3 production was 1 microM. PTH added to renal slices also increased renal 1,25-(OH)2-D3 production, but the effects of PTH and forskolin were not additive. Inclusion of 1,25-(OH)2-D3 in the incubation medium blocked the effect of forskolin on 1,25-(OH)2-D3 production, but it did not block the effect of forskolin on tissue cAMP content. These studies support the concept that forskolin and PTH modulate renal 25OHD3 metabolism though a cAMP-dependent pathway. However, this pathway may be further regulated at sites distal to cAMP production by compounds such as 1,25-(OH)2-D3.

Effect of PTH and 1,25(OH)2D3 on renal 25(OH)D3 metabolism, adenylate cyclase, and protein kinase.

The purpose of these studies was to characterize the action of PTH and 1,25(OH)2D3 on the renal metabolism of 25(OH)D3 to 1,25(OH)2D3 and 24,25(OH)2D3. Renal metabolism of 25(OH)D3, adenylate cyclase, and protein kinase activity were measured using isolated renal slices from rats fed a vitamin D-deficient, low-calcium diet and thyroparathyroidectomized. PTH added to renal slices for 4 h in vitro maximally increased 1,25(OH)2D3 production by 67% and decreased 24,25(OH)2D3 production by 24% over the concentration range 0.05-5.0 U/ml. Parathyroid hormone (PTH) (0.05 U/ml) added to renal slices for 5 min produced a significant increase in tissue cAMP and a near-maximal increase in cAMP-dependent protein kinase activity. Preincubation of renal slices with 50 nM 1,25(OH)2D3 decreased renal 1,25(OH)2D3 production by 26% and increased 24,25(OH)2D3 production by 55%. 1,25(OH)2D3 also blocked the effect of PTH (5.0 U/ml) on renal 25(OH)D3 metabolism. However, PTH-stimulated adenylate cyclase and protein kinase activity was not blocked by preincubation with 1,25(OH)2D3. These studies demonstrate that PTH may act directly on the kidney to modulate renal 25(OH)D3 metabolism and that this action can be inhibited by 1,25(OH)2D3. This inhibition by 1,25(OH)2D3 occurs at a site distal to or separate from PTH-stimulated protein kinase activity.