Experssion of chronic thyroparathyroidectomy on phosphate transport in whole kidney and proximal luminal membranes during phosphate deprivation

Both parathyroid hormone (PTH) and the dietary supply of inorganic phosphate (Pi) influence the tubular Pi reabsorptive capacity. In this study we have assessed how removal of parathyroid gland affects the response of Pi transport to Pi restriction in the whole kidney and in brush border membrane vesicles (BBMV) isolated from the renal cortex. Intact and thyroparathyroidectomized (TPTX) rats were switched from normal to low Pi diet. The maximal tubular Pi reabsorption per volume of glomerular filtrate (max. TRPi/ml GF) and the initial (3, 6 and 9 s) Na⁺-dependent Pi uptake by BBMV were determined before and after 1, 2, 3 and 8 days of Pi restriction. The results indicate that before Pi restriction max. TRPi/ml GF was 1.94±0.04 and 4.03±0.14 mol/ml GF in intact and TPTX rats respectively. The corresponding Na⁺-dependent Pi uptake (3 s value) was 137±23 in sham and 272±41 pmol/mg protein in TPTX rats. During Pi restriction the initial difference in max. TRPi/ml GF remained constant throughout the time course study. At 8 days max. TRPi/ml GF was in intact 3.08±0.09 and in TPTX 5.04±0.18 mol/ml GF. In sharp contrast, in BBMV the initial difference between intact and TPTX became completely abolished 8 days after starting the Pi restriction (SHAM=425±69; TPTX=434±pmol/mg protein). In conclusion, the overall tubular Pi transport capacity and the Na⁺-dependent Pi transport system located in the luminal membrane of the proximal tubule appear to be quite differently affected by changes in the PTH status during Pi restriction.     

Phosphate transport in brush border membrane vesicles isolated from renal cortex of young growing and adult rats

Recent clearance studies have demonstrated that the maximal tubular reabsorption of inorganic phosphate (Pi) per ml of glomerular filtrate (max. TRPi/ml GF) of the whole kidney is markedly lower in adult than in young growing rats fed either normal (0.8 g%) or low (0.2 g%) phosphorus diet. In addition, in adult rats clearance studies indicate that enhancement of max. TRPi/ml GF is observed 21 days but not 8 days after starting the low (0.2%) phosphorus diet. In the present work we have studied in the same experimental condition the Na⁺-dependent Pi uptake in brush border membrane vesicles (BBMV) isolated from renal cortex of either young growing or adult rats. The results of this study indicate that under the low (0.2%) but not under the normal (0.8%) phosphorus diet the Na⁺-dependent Pi uptake by BBMV was significantly depressed in adult as compared to young growing rats. In adult rats the Pi transport response to Pi restriction monitored at the brush border membrane level was different from that observed by clearance studies in the whole kidney. Indeed, the Pi uptake by BBMV was already enhanced after 8 days of Pi restriction and it did not increase further when studied 21 days after starting the low (0.2%) phosphorus diet. These results suggest that the regulation of the overall transfer of Pi across the renal epithelium may involve other additional modulating factors than the Na⁺-dependent Pi transport system present in the luminal membrane of the proximal tubule.     

Mechanism of rapid phosphate (Pi) transport adaptation to a single low Pi meal in rat renal brush border membrane

Previous studies have shown that the adaptive response of tubular inorganic phosphate (Pi) transport to Pi deprivation is detectable in the whole kidney 24 h after switching rats from a high (HPD) to a low (LPD) Pi diet. In the present work we report on a more rapid adaptive response of the sodium (Na)-dependent Pi transport system located in the luminal membrane of the proximal tubule and its relation with changes in phosphatemia an parathyroid hormone status. Rats were fed HPD and trained to eat their daily ration within 1 h. After two weeks of equilibration half of the animals received a single LPD ration. 1, 2 and 4 h after the end of food consumption the animals were either sacrificed for renal cortical brush border membrane vesicle (BBMV) isolation or used for determining plasma Pi concentration, urinary excretion of Pi and cAMP. The results indicate that 2 and 4 h after the end of feeding, the Na-dependent Pi transport in BBMV was stimulated by 70 and 140% respectively in intact rats exposed for the first time to LPD. This response was preceded by a significant fall in plasma Pi concentration (HPD: 2.46±0.03, LPD: 2.04±0.05 mM), in the urinary excretion of Pi (HPD: 899.0±68.1; LPD: 6.5±3.3 mol/ml GFR) and cAMP (HPD: 76.9±7.4, LPD: 48.2±1.4 pmol/ml GF). This last result suggested a rapid inhibition of PTH after one single LPD feeding. In thyroparathyroidectomized (TPTX) rats the Na-dependent Pi transport system was also stimulated 4 h after LPD, but to a slightly less extent than in intact rats.In conclusion, the Na-dependent Pi transport located in the luminal membrane of the proximal tubule reacts within hours to dietary Pi restriction. This specific tubular response coupled with the hypophosphatemia should account for the rapid decrease in urinary Pi excretion. Although the adaptive response at the BBMV level is also expressed in TPTX rats, inhibition of the PTH-cAMP system could contribute to the rapid adaptation observed in intact animals.     

