The influence of nephrectomy, ureteral ligation, and of estradiol on plasma renin substrate in unilaterally nephrectomized rats

The effects of three experimental conditions on the concentration of plasma renin substrate were studied with special reference to plasma renin concentration in unilaterally nephrectomized rats. After simultaneous bilateral nephrectomy the maximum increase in plasma renin substrate was 17 times higher than normal within 24 h, while in rats which were unilaterally nephrectomized 10 days previously, followed by the removal of the remaining kidney (two-step bilateral nephrectomy), the maximum increase in plasma renin substrate was markedly suppressed (6-fold of normal). The maximum increases in plasma renin substrate by estradiol treatment in normal and unilaterally nephrectomized rats were about the same, associated with similarly decreased plasma renin concentrations. The similar increase in plasma renin substrate was found after ureteral ligation in unilaterally nephrectomized rats and bilateral ligation of the ureters in normal rats. This was the case where the plasma renin concentrations changed differently after ureteral ligation. After two-step bilateral nephrectomy plus estradiol treatment the maximum increase in plasma renin substrate was found to be higher than that found after two-step bilateral nephrectomy, but was lower than that after simultaneous bilateral nephrectomy. It is suggested that under the pathological conditions that stimulate renin substrate production, the plasma rein substrate concentration is less affected by circulating renin.     

Effect of 1,25 (OH)2 vitamin D3 on glomerulosclerosis in subtotally nephrectomized rats

In the past, there has been considerable concern that treatment with active vitamin D might accelerate progression independent of hypercalcemia and hypercalcuria. Nevertheless, 1,25(OH)2D3 has known antiproliferative properties and has also been shown to inhibit renal growth. Since glomerular growth is a permissive factor for the development of glomerulosclerosis, we reasoned that 1,25(OH)2D3 might even attenuate progression. To test this working hypothesis we performed two experiments of 8 and 16 weeks duration, respectively, to compare subtotally nephrectomized (SNX) rats treated with ethanol and SNX treated with 1,25(OH)2D3. Control animals were sham operated and pair-fed with SNX animals. 1,25(OH)2D3 (3 ng/100 g body wt/day) was administered by osmotic minipump. 1,25(OH)2D3 had no significant effect on systolic blood pressure and only a transient effect on weight gain. SNX reduced the number of glomeruli (left kidney) from an average of 3.3 x 10(4) to 1.2 x 10(4) per kidney. Mean glomerular volume was 3.87 +/- 0.71 x 10(6) microns 3 in sham operated animals and significantly (P < 0.05) higher (10.1 +/- 1.75 x 10(6) microns 3) in untreated animals 16 weeks after SNX. Glomerular volume was significantly (P < 0.05) less in 1,25(OH)2D3 treated SNX [10.1 +/- 1.75 in ethanol vs. 7.04 +/- 1.78 in 1,25(OH)2D3 treated SNX]. In parallel, there was significantly (P < 0.01) less glomerulosclerosis [glomerulosclerosis index 1.16 +/- 0.14 in the ethanol treated SNX vs. 0.80 +/- 0.16 in SNX treated with 1,25(OH)2D3] in the eight week experiment. Albuminuria was significantly (P < 0.01) lower in 1,25(OH)2D3 treated than in ethanol treated SNX (mean 0.785 mg/24 hr, range 0.43 to 1.80, vs. 3.75 mg/24 hr, 1.29 to 14.2). The morphological data were directionally analogous in a second 16 week experiment. Only slight changes of the vascular sclerosis index and tubulointerstitial index were seen in SNX and were not affected by 1,25(OH)2D3 further. To prove that the effect of 1,25(OH)2D3 was independent of PTH, parathyreoidectomized SNX rats without or with 1,25(OH)2D3 treatment were examined seven days post-SNX. PCNA staining showed suppression of cell proliferation. Furthermore, in situ hybridization for transforming growth factor-B (TGF-beta) showed less vascular and tubular expression in 1,25(OH)2D3 treated rats. We conclude that 1,25(OH)2D3 has antiproliferative actions during the compensatory growth of nephrons in response to subtotal nephrectomy. These effects are independent of PTH. The data document that 1,25(OH)2D3 reduces renal cell proliferation and glomerular growth as well as glomerulosclerosis and albuminuria as indicators of progressive glomerular damage.     

