Palle K. Nielsen

Cloning and Characterization of a Calcium-Sensing Receptor from the Hypercalcemic New Zealand White Rabbit Reveals Unaltered Responsiveness to Extracellular Calcium

The extracellular Ca2+ (Ca  02+)‐sensing receptor (CaR) recently cloned from mammalian parathyroid, kidney, brain, and thyroid plays a central role in maintaining near constancy of Ca  02+. We previously showed that the hypercalcemia normally present in New Zealand white rabbits is associated with an elevated set point for Ca  02+‐regulated PTH release (the level of Ca  02+ half‐maximally inhibiting hormonal secretion). This observation suggested an alteration in the Ca  02+‐sensing mechanism in the rabbit parathyroid, a possibility we have now pursued by isolating and characterizing the rabbit homolog of the CaR. The cloned rabbit kidney CaR (RabCaR) shares a high degree of overall homology (>90% amino acid identity) with the bovine, human, and rat CaRs, although it differs slightly in several regions of the extracellular domain potentially involved in binding ligands. By Northern analysis and/or immunohistochemistry, a similar or identical receptor is also expressed in parathyroid, thyroid C cells, small and large intestine, and in the thick ascending limb and collecting ducts of the kidney. When expressed transiently in HEK293 cells and assayed functionally through CaR agonist‐evoked increases in Ca  i2+, the rabbit CaR shows apparent affinities for Ca  02+, Mg  02+, and Gd  03+ that are indistinguishable from those observed in studies carried out concomitantly using the human CaR. Therefore, at least as assessed by its ability to increase Ca  i2+ when expressed in HEK293 cells, the intrinsic functional properties of the rabbit CaR cannot explain the hypercalcemia observed in vivo in the New Zealand white rabbit.

Microalbuminuria as an early indicator of osteopenia in male insulin-dependent diabetic patients.

Reduced bone mineral density (BMD), termed diabetic osteopenia, has been reported in patients with insulin‐dependent (Type 1) diabetes mellitus (IDDM). To examine BMD in long‐term IDDM patients with normal kidney function, but with different degrees of urinary albumin excretion rate (UAER), compared to that of patients with elevated plasma creatinine, 36 IDDM male patients (mean duration 27 years) were subdivided according to UAER (<30, 30–300, >300, >300 mg 24 h−1 and plasma creatinine 0.120–0.350 mmol l−1) and 15 controls were recruited. BMD was measured by dual energy X‐ray absorptiometry and UAER by enzyme linked immunosorbent assay. BMD was normal in IDDM patients with normal UAER and reduced in the femoral neck, the trochanter major, and the Wards triangle in patients with increased UAER (p < 0.01, p < 0.05, p < 0.02). BMD correlated to creatinine clearance in both cortical and cancellous bone sites (p < 0.001, p < 0.0001), and inversely to the levels of plasma PTH (p < 0.0005). We conclude that BMD is normal in long‐term IDDM male patients with normal kidney function and normal UAER and reduced in patients with increased UAER. Diabetic osteopenia seems to be a progressive phenomenon related to diabetic nephropathy and associated with the decrease in creatinine clearance and with the resulting rise in plasma PTH.

Ketamine in the treatment of bronchospasm during mechanical ventilation

The effect of ketamine on bronchospasm during mechanical ventilation was evaluated in a prospective, placebo-controlled, double-blind trial. Fourteen mechanically ventilated patients with bronchospasm were randomly allocated to either ketamine 1 mg/kg or saline placebo. In the ketamine-treated patients, PO2 increased from 10.5 (+/- 0.5) kPa to 16.4 (+/- 2.7) kPa (P < .05), whereas PO2 in the placebo-treated patients remained unchanged. The PCO2 was constant in the ketamine group, although it increased from 5.6 (+/- 0.9) kPa to 6.1 (+/- 0.9) kPa in the placebo group (P < .05). The pulmonary stethoscopic bronchospasm improved immediately after the administration of ketamine, whereas the thoracic compliance remained unchanged. In conclusion, the ketamine-treated patients showed an improvement by stethoscopic examination, in PO2 and in PCO2, suggesting that ketamine might be useful in the treatment of bronchospasm during mechanical ventilation. However, further studies are required to decide whether ketamine should be considered the drug of choice in patients with severe bronchospasm during ventilator treatment.

The calcium/parathyroid hormone concept of the parathyroid glands.