Regulation of Na-dependent phosphate influx across the mucosal border of duodenum by 1,25-dihydroxycholecalciferol

Animals treated with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), at doses which decrease the renal production and/or the plasma levels of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃], display a reduced net absorption of phosphate. In this study we investigated whether EHDP-treatment and administration of 1,25(OH)₂D₃ to EHDP-treated animals affected the phosphate influx across the mucosal border of rabbit duodenum. The initial rate of phosphate influx into mucosal cells was measured in isolated intestine. In control, untreated rabbits, the phosphate influx shows a saturable, Na-dependent component and a diffusional, Na-independent uptake. In tissue from rabbits treated for 3 days with EHDP, the phosphate influx was found to be strongly reduced. EHDP-treatment decreased the Na-dependent, carrier mediated phosphate influx in duodenum. Administration of 1,25(OH)₂D₃ to EHDP-treated animals reversed the reduced phosphate influx. These effects were mainly apparent through changes in theJ _mc ^max of the phosphate influx, which was decreased from 211±38.7 nmole/cm² h in controls to 42.1±18.1 nmole/cm² h in the EHDP-treated group and increased to 413±43.6 nmole/cm² h by 1,25(OH)₂D₃. The treatment did not appear to affect the diffusional, Na-independent phosphate influx. EHDP-treatment did not affect the influx of alanine in this segment suggesting that EHDP-treatment affects only 1,25(OH)₂D₃-dependent transport mechanisms. The results suggest that 1,25(OH)₂D₃ modulates the number of carrier sites available at the mucosal membrane for Na-dependent phosphate entry.     

Phosphatemic action of 1,25-dihydroxyvitamin D3.

The action of a single intraperitoneal injection of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was investigated in thyroparathyroidectomized (TPTX) vitamin D-deficient phosphate-depleted rats. After 14 h, plasma inorganic phosphorus (Pi) was significantly greater in animals receiving 1,25(OH)2D3 than in D-deficient controls, but urinary Pi excretion was very low in both groups and not significantly different in the rats given 1,25(OH)2D3. Clearance studies indicated that the D-deficient controls reabsorbed more than 99% of their filtered Pi. Avid Pi reabsorption continued even after the infusion of sufficient phosphate to raise the plasma and filtered Pi to approximately 3 times normal. Fractional calcium excretion (FECa) exceeded fractional sodium excretion (FENa) by severalfold, but FECa decreased strikingly during phosphate infusion. In animals that manifested a substantial elevation of plasma Pi after 1,25(OH)2D3, FECa was significantly less than in D-deficient controls. Therefore, the increase in plasma Pi following 1,25(OH)2D3 administration occurs independently of any effect on renal Pi reabsorption and may be responsible, at least in part, for the amelioration of hypercalciuria after 1,25(OH)2D3 treatment.     