Inhibition of transmembrane K transfer in ureter-ligated dogs infused with KCl

In anuric dogs loaded with K by infusion with 2 meq KCl/kg per h until prelethal hyperkalemic cardiotoxicity appears, the extent of transmembrane K transfer depends on the origin of the anuria. Animals with bilateral ureter ligation transfer a mean of 1.2 meq/kg to intracellular fluid, while those with bilateral nephrectomy transfer more than 2.5 times as much (3.1 meq/kg). Further, if dogs with functioning kidneys are ureter ligated or nephrectomized after approximately 45 min of K loading, K transfer ultimately falls as infusion continues. The fall is precipitate and over 90% in ligated animals; but it is gradual, and only 10% in those that are nephrectomized. Finally, K transfer, because of the absence of insulin, is negligible in K-loaded pancreatectomized dogs with bilateral ureter ligation, but fairly substantial in pancreatectomized animals with bilateral nephrectomy. The data suggest that ureter ligation and hyperkalemia activate a renal mechanism that interferes with the transfer of infused K to intracellular fluid. The mechanism may involve the renin-angiotensin II-aldosterone system to a limited degree.     

Serum 1alpha,25-dihydroxyvitamin D3 accumulates into the fracture callus during rat femoral fracture healing

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is thought to be an important systemic factor in the fracture repair process, but the mechanism of action of 1,25(OH)2D3 has not been clearly defined. In this study, the role of 1,25(OH)2D3 in the fracture repair process was analyzed in a rat closed femoral fracture model. The plasma concentration of 1,25(OH)2D3 rapidly decreased on day 3 and continued to decrease to 10 days after fracture. We assessed whether this decrease was based on the accelerated degradation or retardation of the synthesis rate of 1,25(OH)2D3, from 25(OH)D3. After radiolabeled 3H-1,25(OH)2D3 or 3H-25(OH)D3 was injected i.v. into fractured or control (unfractured) rats, the concentrations of 25(OH)D3 and 1,25(OH)2D3 metabolites were measured by HPLC. The plasma concentrations of these radiolabeled metabolites in fractured group were similar to those in control rats early after operation. However, radioactivity in the femurs of fractured rats was higher than that of the control group. Furthermore, the radioactivity was concentrated in the callus of the fractured group analyzed by autoradiography. 1,25(OH)2D3 receptor gene expression was detected early after fracture and, additionally, both in the soft and hard callus on days 7 and 13 after fracture. These results showed that the rapid disappearance of 1,25(OH)2D3 in the early stages after fracture was not due to either increased degradation or decreased synthesis of 1,25(OH)2D3, but rather to increased consumption. Further, these results suggest the possibility that plasma 1,25(OH)2D3 becomes localized in the callus and may regulate cellular events in the process of fracture healing.     

