Martin Egfjord

Renal and intestinal calcium-binding proteins in normal and uraemic rats

The effect of chronic uraemia on the concentrations of the 28 kDa renal and 9 kDa intestinal calcium-binding proteins (calbindin-D28K and calbindin-D9K) was investigated in rats. Calbindin-D9K was measured by a competitive enzyme-linked immunoadsorbent assay and calbindin-D28K by rocket immunoelectrophoresis. Chronic uraemia was induced by 5/6 nephrectomy and the results were compared to sham-operated animals. Rats were fed on a diet containing 0.9% calcium and 1.2% phosphorous. Plasma creatinine and plasma urea were elevated in the nephrectomized rats (p less than 0.001), while plasma-1,25-dihydroxycalciferol vitamin D and fractional calcium absorption were unchanged. Plasma parathyroid hormone was significantly elevated in the uraemic rats. The concentration of calbindin-D28K in renal tissue was increased (p less than 0.001) in rats with chronic uraemia and a direct correlation was found between renal calbindin-D28K and plasma urea (p less than 0.05). Intestinal calbindin-D9K correlated inversely with plasma creatinine (p less than 0.05), but the mean level of calbindin-D9K was unchanged in this model of moderate chronic uraemia. Thus, different regulatory mechanisms control levels of calbindin-D9K and calbindin-D28K.

Comparison between 1α(OH)D3 and 1,25(OH)2D3 on the Suppression of Plasma PTH Levels in Uremic Patients, Evaluated by the ‘Whole’ and ‘Intact’ PTH Assays

Background/Aims: The aim was to evaluate the acute effects of intravenous 1α(OH)D₃ and 1,25(OH)₂D₃ on (1) plasma parathyroid hormone (PTH) and Ca²⁺ levels in chronic uremic patients and (2) circulating large C-terminal PTH fragments as measured by the ‘whole PTH’ assay compared to two different ‘intact PTH’ assays. Methods: 11 patients on chronic hemodialysis with plasma intact PTH >90 pg/ml were studied. At time zero 10 µg 1,25(OH)₂D₃ (Calcijex, Abbott, USA), or 10 µg 1α(OH)D₃ (Etalpha, LEO, Denmark) or 10 ml of isotonic saline was injected as a bolus. Blood samples for analyses of plasma Ca²⁺ and plasma PTH were drawn at 0, 6, 12, 24, 48 and 72 h. The same patient was studied 3 times in a random fashion with an interval of 3 weeks. Further, 7 of the patients were studied after an injection of 6 µg 1,25(OH)₂D₃ intravenously. Results: No significant changes in plasma Ca²⁺ and PTH were seen after administration of saline. Twenty-four hours after administration of 1,25(OH)₂D₃, plasma PTH decreased from a maximum level of PTH_WHOLE 151 ± 27 to a minimum of 58 ± 13 pg/ml; from a maximum level of PTH_TOTAL 247 ± 40 to a minimum of 99 ± 26 pg/ml and from a maximum level of PTH_INTACT 205 ± 29 to a minimum of 83 ± 18 pg/ml (p < 0.001). Twenty-four hours after administration of 1α(OH)D₃, plasma PTH levels decreased from a maximum level of PTH_WHOLE 155 ± 21 to a minimum of 116 ± 15 pg/ml; from a maximum level of PTH_TOTAL 265 ± 33 to a minimum of 221 ± 35 pg/ml and from a maximum level of PTH_INTACT 222 ± 26 to a minimum of 182 ± 23 pg/ml (p <0.05). Regardless of which of the three assays that was applied, the percentage suppression of PTH following administration of 1,25(OH)₂D₃ was approximately 60% and following administration of 1α(OH)D₃ approximately 20%. Significant correlations were demonstrated between the Whole and the intact PTH assays, and as expected between the 2 intact assays (‘Whole’/‘Intact’, r = 0.92, p < 0.0001, ‘Whole’/‘Total’, r = 0.94, p < 0.0001, ‘Intact’/‘Total’, r = 0.97, p < 0.0001) with no influence of the two vitamin D analogs administered. Plasma Ca²⁺ remained stable after administration of saline. After 24 h, no increase in plasma Ca²⁺ was observed after administration of 1α(OH)D₃ or after administration of 6 µg 1,25(OH)₂D₃, while plasma Ca²⁺ after administration of 10 µg 1,25(OH)₂D₃ increased to 1.31 ± 0.03 mmol/l (p < 0.008). After 72 h, 1α(OH)D₃ increased plasma Ca²⁺ to 1.22 ± 0.02 mmol/l (p < 0.05) and 10 µg 1,25(OH)₂D₃ to 1.27 ± 0.03 mmol/l. Plasma phosphate was within the normal range before administration of saline (1.24 ± 0.13 mmol/l), 1,25(OH)₂D₃ (1.28 ± 0.12 mmol/l) and 1α(OH)D₃ (1.46 ± 0.21 mmol/l). Plasma phosphate increased significantly after 24, 48 and 72 h to a maximum of 2.06 ± 0.27 mmol/l after administration of 1,25(OH)₂D₃ and a maximum of 1.94 ± 0.31 mmol/l after administration of 1α(OH)D₃. Plasma phosphate was significantly higher after 1,25(OH)₂D₃ than after 1α(OH)D₃ at 48 (p = 0.016) and 72 h (p < 0.010). Conclusion: A single intravenous dose of both 10 µg 1,25(OH)₂D₃ and 1α(OH)D₃ significantly suppressed plasma PTH. The acute suppressive effect of 1,25(OH)₂D₃ was 3 times greater than that of 1α(OH)D₃. The increase in plasma Ca²⁺ after intravenous administration of 10 µg of 1,25(OH)₂D₃ was, however, significantly greater than that of 10 µg of 1α(OH)D₃ (p < 0.005). The PTH response to acute administration of 10 µg of the two vitamin D analogs was in principle the same, when measured by the three different assays and resulted in a parallel shift of the PTH response curves. Thus, circulating levels of large C-terminal PTH fragments were not influenced by differences in plasma Ca²⁺ or by the vitamin D analog given.