Effect of diphosphonate treatment on phosphate transport by renal brush border vesicles

Treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) given in doses of 10 mg P/kg s.c. for 7 days inhibits bone mineral retention and decreases the tubular capacity to reabsorb inorganic phosphate (Pi) in thyroparathyroidectomized (TPTX) rats. In the present work we show that pretreatment with EHDP depressed Na+-dependent Pi uptake by brush border membrane vesicles (BBMV) isolated from renal cortex of TPTX rats. The effect of EHDP was observed after feeding both high (1.2%) and low (0.2%) phosphorus diets. The EHDP-induced changes observed in vitro at the brush border level parallel the variations in the overall tubular Pi transport capacity as assessed by clearance techniques in conscious rats. Na+-dependent glucose uptake by BBMV, as well as alkaline phosphatase activity in cortical homogenates and in the BBMV were not affected by EHDP treatment. Accordingly, EHDP, given in doses that block bone mineral retention, appears to specifically affect the Na+-dependent transport of Pi across the luminal membrane of proximal tubules of renal cortex.     

Regulation of mineral metabolism by lithium

Lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), is widely used for the treatment of mood disorders. Side effects of lithium include nephrogenic diabetes insipidus, leading to renal water loss. Dehydration has in turn been shown to downregulate Klotho, which is required as co-receptor for the downregulation of 1,25(OH)₂D₃ formation by fibroblast growth factor 23 (FGF23). FGF23 decreases and 1,25(OH)₂D₃ stimulates renal tubular phosphate reabsorption. The present study explored whether lithium influences renal Klotho expression, FGF23 serum levels, 1,25(OH)₂D₃ formation, and renal phosphate excretion. To this end, mice were analyzed after a 14-day period of sham treatment or of treatment with lithium (200 mg/kg/day subcutaneously). Serum antidiuretic hormone (ADH), FGF23, and 1,25(OH)₂D₃ concentrations were determined by ELISA or EIA, renal Klotho protein abundance and GSK3 phosphorylation were analyzed by Western blotting, and serum phosphate and calcium concentration by photometry. Lithium treatment significantly increased renal GSK3 phosphorylation, enhanced serum ADH and FGF23 concentrations, downregulated renal Klotho expression, stimulated renal calcium and phosphate excretion, and decreased serum 1,25(OH)₂D₃ and phosphate concentrations. In conclusion, lithium treatment upregulates FGF23 formation, an effect paralleled by substantial decrease of serum 1,25(OH)₂D₃, and phosphate concentrations and thus possibly affecting tissue calcification.     

Glutathione-dependent inactivation of sodium-dependent phosphate transport across rat renal brush-border membrane

Thiol/disulfide is fundamental in protein function; we previously observed an inhibitory effect of thiol oxidants on the Na-dependent phosphate (Pi) uptake into renal brush border membrane vesicles (BBMV). We examined whether oxidation of glutathione (GSH) is involved in the mechanism. Vesicular thiols were measured by liquid chromatography. BBMV were incubated with reagents before an influx of Pi. Diamide (5 mM) reduced the capacity of the Pi uptake. Subsequent treatment with dithiothreitol (5 mM) blocked the inhibitory effect of diamide. Vesicular GSH was not modified only by the incubation, whereas it was oxidized by the treatment with diamide, and reduced by dithiothreitol. Furthermore, in vivo treatment with cAMP provided GSH-depleted BBMV without any influence on Pi uptake. Diamide did not inhibit the transport of Pi into GSH-depleted vesicles, but it did inhibit the uptake when GSH was introduced into the vesicles. In conclusion, a GSH-dependent mechanism is involved in the inhibitory effect of diamide on sodium-dependent Pi transport across the renal brush-border membrane.     

Short-Term Effects of Vitamin D3 and 1,25-Dihydroxyvitamin D3 on Osteomalacia in Uremic Rats Fed a Low-Calcium-Low-Phosphorus Diet