Homologous up-regulation of vitamin D receptors is tissue specific in the rat

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors (VDR) are expressed in multiple tissues within the body. VDR levels are increased by 1,25(OH)2D3 in intestine and kidney and in numerous cell models. The ability of 1,25(OH)2D3 to affect VDR levels in other target tissues in vivo was studied by assessing VDR levels by the 3H-1,25(OH)2D3 binding assay under varied physiological conditions in the rat. When compared with vitamin D-deficient (-D) controls, rats raised on a normal vitamin D-sufficient (+D) diet showed elevated VDR levels in kidney (391 +/- 53 vs. 913 +/- 76 fmol/g of tissue;p < 0.05), but not in testis, heart, or lung. Up-regulation of the VDR also occurred in kidney of +D rats 1 day after a single 100-ng dose of 1,25(OH)2D3 (454 +/- 43 vs. 746 +/- 113 fmol/mg of DNA; p < 0.05), but no changes were seen in intestine, testis, or lung. Because 1,25(OH)2D3-induced hypercalcemia may independently affect VDR regulation, 1,25(OH)2D3 was infused into -D rats, and normocalcemia was maintained by reduced dietary calcium intake. In this model, the renal VDR was again up-regulated (446 +/- 115 vs. 778 +/- 58 fmol/mg of DNA; p < 0.05), but VDR levels in testis and lung were unaffected. Scatchard analysis and tests of 1,25(OH)2D3 dose (1-100 ng/day for 7 days) and temporal (100 ng/day for 1-7 days) responsiveness further supported the tissue-specific nature of the homologous VDR regulation. Assay of VDR levels by L-1-tosylamido-2-phenylethyl chloromethyl ketone-3H-1,25(OH)2D3 exchange assay ruled out differences in endogenous 1,25(OH)2D3 occupancy as the basis for the observed differences in VDR regulation. Finally, coidentity of the VDR-like sites in kidney versus testis was confirmed by competitive binding analysis comparing their relative affinities for 25(OH)D3 versus 1,25(OH)2D3 (30.5 +/- 6.4 vs. 35.6 +/- 3.6 in kidney and testis, respectively) and by immunoblot analysis using a highly specific monoclonal anti-rat VDR antibody. Thus, under a wide variety of experimental conditions, homologous up-regulation of the VDR occurs in the rat kidney in vivo, but not in several other target tissues which do not regulate plasma calcium homeostasis. Moreover, this differential VDR regulation did not result from secondary changes in plasma calcium, from differential 1,25(OH)2D3 responsiveness in the various tissues, nor from differences in endogenous 1,25(OH)2D3 occupancy of the VDR. These studies thus establish that, in contrast to observations in vitro, the widely described phenomenon of homologous VDR up-regulation in kidney and intestine is not a universal property of 1,25(OH)2D3 target tissues in vivo in the rat.     

EB 1089, a novel vitamin D analogue, has strong antiproliferative and differentiation inducing effects on cancer cells

EB 1089 is a novel vitamin D analogue which in vitro strongly inhibits the proliferation of U937 histiocytic lymphoma cells and MCF-7 breast cancer cells, with a potency of 50 to 100 times that of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Studies of c-myc and c-fos expression in MCF-7 cells and of differentiation markers in U937 cells show that growth inhibition by EB 1089 is accompanied by induction of differentiation. The ability of EB 1089 to affect calcium metabolism in vivo in rats is decreased, compared to 1,25(OH)2D3. This low calcemic effect combined with the strong biological effect on cancer cells in vitro, makes EB 1089 an interesting candidate for treatment of cancer.     

Posttraumatic hypothermia in the treatment of axonal damage in an animal model of traumatic axonal injury

Twenty-one-day-old BALB/c mice were shaved on the back to synchronize hair growth. On day 30 or 31, when at least 90% of mice exhibited hair regrowth in the shaved area, 1,25(OH)2D3 was applied topically to the shaved area daily for 5 days. On the 6th day, cyclophosphamide (Cytoxan, CTX) was injected i.p. to induce hair loss in the shaved area. Alopecia was induced in a dose-dependent manner by CTX treatment within 1 to 2 weeks. This effect was reduced significantly if mice were pre-treated with 1,25(OH)2D3, though only slight protection was observed in female mice. Interestingly, this 1,25(OH)2D3-mediated protection against hair loss was attenuated in male mice but became more significant in female mice when they were inoculated with the EMT-6 murine mammary tumor prior to treatment. More importantly, topical treatment with 1,25(OH)2D3 alone was able to inhibit EMT-6 tumor growth in both male and female BALB/c mice. Furthermore, 1,25(OH)2D3 pre-treatment also augmented the anti-tumor effect of CTX. Our results demonstrate that topical application of 1,25(OH)2D3 can protect against CTX-induced alopecia both in tumor-free and in tumor-bearing mice in a sex-dependent manner. Moreover, 1,25(OH)2D3 was shown, either alone or in combination with CTX, to inhibit tumor growth.     