Plasma ionized calcium is the major determinant of parathyroid hormone (PTH) secretion. The minute-to-minute secretory response of the parathyroids to changes in plasma ionized calcium is described by the calcium/PTH concept, but the detailed mechanism is not yet well understood. The recent cloning of a calcium-sensing receptor in the plasma membrane of the parathyroid cells will probably yield important information concerning the mechanisms by which calcium and other ions control the parathyroid function. It is likely that autocrine and paracrine factors also participate in the regulation of PTH secretion. PTH, chromogranin A, chromogranin A-related peptides and endothelin-1 have been suggested as autocrine factors. More documentation is needed on the impact of these factors in the physiology of the parathyroid gland. In-vivo investigations of the parathyroid function are difficult to interpret because of the complexity of the PTH secretory response to hypo- and hypercalcaemia. Rate dependency and the ability of the parathyroids to sense the direction of changes in calcium make the existing models for investigating the calcium/PTH relationship inappropriate. In vitro, the models are compromised by a rapid drop in the expression of the calcium-sensing receptor of the cultured parathyroid cells. We, therefore, recommend caution when using the calcium/PTH concept in clinical or experimental investigations.

Long-Term Effects of Intravenous 1α(OH)D3 Combined with CaCO3 and Low-Calcium Dialysis on Secondary Hyperparathyroidism and Biochemical Bone Markers in Patients on Chronic Hemodialysis

The effects of intravenous administration of 1 α-hydroxycholecalciferol [lα(OH)D3] in combination with CaCO3 and ‘low-calcium dialysis’ (1.25 mmol/l) on plasma (p) parathyroid ho

Long-Term Suppression of Secondary Hyperparathyroidism by Intravenous 1α-Hydroxyvitamin D3 in Patients on Chronic Hemodialysis

The effect of intravenous 1 alpha-hydroxyvitamin D3 [1 alpha(OH)D3] on circulating levels of intact parathyroid hormone (PTH 1-84) and COOH-terminal immunoreactive PTH(PTH 53-84) was examined in 13 patients on chronic hemodialysis. Thirteen patients were treated for 300 days (10 months), 9 patients for 520 days (14 months) and 6 patients for 720 days (2 years) with increasing doses of 1 alpha(OH)D3 intravenously under careful control of plasma Ca2+. Blood samples were obtained 1 week before start of treatment and then at every 2nd week. None of the patients had previously been treated with oral vitamin D metabolites. Intact PTH levels were maximally suppressed after 27-33 weeks of treatment by approximately 73%. At the end of the study periods, PTH 1-84 was still suppressed by 78 +/- 4.3% after 300 days, 78 +/- 8.8% after 520 days and 85 +/- 6.5% after 720 days. Plasma Ca2+ was kept within normal levels, but showed an initial increase from 1.14 +/- 0.03 to 1.27 +/- 0.15 mmol/l, and an adjustment of the doses of 1 alpha(OH)D3 was necessary. The present investigation demonstrated (1) that intravenous administration of the 1-hydroxylated vitamin D metabolite 1 alpha(OH)D3 induced a significant decrease in circulating levels of biologically active intact PTH, and (2) that it was possible to maintain the marked suppression of PTH secretion by intravenous treatment of 1 alpha (OH)D3 for up to 2 years. Hypercalcemia could be avoided by careful monitoring of plasma Ca2+ and adjustment of the doses of 1 alpha(OH)D3.

Effect of Vitamin D Metabolites and Analogs on Renal and Intestinal Calbindin-D in the Rat