The objectives of this study were to evaluate the effects of vitamin D₃ (D₃) and 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on uremic bone disease independent of their action on the intestine. The histomorphology of tibial metaphyses in uremic (5/6 nephrectomized [5/6 Nx]) rats fed a low-calcium-low-phosphorus (LCLP) diet was compared with sham-operated (SO) rats fed an LCLP diet and 5/6 Nx rats fed an LCLP diet and given 15,000 IU D₃ or 5 units (135 ng) 1,25-(OH)₂D₃ daily for 7 days. A marked osteomalacia characterized by an increased percentage of active and inactive trabecular osteoid surface and thickened growth plates developed in proximal tibial metaphyses in 5/6 Nx rats given the placebo, compared with SO rats. These bone changes were associated with relative hypophosphatemia, hypophosphaturia, and hypercalciuria in 5/6 Nx rats. In 5/6 Nx rats treated with D₃ or 1,25-(OH)₂D₃ the growth plates had undergone mineralization and vascular invasion and were markedly reduced in thickness. Other parameters of osteomalacia in trabecular bone were not different from 5/6 Nx rats given the placebo. There was a significant decrease in osteoclasts per millimeter of trabecular surface perimeter in D₃- and 1,25-(OH)₂D₃-treated rats. These bone changes were associated with hypercalcemia, hyperphosphatemia, and hyperphosphaturia, compared with 5/6 Nx rats given the placebo. It was concluded that in uremic rats fed the LCLP diet, shortterm treatment with either pharmacologic levels of D₃ or 1,25-(OH)₂D₃ corrected only lesions in the growth plate. Osteoid seams were not reduced in treated rats, although the serum calcium-phosphorus product was elevated. The 5/6 Nx rat fed the LCLP diet appears to be a useful model for the rapid induction of uremic osteomalacia in adult animals.     

1,25(OH)2 vitamin D3 sites of action in spinal cord and sensory ganglion

Summary Autoradiographic studies revealed concentration of ³H 1,25(OH)₂ vitamin D₃ in nuclei of certain neurons in the spinal cord of adult and neonatal mice, fed a normal or a vitamin D deficient diet. Nuclear uptake and retention was strongest in motor neurons in lamina IX. Nuclear concentration also existed in neurons of lamina II, lamina VIII, lamina X and intermediate nucleus of the lateral column. The results indicate that these neurons are target neurons which contain nuclear receptors for 1,25(OH)₂ vitamin D₃. this suggests that 1,25(OH)₂ vitamin D₃ has direct genomic actions on the innervation of skeletal muscle by exerting related trophic, secretory, and electrophysiological effects. In addition, these data point to direct genomic actions of 1,25(OH)₂ vitamin D₃ on spinal sensory perception, and on certain autonomic functions. Nuclear binding in certain neurons in the peripheral ganglion of the trigeminal nerve further suggests that sensory perception is influenced by 1,25(OH)₂ vitamin D₃ not only at the level of the substantia gelatinosa, but also at the level of spinal ganglia.Supported in part by USPHS grants NS 09914 and HD 03110     

Chronic thyroparathyroidectomy and tubular handling of phosphate

The fractional reabsorption (FR) of inorganic phosphate (Pi) along the proximal tubule depends upon both the filtered load of Pi (FLPi) and the tubular reabsorptive capacity of the Pi transporting system. To assess the actual effect of parathyroid hormone on the reabsorptive capacity only, the influence of Pi load has to be eliminated. In this study FRPi was determined by free-flow micropuncture along superficial nephrons of chronically (48 h) thyroparathyroidectomized (TPTX) and pair-fed sham-operated (SHAM) rats at identical FLPi [TPTX 3.07±0.14 (n=26) and SHAM 3.07±0.11 (n=26) mol/min±SEM]. The micropuncture results indicate that in the ranges of tubular fluid over plasma inulin concentration [TF/P)_In] 1.00–1.49 and 1.50–1.99, no difference in FRPi between TPTX and SHAM could be detected. It is only between a TF/P_In of 2.0 and 2.49 that chronic TPTX resulted in a significant increase in FRPi. Accordingly the present study indicates that chronic TPTX increases FRPi in late but not in early portions of proximal tubule. Thus in the early proximal tubule the tubular reabsorptive capacity of the Pi transporting system appears to be unaffected by chronic removal of the parathyroid glands. From this result it can be inferred that the increased plasma concentration of Pi which follows the removal of the parathyroid glands, particularly in the chronic stage, will lead to an apparently paradoxical decrease in FRPi in early proximal tubule as a mere consequence of the increased filtered load of Pi.     