Influence of vitamin D3 infusion and dietary calcium on secretion of interleukin 1, interleukin 6, and tumor necrosis factor in mice infected with Mycobacterium paratuberculosis

OBJECTIVE: To study the effects of 1,25(OH)2D3 and calcium (Ca) on splenocyte cytokine secretion during Mycobacterium paratuberculosis infection. DESIGN: Mice were assigned to the following treatments: 1-noninfected, 2-infected, 3-noninfected/1,25(OH)2D3, 4-infected/1,25(OH)2D3, and 5-infected/low-Ca diet (0.15%). ANIMALS--Male beige mice averaging 6 weeks of age and 20 g in body weight. PROCEDURE: After acclimation to their diets, mice in treatments 2, 4, and 5 were inoculated IV with 10(8) colony-forming units of M paratuberculosis. At 1, 6, and 12 months after infection, mice in treatment groups 3 and 4 had miniosmotic pumps implanted subcutaneously that delivered 13 ng of 1,25(OH)2D3/day for 14 days. Treatment 5 was included as a control for comparison with treatment 4 because low dietary Ca should increase endogenous 1,25(OH)2D3 values. Splenocytes were isolated from mice at 1, 6, and 12 months and stimulated in vitro with medium alone (nonstimulated), lipopolysaccharide (LPS), concanavalin A, and M paratuberculosis whole-cell sonicate (MpS). RESULTS: Production of interleukin 6 after stimulation with LPS, concanavalin A, or MpS was higher (P < 0.05) for splenocytes isolated from mice fed the low-Ca diet, compared with control infected mice 1 and 6 months after infection. Interleukin 1 and tumor necrosis factor activities were increased (P < 0.05) in splenocytes cultured with LPS and MpS after isolation from mice of the low-Ca group. Mice infused with 1,25(OH)2D3 had higher (P < 0.05) interleukin 1 secretion after stimulation of splenocytes with LPS and higher (P < 0.05) tumor necrosis factor production after incubation with MpS. CONCLUSION: 1,25(OH)2D3 and low dietary Ca increase cytokine secretion in mice infected with M paratuberculosis.     

In vivo treatment with calcitriol (1,25(OH)2D3) reverses age-dependent alterations of intestinal calcium uptake in rat enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D3; [1, 25(OH)2D3] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)2D3 stimulation of Ca2+ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20-24-month-old rats given 1,25(OH)2D3 for 3 days (30 ng/100 g bw/day), calcitriol (10(-10) M, 3-5 minutes) stimulated Ca2&plus uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)2D3. When the aged rats were injected with 25(OH)D3, similar Ca2+ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1, 25(OH)2D3-dependent changes in Ca2+ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)2D3 stimulation of duodenal cell Ca2+ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)2D3 or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system.     

Growth and nutrition of uremic piglets

Piglets aged 6 days were rendered uremic by subtotal nephrectomy and their growth and dietary intakes studied over the next 21 days. Eleven control piglets fed a voluntary intake of a sow's milk substitute (group A), 11 nephrectomized piglets fed a voluntary intake of the same feed (group B), 6 nephrectomized piglets tube fed the same milk (group C), and 11 nephrectomized piglets fed a voluntary intake of a low protein, isocaloric food (group D) were studied. After nephrectomy the piglets had an initial rapid rise in blood urea concentration which had fallen by day 7 and then leveled out around 13 mmol/liter in group B and 8 mmol/liter in group D. After operation control piglets (group A) ate more from day 4 and were larger from day 7 than the nephrectomized piglets (group B). Those piglets tube fed (group C) were of a similar size to the controls but all died between day 7 and day 11 with associated high blood urea concentrations. Piglets fed the low protein, isocaloric feed (group D) were smaller than both the controls and group B. They also ate less food than the controls and those nephrectomized piglets in group B which were on a voluntary intake of the normal feed.     