Effects of methylprednisolone and uremia on renal and intestinal calbindin-D in the rat.

The effects of glucocorticoids on renal and intestinal calcium binding protein (calbindin-D28K and calbindin-D9K) were examined in normal and uremic rats. Chronic uremic rats and normal controls were treated with either methylprednisolone (MP) 1.3 mg/kg/d or isotonic saline given as a continuous intraperitoneal infusion for 1 week before sacrifice. Renal calbindin-D28K was measured by rocket immunoelectrophoresis and intestinal calbindin-D9K was measured by an enzyme-linked immunoadsorbent assay. Methylprednisolone treatment of chronic uremic rats increased plasma phosphate levels (P < 0.05), but plasma calcium and 1,25-dihydroxyvitamin D3 were unchanged in all groups. MP treatment did not affect the renal calbindin-D28K in either normal or uremic rats. In normal rats, MP treatment reduced intestinal calbindin-D9K by 28% when compared to placebo (P < 0.05). In contrast, chronic uremia increased renal calbindin-D28K by 51% and 38% (P < 0.001) in placebo and MP treated uremic rats, respectively, while intestinal calbindin-D9K was unchanged. Thus, MP treatment and chronic uremia induced different changes in renal and intestinal calbindin-D of the rat suggesting that different mechanisms are involved in the regulation of these vitamin D dependent proteins.

Calcium metabolic changes and calbindin-D in experimental hypertension.

Objective To examine renal and intestinal calbindin-D in relation to calcium metabolic changes in three different models of experimental hypertension. Design: Spontaneously hypertensive rats (SHR), hypertension-prone Dahl salt-sensitive (Dahl-S) rats and the Goldblatt two-kidney, one clip rat model of renovascular hypertension were examined. Results Both prehypertensive and hypertensive SHR had significantly lower concentrations of both renal calbindin-D28k and intestinal calbindin-D9k than Wistar control rats. This was accompanied by hypocalcaemia, hypomagnesaemia and increased plasma 1,25(OH)2 vitamin D levels. Induction of hypertension in Dahl-S rats reduced intestinal calbindin-D9k and increased plasma levels of 1,25(OH)2 vitamin D, while renal calbindin-D28k levels, plasma calcium levels and plasma magnesium levels were unchanged. Renovascular hypertension was associated with a significant increase in the intestinal calbindin-D9k, plasma 1,25(OH)2 vitamin D, parathyroid hormone and magnesium levels, while renal calbindin-D28k, plasma calcium and phosphorus levels were unaffected. Conclusions These three models of experimental hypertension have clearly demonstrated three separate patterns in the regulation of renal and intestinal calbindin-D, which relate to different alterations of factors involved in calcium and magnesium metabolism. In all three models hypertension was accompanied by a significant increase in plasma concentrations of 1,25(OH)2 vitamin D. Only rats with renovascular hypertension showed increased intestinal calbindin-D9k levels, whereas reduced concentrations were found in the SHR and in the hypertensive Dahl-S rats. This indicates the existence of a resistance at the cellular level to 1,25(OH)2 vitamin D affecting the expression of calbindin-D in both SHR and Dahl-S rats.

Intestinal and renal calcium-binding protein in rats with experimental short bowel syndrome

Intestinal calcium-binding protein (calbindin-D9K) and renal calcium-binding protein (calbindin-D28K) levels were measured by enzyme-linked immunoadsorbent assays in rats with short bowel syndrome induced by resection of about 85% of the small intestine. Rats with short bowel syndrome had significantly lower mucosal concentrations of calbindin-D9K (P less than 0.001) and a parallel reduction of both intestinal calcium absorption (P less than 0.001) and p-1,25-dihydroxyvitamin D (P less than 0.01) in spite of a general hypertrophy of the duodenal mucosa. Interestingly, the renal concentration of calbindin-D28K was significantly elevated (P less than 0.05) in rats with short bowel syndrome, a change apparently related to factors independent of vitamin D.