Renal handling of calcium and phosphate

Summary In glomerular filtrate, calcium concentration (UF) is some 50–70% of total plasma calcium concentration. About 60% of filtered calcium is reabsorbed in the proximal convoluted tubule, some 15% in pars recta, 15% in the thick ascending limb, and 9% in the distal convoluted tubule including the granular portion of the cortical collecting duct. About 1% is excreted. Approximately 2/3 of proximal tubular calcium transport is passive, driven by a chemical (TF/UF=1.1) and electrical (2 mV, lumen positive) gradient; 1/3 is active, whereby calcium enters the cell at the brush border membrane by passive diffusion and is pumped out at the basolateral membrane in exchange for sodium. Transport in distal convoluted tubules is entirely active and operates both against a chemical (TF/UF 0.3–0.7) and electrical (10–70 mV, lumen negative) gradient. Although saturability of transport is not detectable in microperfusion studies, enhancement of plasma calcium leads to calciuria. Factors stimulating calcium reabsorption are parathyrin, 1.25(OH)₂D₃, thiazides, and alkalosis. Factors inhibiting calcium reabsorption are calcitonin, growth hormone, thyroid hormone, chronic application of mineralo- and glucocorticoids, insulin, glucose, acidosis, sodium infusion, acetazolamide, furosemide, and mannitol.Phosphate concentration in ultrafiltrate is some 90% of total plasma concentration. In intact animals about 2/3 of filtered phosphate is reabsorbed in proximal convoluted tubules, and some 20% is excreted in urine. Shortly after thyroparathreoidectomy (TPTX), 80% of filtered phosphate is reabsorbed in the proximal convoluted tubule, 15% in the pars recta, and 2% is excreted in the urine. Thus, less phosphate is excreted in urine (20% in intact, 2% in TPTX animals) as is recovered in late distal convoluted tubules of superficial nephrons (35% and 5%, respectively). The discrepancy is at least partially due to more avid reabsorption in deep nephrons. Furthermore, evidence exists in favor of phosphate reabsorption in the arcades or collecting duct. Phosphate reabsorption in proximal convoluted tubule and pars recta is active. Uptake of phosphate at the brush border membrane is uphill and is driven by coupling with two sodium ions, whereas exit at the basolateral membrane may be entirely passive. At increasing plasma phosphate concentrations, reabsorption is saturated and excess filtered phosphate excreted. Factors stimulating phosphate transport are phosphate depletion, acute 1,25(OH)₂D₃, thyroxin, growth hormone, magnesium, lithium, and metabolic acidosis. Factors inhibiting phosphate transport are high phosphate or high calcium diets, parathyrin, calcitonin, chronic 1,25(OH)₂D₃, hypocalcemia, magnesium deficiency, respiratory acidosis, metabolic alkalosis, glucose, colchicine, NaCl-infusions, thiazides, furosemide, ethacrynic acid, mannitol, and acetazolamide.     

Relation between intestinal alkaline phosphatase activity and brush border membrane transport of inorganic phosphate,D-glucose,andD-glucose-6-phosphate

In order to evaluate the role of the alkaline phosphatase in intestinal transport processes, we studied the influence of known modulators of the alkaline phosphatase (polyclonal anti-calf AP antibodies, theophylline and zinc ions) on the absorption rate of glucose, inorganic phosphate and glucose liberated from glucose-6-phosphate into calf duodenal brush border membrane vesicles. Our results allow the following conclusions: First a direct involvement of the AP in the Na⁺-dependent glucose absorption is unlikely. Indeed, theophylline inhibits strongly the AP activity but rather stimulates the glucose uptake; second the AP is indirectly involved in glucose absorption from glucose-6-phosphate, if its enzymatic hydrolysis is the only source of glucose. In that case the Na⁺-dependent uptake of glucose was completely suppressed either by phosphatase specific antibodies or by theophylline; third the positive correlation found with calf intestinal BBMV between the inhibition of AP by AP antibodies or by theophylline and the decrease of rate of Na⁺-dependent P_i uptake rate suggests that the enzyme plays some role in the P_i absorption.It appears from the present study that the AP is probably not a carrier protein itself, but its hydrolytic activity might nevertheless be important for intestinal absorption. After hydrolysis of phosphoric esters the alcohol residues and P_i can be supplied to their specific carriers. Furthermore, the high P_i affinity of the enzyme at physiological pH values, could even favour a transient sequestration of phosphate, which then could be transferred to the P_i carrier.Abbreviations used AP alkaline phosphatase - pNPP paranitrophenylphosphate - Gle-6-ph glucose-6-phosphate - P phosphate ion - IgG protein A purified immunoglobulins G - TCA trichloracetic acid - DEA diethanolamine - MgAc₂ magnesium acetate - ZnAc₂ zinc acetate - BBMV brush border membrane vesicles - 1,25(OH)₂D₃ 1,25-dihydroxycholecalciferol - Mannitol-buffer buffer-containing per liter 300 mmol mannitol and 10 mmol HEPES/Tris, pH 7.4     