Intermittent versus continuous administration of 1,25-dihydroxyvitamin D3 in experimental renal hyperparathyroidism

Conflicting results have been reported regarding the efficacy of intermittent versus continuous administration of 1,25(OH)2D3 in renal secondary hyperparathyroidism. To address this issue we examined sham-operated control rats and hyperparathyroid rats with subtotal (5/6) nephrectomy (Nx). The Nx animals (20 to 22 animals per group) were subjected to three treatment protocols: (i) solvent treatment (Nx-solvent); (ii) two i.p. injections of 35 pmol 1,25(OH)2D3 on days 0 and 4 (Nx-bolus); and (iii) continuous infusion of 70 pmol 1,25(OH)2D3 over six days via osmotic minipump (Nx-infusion). All measurements were performed six days after start of treatment. As compared to sham-operated controls, the pre-pro-PTH/beta-actin mRNA ratio was 2.04-fold higher in Nx-solvent. Both modes of administration of 1,25(OH)2D3 resulted in inhibition of PTH mRNA concentrations relative to Nx-solvent. The pre-pro-PTH/beta-actin mRNA ratio was, however, significantly lower (P < 0.05) in Nx-bolus than in Nx-infusion (Nx-bolus 1.26 higher than sham-operated controls; Nx-infusion 1.65 higher than sham-operated controls). Aminoterminal PTH (N-PTH) serum concentrations were higher in Nx-solvent (52 +/- 4 pg/ml) than in sham-operated controls (32 +/- 3 pg/ml, P < 0.01). N-PTH concentrations in Nx-bolus (38 +/- 4 pg/ml) were significantly lower than in Nx-solvent (P < 0.01) and in Nx-infusion (46 +/- 4 pg/ml, P < 0.05). Parathyroid gland weight (microgram/g body wt) was higher in Nx-solvent (1.30 +/- 0.08 pg/ml) than in sham-operated controls (0.79 +/- 0.04 pg/ml, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial prevention of active Heymann nephritis by 1 alpha, 25 dihydroxyvitamin D3

The hormone 1 alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3) has potent immunosuppressive effects in vitro. Recent publications also described a protective effect of the hormone in various animal models of immune-mediated diseases. To test its in vivo activity we induced active Heymann nephritis in Lewis rats that were either untreated or treated with 1,25(OH)2D3 or its synthetic 20-epi analogue, KH1060. Treatment with cyclosporine A (CsA) was used as an immunosuppressive control. In this nephrotic model the administration of 1,25(OH)2D3 (0.5 microgram/kg body weight) given on alternate days during the first 13 days after active immunization significantly reduced the proteinuria as measured by weeks 7-9. This reduction was comparable to the reduction observed in rats treated with CsA (20 mg/kg) on alternate days. A second series of experiments with 1,25(OH)2D3 confirmed these findings. The level of autoantibodies was found to be significantly suppressed during the treatment time in the CsA (20 mg/kg) group, whereas the limit of significance (P = 0.06) was reached in the 1,25(OH)2D3 (0.5 microgram/kg) group. The size of the immune deposits also was found to be substantially smaller in the groups that developed less proteinuria. The administration of 1,25(OH)2D3 transiently increased the mean serum calcium concentration with 2.5 mg/dl above the pretreatment values, and the urinary calcium excretion by a factor of 3-5 during the short treatment time. Treatment with the analogue KH1060 did not reduce the proteinuria significantly. Our experiments add evidence to the hypothesis that 1,25(OH)2D3 in pharmacological doses has immunosuppressive potency.     