Effect of uremia on aldosterone metabolism in the isolated perfused rat liver

The effect of uremia on hepatic metabolism of aldosterone was studied in the isolated perfused liver of female Wistar rats. Uremia was induced by five-sixths partial nephrectomy 4 weeks before experiments. Isolated livers of normal and uremic rats were perfused at a constant flow rate with a hemoglobin-free medium, to which 4-14C-D-aldosterone was added at 3 nmol/L. Aldosterone was analyzed by radioimmunoassay (RIA) and 4-14C-D-aldosterone radiometabolites in perfusate and bile were assayed by high-performance liquid chromatography (HPLC). Uremic rats had a 10% lower body weight (P < .01) and increased plasma urea, creatinine, and parathyroid hormone (PTH) levels (258%, 200%, and 208%, respectively; P < .01-.001). Blood pressure and plasma K+, Na+, and aldosterone levels were similar. Plasma renin activity was suppressed by 68% in uremic rats (P < .001). Liver wet weight and hepatic function were similar in livers of both groups of rats. Hepatic elimination of aldosterone was compatible with a first-order kinetics. Hepatic clearance of aldosterone per liver and per gram liver was similar; however, when expressed per 100 g rat body weight, a 21% higher value was observed in uremic rats (11.6 +/- 1.8 mL/min) compared with normal rats (9.6 +/- 1.5 mL/min, P < .01). Polar aldosterone radiometabolites accumulated in the perfusate to approximately 40% of the initial 14C added at 15 minutes, and were eliminated in bile at a similar rate in both groups. No qualitative difference was found in the pattern of radiometabolites of aldosterone in perfusate and bile.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple myeloma in a kidney transplanted patient primarily diagnosed with monoclonal gammopathy of unknown significance (MGUS)-related nephropathy

A 36-year-old female was referred with renal impairment, creatinine clearance 0.4 mL/s and proteinuria 4.7 g/day. Physical examination was unremarkable. Biochemistry showed normochromic normocytic anaemia, slight hypoalbuminaemia and hypercalcaemia. Anti-neutrophil cytoplasmic antibodies, anti-nuclear antibodies, anti-glomerular basement membrane, anti-phospholipid antibodies, and viral screening were negative. IgG-kappa M-protein was found in plasma (4.8 g/L) and urine (<0.02 g/L), while plasma immunoglobulins and liver enzymes were normal. Bone marrow, skeleton scintigraphy and MR scan were normal. An ultrasound showed normal-sized kidneys. Kidney biopsy presented focal interstitial nephritis, light grade

Influence of glucocorticoid on the metabolism of aldosterone in the isolated perfused rat liver and kidney

The effect of glucocorticoid deficiency and excess on the extraadrenal metabolism of D-[4-14C]aldosterone (at 4 nM) was studied by radioimmunoassay and by high-performance liquid chromatography in the isolated perfused liver and kidney of adult Wistar rats. Bilateral adrenalectomy was performed 3 weeks before experiments. In nonadrenalectomized rats, 0.3 mg/kg/day dexamethasone was continuously infused subcutaneously for 1 week before experiments. Adrenalectomy did not affect hepatic or renal metabolism of aldosterone. Dexamethasone treatment did not change the renal handling of aldosterone. However, the hepatic clearance of aldosterone was 19% lower (P less than 0.05) in livers of dexamethasone treated rats than in livers of normal rats. After 5 minutes, perfusate [4-14C]aldosterone metabolites were lower in livers of dexamethasone-treated than in livers of normal rats (P less than 0.05). Similar perfusate levels were then obtained. Radiometabolite peaks with similar relative retention times were found in the hepatic perfusate of all groups. However, the ratio between circulating polar metabolites of aldosterone and the metabolites less polar than tetrahydroaldosterone, after 5 and 15 minutes, was highest in livers of dexamethasone-treated rats. Biliary elimination of 14C was similar in all groups. Significant amounts of conjugated tetrahydroaldosterone were only excreted in the bile of dexamethasone-treated rats. In conclusion, glucocorticoid excess reduced the hepatic clearance of aldosterone and changed the pattern of the hepatic metabolites of aldosterone both in circulation and in bile.

Enhancement of the Stimulatory Effect of Ca++ on Aldosterone Secretion by PTH

In previous clinical investigations we found a stimulatory effect of the presence of hyperparathyroidism on the Ca++ induced secretion of aldosterone and vasopressin. The present investigation, therefore, examined the possible effect of PTH on the Ca++ mediated aldosterone secretion from purified rat zona glomerulosa cells.