Compensatory regulation of the sodium/phosphate cotransporters NaPi-IIc (SCL34A3) and Pit-2 (SLC20A2) during Pi deprivation and acidosis

The role of four Pi transporters in the renal handling of Pi was analyzed using functional and molecular methods. The abundance of NaPi-IIa, NaPi-IIc, and Pit-2 was increased by 100% in kidney from rats on a 0.1% Pi diet, compared to a 0.6% Pi diet. Pit-1 was not modified. Type II-mediated Pi uptake in Xenopus oocytes increased as the pH of the uptake medium increased, and the opposite occurred with Pit-1 and Pit-2. At pH 6.0, Pi uptake mediated through type II was ≈10% of the uptake at pH 7.5, but the uptake through Pit-2 was 250% of the activity at pH 7.5. Real brush-border membrane vesicles (BBMV) responded to pH changes following the same pattern as type II transporters. Adaptation to a 0.1% Pi diet was accompanied by a 65% increase in the V _max of BBMV Pi transport at pH 7.5, compared to a 0.6% Pi diet. The increase was only 11% at pH 6.0. Metabolic acidosis increased the expression of NaPi-IIc and Pit-2 in animals adapted to a low Pi diet, and phosphaturia was only observed in control diet animals. The combination of the pH effect, Pi adaptation, and metabolic acidosis suggests very modest involvement of Pit-2 in renal Pi handling. Real-time PCR and mathematical analyses of transport findings suggest that NaPi-IIa RNA accounts for 95% of all Pi transporters and that type II handles 97% of Pi transport at pH 7.5 and 60% of Pi transport at pH 6.0, depending on the pH and the physiological conditions.     

Decreased immunosuppressive actions of 1α, 25-dihydroxyvitamin D3 in patients with immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease. 1α, 25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and vitamin D receptor (VDR) play important immune-suppressive roles in immune system. It has been reported that serum 1,25(OH)₂D₃ were lower in ITP patients. In this study, we evaluated local 1,25(OH)₂D₃ level and VDR mRNA expression further, and determined whether 1,25(OH)₂D₃/VDR were correlated with T cell dysfunction in ITP patients. We found that 1,25(OH)₂D₃/VDR levels were decreased in active ITP patients, and 1,25(OH)₂D₃ had significant anti-inflammatory effects on ITP patients, including both anti-proliferation of peripheral blood mononuclear cells (PBMCs) and reversing the abnormal T cells polarization. 1,25(OH)₂D₃ inhibited the differentiation of T helper (Th)1 and Tc1 cells but induced the differentiation of Th2, Tc2 and T regulatory (Treg) cells in ITP patients. However, the percentage of Th17 cells were not affected obviously with 1,25(OH)₂D₃. In addition, 1,25(OH)₂D₃ also suppressed pro-inflammatory cytokines (INF-γ and IL-17A) but promoted anti-inflammatory cytokine (IL-10) secretion in ITP patients. In conclusion, decreased 1,25(OH)₂D₃/VDR might participate in the pathogenesis of ITP, and appropriate supplement of 1,25(OH)₂D₃ may be a promising treatment.     