Pathogenesis and characterization of hyperglucagonemia in the uremic rat

The pathogenesis of hyperglucagonemia and of the alterations in the pattern of circulating immunoreactive glucagon (IRG) associated with renal insufficiency was studied in rats in which a comparable degree of uremia was induced by three different methods, i.e., bilateral nephrectomy, bilateral ureteral ligation, and urine autoinfusion. Nephrectomized and ureteral-ligated rats were markedly hyperglucagonemic (575 +/- 95 pg/ml and 492 +/- 54 pg/ml, respectively), while IRG levels of urine autoinfused animals (208 +/- 35 pg/ml) were similar to those of control rats (180 +/- 26 pg/ml), indicating that uremia per se does not account for the hyperglucagonemia observed in renal failure. Similarly, plasma IRG composition in this group of animals was indistinguishable from that of controls, in which 88.2 +/- 5.9% of total IRG consisted of the 3,500-mol wt fraction. The same component was almost entirely responsible (82.6 +/- 4.1%) for the hyperglucagonemia observed in ligated rats, while it accounted for only 57.6 +/- 5.0% of the circulating IRG in nephrectomized animals. In the latter group, 36.8 +/- 6.6% of total IRG had a mol wt of approximately 9,000, consistent with a glucagon precursor. This peak was present in samples obtained as early as 2 h after renal ablation and its concentration continued to increase with time reaching maximal levels at 24 h. These results confirm that the kidney is a major site of glucagon metabolism and provide evidence that the renal handling of the various circulating IRG components may involve different mechanisms. Thus, the metabolism of the 3,500-mol wt fraction is dependent upon glomerular filtration, while the uptake of the 9,000-mol wt material can proceed in its absence, as long as renal tissue remains adequately perfused. This finding suggests that the 9,000-mol wt component may be handled by peritubular uptake.     

In vivo regulation of chromogranin A messenger ribonucleic acid in the parathyroid by 1,25-dihydroxyvitamin D: studies in normal rats and in chronic renal insufficiency

Chromogranin-A (CgA) and PTH are the two major secretory products of the parathyroid gland. In vitro, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases CgA, but decreases PTH messenger RNA (mRNA) levels. We investigated the physiological significance of the induced changes in CgA expression by examining the effects of 1,25-(OH)2D3 on parathyroid CgA mRNA levels in vivo. Normal rats were injected with 1,25-(OH)2D3 at 48 and 24 h before blood sampling and isolation of both parathyroid glands. Parathyroid total RNA was extracted and CgA and PTH mRNA quantified by Northern blot analysis. CgA mRNA levels increased 1.6-, 3.2- and 5.6-fold, whereas PTH mRNA levels decreased by 37, 63 and 97%, respectively, with 1,25-(OH)2D3 doses of 10, 50, and 250 pmol/100 g BW. Parathyroid gland CgA expression also was examined in rats with mild chronic renal insufficiency, induced by a 5/6 nephrectomy 5 weeks earlier. Chronic renal insufficiency rats, fed normal chow, had elevated serum urea, creatinine, and PTH levels and reduced 1,25-(OH)2D3 but normal serum levels of calcium and phosphate. PTH mRNA levels were elevated 4-fold and CgA mRNA levels were 50% lower in the uremic animals. This indicates that the regulation of CgA expression in normocalcemic rats occurs at physiological 1,25-(OH)2D3 concentrations. In summary, increases and decreases in serum 1,25-(OH)2D3 levels are associated with corresponding increases and decreases in CgA mRNA levels in the parathyroid glands of rats. Therefore, this study is the first to demonstrate the physiological relevance of the earlier in vitro observations.     

Human and murine osteocalcin gene expression: conserved tissue restricted expression and divergent responses to 1,25-dihydroxyvitamin D3 in vivo

Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)2D3 treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)2D3 injection (23.6+/-3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2+/-0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)2D3 treatment in animals on a low calcium diet (25.2+/-7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)2D3 production (7.5+/-0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)2D3 injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)2D3. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)2D3 is not. The mouse osteocalcin genes do not respond to 1,25-(OH)2D3 treatment, but the human osteocalcin-directed transgene is markedly upregulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)2D3 are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.     