Abstract. The effects on renal and intestinal calbindin-D of vitamin D3 metabolites and synthetic 20-epi-vitamin D3 analogs with different calcemic actions were examined in Wistar rats. The compounds were administered intraperitoneally once daily for 5 days. The dosages of the metabolites were 1,25-(OH)2D3 0.01, 0.05, 0.1, and 0.4 μg/kg × d, 24,25-(OH)2D3 0.1, 1 and 10 μg/kg × d, and 25-(OH)D3 10 and 400 μg/kg × d. The dosage of the synthetic analogs were MC903 0.1, 10, and 100 μg/kg × d, EB1213 0.1 and 10 μg/kg × d, KH1060 0.1 and 0.4 μg/kg × d, and GS1725 0.01 and 0.1 μg/kg × d. Two control groups had either vehicle alone or no treatment. N= 8 in each group. 1,25-(OH)2D3 increased renal and intestinal calbindin-D levels, induced hypercalcemia, and suppressed plasma PTH and magnesium concentrations. 24,25-(OH)2D3 increased intestinal calbindin-D9k and plasma calcium, but had no effect on renal calbindin-D28k, plasma PTH, and magnesium. The dosage of 24,25-(OH)2D3 that was required to increase plasma calcium was larger than the dosage required to increase intestinal calbindin-D9k. 25-(OH)D3 did not change the calcium metabolic parameters. MC903, a low calcemic analog with a relative high affinity for the vitamin D receptor and a short half-life, increased renal calbindin-D28k without increasing ionized calcium or intestinal calbindin-D9k. EB1213, an analog with a reduced calcemic action and short half-life, increased renal calbindin-D28k and ionized calcium without increasing intestinal calbindin-D9k. The effect of the high calcemic vitamin D analogs KH1060 and GS1725 on calbindin-D was directly related to their calcemic activity. In conclusion, these results demonstrate that 24,25-(OH)2D3 increases intestinal calbindin-D9k, but has no effect on renal calbindin-D28k, that low calcemic analogs may increase renal calbindin-D28k without increasing intestinal calbindin-D9k, and that the effect of high calcemic analogs on calbindin-D is directly related to their calcemic activity.

Dual energy X-ray absorptiometry in small rats with low bone mineral density.

Abstract. The feasibility of dual energy X-ray absorptiometry (DXA) using the Norland XR-26 Mark II bone densitometer for measurements of bone mineral content (BMC) and bone mineral density (BMD) in small rats was evaluated. Thirty-two young, isogenic, Lewis rats (weights from 119 g to 227 g) were used; normal rats (n = 7) and rats with low BMD obtained from three different vitamin D-depleted models (n = 25). DXA measurements were performed using the special software for small animals. Duplicate scans of excised femurs performed at 2 mm/second (pixel size of 0.5 mm × 0.5 mm) were very precise measurements with a coefficient of variation (CV) below 1.6% in animals with normal BMD; in rats with low BMD, the CV was significantly higher (P= 0.02–0.04), 7.8% and 4.4% for BMC and BMD, respectively. Regression analysis demonstrated that these measurements were related to the ash weight (R2 > 98.6%). The CV for measurements of the lumbar spine at 10 mm/second (pixel size 0.5 mm × 0.5 mm) was 2.6% and 2.2% for BMC and BMD, respectively in rats with normal BMD, and again higher (P= 0.03–0.14) in rats with low BMD, 7.3% and 4.7%, respectively, for BMC and BMD. Even though low CVs were obtained for total body duplicate scans (scan speed of 20 mm/second and a pixel size of 1.5 mm × 1.5 mm), the measurements were problematic for accuracy because of an overestimation of both BMC and the area of bone. Using these scan parameters the measurements of total body bone mineral could not be recommended in small rats with low BMD.

The Plasma Leptin Concentration Is Closely Associated with the Body Fat Mass in Nondiabetic Uremic Patients

Plasma leptin is associated with the body mass index and, more precisely, with the body fat mass. Plasma leptin has been found to be elevated in uremic patients. This study aimed at investigating the plasma leptin concentration and associations between plasma leptin, body fat mass, and glomerular filtration rate in nondiabetic predialysis uremic patients and in nondiabetic patients on chronic hemodialysis. Plasma leptin, body fat mass, and creatinine clearance were measured in 22 predialysis uremic patients, 18 hemodialysis patients, and 24 healthy control subjects. The logarithmically transformed plasma leptin concentration was closely associated with the body fat mass in all groups (r = 0.93, r = 0.83, and r = 0.72, respectively; p < 0.000001, < 0.000002 and p < 0.001, respectively). In predialysis uremic patients the plasma leptin concentration was slightly elevated as compared with controls 10.4 (3.1–59.5) ng/ml versus 5.4 (1.6–47.5) ng/ml (median and range in parentheses; p < 0.05), whereas the plasma leptin concentration was normal in hemodialysis patients. Plasma leptin was not significantly associated with the creatinine clearance in predialysis patients. In conclusion; the glomerular filtration rate seemed to have a limited influence on the plasma leptin concentration in nondiabetic uremic subjects matched by body fat mass to controls. The plasma leptin concentration was closely associated with the body fat mass, and the leptin level might, therefore, be useful as an indicator of the fat mass in nondiabetic uremic patients.