Blockade of the renal tubular effects of vitamin D by cycloheximide in the rat

To further characterize the mechanisms by which 25(OH)vitamin D₃ (25(OH)D₃) and 1.25(OH)₂ vitamin D₃ (1,25(OH)₂D₃) suppress the phosphaturic action of parathyroid hormone (PTH) we have studied the effects of cycloheximide (cyclohex), a protein synthesis inhibitor, on the interaction between PTH and vitamin D metabolites in parathyroidectomized (PTX) rats, both in vivo and in vitro experiments. In clearance studies PTX PTH-infused rats were pretreated with cyclohex 2 h before the administration of vitamin D. In control, PTX PTH-infused rats not pretreated with cyclohex, the administration of 25(OH)D₃ and 1,25(OH)₂D₃ was associated with a fall in fractional excretion of phosphate (CP/CIN) from 0.30±0.05 to 0.16±0.02 and from 0.31±0.05 to 0.13±0.01 (P<0.005) respectively. Cyclohex-pretreated PTX PTH-infused rats failed to respond to both 25(OH)D₃ and 1,25(OH)₂D₃, and CP/CIN, which rose after PTH, remained 0.32±0.05 and 0.29±0.03 respectively. In vitro, both 25(OH)D₃ and 1,25(OH)₂D₃ inhibited the PTH-induced activation of adenylate cyclase in the renal isolated membrane fractions. Pretreatment with cyclohex abolished this effect of vitamin D metabolites. These results show that cyclohex blocks the antiphosphaturic effects of both 25(OH)D₃ and 1,25(OH)₂D₃ but does not alter the response to PTH. These findings are consistent with the possibility that the acute renal action of vitamin D depends on de novo synthesis of protein.An abstract of this work appeared in Clinical Research, 28 (2) A 387, 1980.     

Na,K-ATPase activity and 25(OH)vitamin D3 hydroxylation in rat proximal tubules

The proximal tubule is the target site for parathyroid hormone (PTH), and conversion of 25(OH)vitamin D₃ hormones which impinge on calcium (Ca) homeostatis, as well as a major site for sodium (Na) reabsorption. The effect of changes in PTH and vitamin D status on Na,K-ATPase activity, as a measure of Na transport, were studied in the proximal tubules of adult rat kidneys where Na and Ca reabsorption rates are in parallel. Na,K-ATPase activity and 25(OH)D₃ metabolism were determined in cortical and juxtamedullary proximal tubule segments from normal, parathyroidectomized (PTX), and vitamin D-deficient (-D) rats. Na,K-ATPase activity was highest in cortical segments. PTX led to a decrease in activity in convoluted segments but increased activity in straight segments. In-D rats, Na,K-ATPase activity decreased in cortical segments but increased in juxtamedullary segments. 25(OH)D₃ was metabolized more to 24,25(OH)₂D₃ than to 1,25(OH)₂D₃ in all normal segments. Juxtamedullary segments were more sensitive to PTX and-D conditions. These findings suggest that cortical and juxtamedullary nephrons are inherently different in basal Na,K-ATPase activity, in conversion of 25(OH)D₃ to active metabolites, and in response to altered PTH and vitamin D₃ status.     

Vitamin D-Metabolismus bei Niereninsuffizienz — Störung eines endokrinen Regelkreises

Zusammenfassung In den proximalen Tubulusepithelien der Niere wird der Vitamin D-Metabolit 25(OH)D in das aktive Secosterol 1,25(OH)₂D₃ umgewandelt. Diese Umwandlung ist bei niereninsuffizienten Patienten beeinträchtigt, jedoch sind möglicherweise nicht alle Vitamin D-abhängigen Störungen bei Niereninsuffizienz allein durch den Ausfall der Synthese von 1,25(OH)₂D₃ zu erklären.Bei initialer Niereninsuffizienz, bei der bereits Vitamin D-abhängige Funktionen (calzämische Wirkung von PTH, Calziumabsorption) gestört sind, liegen die 1,25(OH)₂D₃-Spiegel im Serum geringfügig oberhalb des Normalbereichs. Dieser Befund ist mit einer inadäquaten Antwort der 1-alpha-Hydroxylase auf aktivierende Stimuli (Hyperparathyreoidismus, Hypocalzämie, Hypophosphatämie) und/oder einer möglichen Endorganresistenz gegenüber 1,25(OH)₂D₃ vereinbar.Die Osteomalazie bei niereninsuffizienten Patienten ist nicht ausschließlich als Folge der Erniedrigung der Serum-Konzentration eines der bekannten Vitamin D-Metabolite [25(OH)D₃; 24,25(OH)₂D₃; 1,25(OH)₂D₃] zu erklären. Das schlechte Ansprechen der Osteomalazie urämischer Patienten auf 1,25(OH)₂D₃ legt die Frage nach der möglichen Wirkung zusätzlicher Vitamin D-Metabolite oder dem Vorhandensein nicht Vitamin D-abhängiger Zusatzfaktoren nahe. Bislang fehlen Informationen zum Verhalten der 1,25(OH)₂D₃ Rezeptoren und nachgeschalteter Ereignisse an Knochenzellen und Einzelheiten einer möglichen Wechselwirkung zwischen 1,25(OH)₂D₃ und PTH bleiben noch unklar.Obwohl ein spezifischer wachstumsfördernder Effekt von 1,25(OH)₂D₃ auf das Längenwachstum urämischer Kinder beschrieben wurde, zeigten mehrere klinische und experimentelle Untersuchungen keine Normalisierung durch 1,25(OH)₂D₃ resp. keinen Wirkunterschied zwischen Vitamin D und 1,25(OH)₂D₃.Gegenwärtig ist noch unklar, ob Vitamin D-Metabolite, und gegebenenfalls welcher Vitamin D-Metabolit, die PTH-Sekretion der Parathyreoidea hemmen. Die Klärung dieser Frage erscheint dringend für eine optimale medikamentöse Suppression der Parathyreoidea niereninsuffizienter Patienten.Auch außerhalb der Homöostase des Ca-Pi-Stoff-wechsels spielen Vitamin D-Metabolite eine wichtige Rolle in der Funktion einiger Organe, z.B. Muskel, Hoden, Pankreas etc. Der Ausfall dieser Funktionen ist möglicherweise bedeutsam zum Verständnis des urämischen Syndroms und seiner mangelnden Rückbildung unter Hämodialysebehandlung.