Vitamin D increases expression of the tyrosine hydroxylase gene in adrenal medullary cells

We examined expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] receptors in chromaffin cells of the adrenal medulla and the effects of 1,25(OH)2 D3 on expression of the tyrosine hydroxylase (TH) gene. Accumulation of 1,25(OH)2 D3 in the nuclei of adrenal medullary cells, but not in the adrenal cortex, was observed in mice intravenously injected with radioactively labeled hormone. 1,25(OH)2 D3 produced concentration-dependent increases in the TH mRNA levels in cultured bovine adrenal medullary cells (BAMC). The maximal increases (2-3-fold) occurred at 10(-8) M 1,25(OH)2 D3. Combined treatment with 1,25(OH)2 D3 and 20 microM nicotine had no additive effect on TH mRNA levels suggesting that transsynaptic (nicotinic) and vitamin D (hormonal) stimulation of TH gene expression are mediated through converging mechanisms. Induction of TH mRNA by 1,25(OH)2 D3 was not affected by calcium antagonist TMB-8. By increasing expression of the rate limiting enzyme in the catecholamine biosynthetic pathway, 1,25-(OH)2 D3 may participate in the regulation of catecholamine production in adrenal chromaffin cells. This regulation provides mechanisms through which 1,25(OH)2 D3 may control response and adaptation to stress.     

Studies on the bioactive constituents of Aurantii Fructus Immaturus

This study investigated the regulatory activity of 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) on phagocytic cells obtained from normal human peripheral blood. Flow cytometric analysis enabled identification of two discrete populations of cells, one predominantly monocytes ("monocyte" gate) and one containing primarily lymphoid and other cell types ("lymphoid" gate). The monocyte-associated antigens CD13 and CD33 were highly expressed by cells in this monocyte gate and used to monitor this population. Following 5 days of culture, cells in the monocyte gate manifested high phagocytic activity as determined by ingestion of fluorescent carboxylmicrospheres and exhibited high expression of class II HLA-DR products. 1,25-(OH)2D3 profoundly upregulated phagocytic activity while downregulating HLA-DR antigen expression on the cells in the monocyte gate. Moreover, 1,25-(OH)2D3 also reduced cell surface CD13 expression on the cells with low but not high phagocytic activity in this gate. Proportional activities by the 1,24-(OH)2D3 and 24,25-(OH)2D3 metabolites indicated the regulatory effects are likely mediated by the 1,25-(OH)2D3 receptor (VDR). Prostaglandin E2 (PGE2), a known modulator of monocyte/macrophage activity also markedly inhibited HLA-DR expression while enhancing the phagocytic activity of cells in the monocyte gate. In contrast to 1,25-(OH)2D3, PGE2 clearly upregulated CD13 expression in cells with high phagocyte activity. Since indomethacin, an inhibitor of PGE2 synthesis, failed to reverse the 1,25-(OH)2D3 induced inhibitory effect on HLA-DR expression, this effect is apparently not mediated through endogenous PGE2 synthesis. Based on these findings we speculate that 1,25-(OH)2D3 may be capable of acting as both an upregulating agent during natural immunity via the enhancement of phagocytosis by monocyte/macrophage populations and as a "downregulator" during acquired immune responses via an inhibitory effect on MHC class II antigen expression by professional antigen-presenting cells.     

Effect of metabolic acidosis on insulin action and secretion in uremia

BACKGROUND: Metabolic acidosis affects both vitamin D and insulin metabolism. Vitamin D is important in modulation of both insulin secretion and insulin sensitivity in uremia. The present study examines the effect of correction of metabolic acidosis on insulin action and secretion as well as 1,25 vitamin D3 concentrations in uremic patients. METHODS: Eight patients (age 18 +/- 1 year) on maintenance hemodialysis with metabolic acidosis were studied before and after two weeks of oral sodium bicarbonate (NaHCO3) treatment to correct the acidosis. To control for the effect of additional sodium, they were also studied after two weeks of an equivalent amount of oral sodium chloride (NaCl). Controls consisted of 7 healthy controls (age 19 +/- 1 year). Insulin sensitivity was measured by the hyperinsulinemic euglycemic clamp technique. Insulin secretion was measured by the hyperglycemic clamp technique. RESULTS: Oral NaHCO3 treatment led to significant increases in venous pH and serum bicarbonate concentrations but no significant change in intact parathyroid hormone (PTH) concentrations. Circulating 1,25 dihydroxyvitamin [(OH)2] D3 were significantly lower than control values initially and increased significantly after treatment. Oral NaCl did not change any of the biochemical parameters. Before treatment of acidosis, uremic patients had lower insulin sensitivity (insulin resistance) during constant hyperinsulinemia and lower insulin secretion during constant hyperglycemia compared with controls. Following two weeks of NaHCO3 treatment there were significant increases in insulin sensitivity and insulin secretion, although the values did not normalize. There were no changes in insulin sensitivity or insulin secretion following two weeks of NaCl. CONCLUSION: Treatment of metabolic acidosis increased both insulin sensitivity and insulin secretion in patients with uremia. This was accompanied by an increase in the circulating levels of 1,25(OH)2D3 but no change in those of parathyroid hormone.     

The roles of renal catabolism and uremia in modifying the clearance of fibrinogen and its degradative fragments D and E

Elevated levels of fibrinogen/fibrin degradation products (FDP) occur in uremia, and have been thought to be in part related to intravascular coagulation in the kidney. More recent data indicated that delayed catabolism of fibrinogen fragment D occurred in anephric animals. To further evaluate FDP catabolism in the kidney, turnover studies of purified dog 131I-Fg-D and 125I-Fg-E were performed on dogs before and after acute subtotal nephrectomies, and later during chronic uremia. 131I-fibrinogen clearances were also perfomed. Slowed catabolism of Fg-D and Fg-E was observed in both the acute and chronic uremic stages. Altered urinary excretion was not a factor as only minimal amounts of Fg-D and Fg-E were excreted in the urine of the control animals. In the 131I-fibrinogen studies, there were significant changes in plasma volume, fibrinogen t 1/2, and intravascular/extravascular distribution, but not in fractional catabolic rate. To differentiate fully, the effects of uremia from those of loss of catabolic renal tissue, the Fg-D and Fg-E turnover studies were repeated on other animals with intact kidneys whose ureters were diverted into the peritoneum and compared to subsequent studies after total nephrectomy. The control and ureter-severed studies had the same clearance pattern, whereas decreased catabolism occurred in the nephrectomized dogs. The results demonstrate uremia per se does not have a major effect upon the catabolism of fibrinogen, Fg-D, and Fg-E. Loss of renal tissue does impair the clearance of Fg-D and Fg-E, indicating these proteins are normally catabolized in part by the kidneys. Thus elevated plasma FRA in uremic patients may reflect decreased Fg-D and Fg-E catabolism rather than increased FDP production from primary or secondary fibrinolysis.     

The E1A oncogene induces resistance to the effects of 1,25-dihydroxyvitamin D3 on inhibition of growth of mouse keratinocytes

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited DNA synthesis in transformed mouse keratinocytes (Pam212) in a time- and dose-dependent manner as measured by [3H]thymidine incorporation. To investigate the mechanism through which 1,25-(OH)2D3 acts, we examined its effects on Pam212 cells further transformed with the E1A oncogene. Here, we show that transformation of the cells with the E1A oncogene induced resistance to the effects of 1,25-(OH)2D3 on inhibition of growth of Pam212 cells. While 1,25-(OH)2D3 treatment increased the level of expression of vitamin D receptor mRNA 20-fold in parental cells, the E1A-transformed cells failed to express vitamin D receptor mRNA even after treatment with 1,25-(OH)2D3. Transfection of the E1A-transformed cell line with an expression construct encoding the vitamin D receptor restored receptor expression as well as the inhibition of growth by 1,25-(OH)2D3. These results suggest that one of the mechanisms for acquisition of 1,25-(OH)2D3 resistance induced by E1A may involve loss of vitamin D receptor inducibility by 1,25-(OH)2D3.