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Abkürzungen PTH Parathormon - iPTH immunreaktives Parathormon - cAMP zyklisches Adenosinmonophosphat - 1,25(OH)₂D₃ 1,25(OH)₂ Vitamin D₃ - Ca Calcium - Pi Phosphat     

Renal handling of calcium: influence of parathyroid hormone and 1,25-dihydroxyvitamin D3.

The influence of parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the renal Ca handling was studied in vitamin D-replete rats. The relation between plasma concentration ([Ca]P) and urinary Ca (UCaV/ml GF) was ascertained by clearance techniques over the [Ca]P range of 1.4-3.4 mM varied by infusion of Ca gluconate. Chronic thyroparathyroidectomy (TPTX) decreased the plasma Ca threshold from about 2.3 to 1.5 mM. Between [Ca]P 1.4 and 3.4 mM there was a linear increase in UCaV/ml GF corresponding to 35-50% of the increment in filtered load. In TPTX, PTH (2.5 IU/h i.v.) shifted the Ca threshold from 1.5 to 2.3 mM, without changing the slope of UCaV/ml GF on [Ca]P. The effect of TPTX on the renal Ca handling was not corrected by doses of 1,25(OH)2D3, which increased the intestinal Ca absorption of TPTX rats to normal level. In intact and TPTX rats disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) given at doses which inhibit the production of 1,25(OH)2D3 did not change the tubular Ca handling. Furthermore, 1,25(OH)2D3 had no effect in EHDP-treated TPTX rats. Therefore, tubular Ca handling does not appear to be altered in response to chronic endogenous variation or physiologic supplementation of 1,25(OH)2D3 in vitamin D-replete rats. This is in contrast to the marked alteration observed after TPTX or PTH administration.     

1, 25-dihydroxyvitamin D3 decreases adriamycin-induced podocyte apoptosis and loss

Background: Selective proteinuria is frequently observed in glomerular diseases characterized by podocyte injury. Although, 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] has potential therapeutic effects on chronic kidney diseases through decreasing podocyte loss, the mechanism underlying the beneficial effects of 1,25(OH)₂D₃ on podocytes remains still unknown. The present study tested the hypothesis that 1,25(OH)₂D₃ directly reduced podocyte apoptosis and loss.Methods: Sprague-Dawley (SD) rats were randomly assigned into three groups: Adriamycin (ADR) group (n=15), ADR+1,25-(OH)₂D₃ group (n=16), and control group (n=16). Rats in ADR+1,25-(OH)₂D₃ group were treated with 1,25(OH)₂D₃ for 8 weeks. The number of podocytes and foot process width (FPW) were detected by transmission electron microscopy. The number of apoptotic podocytes per glomerulus and that of apoptotic nuclei and caspase-3 activity in cultured podocytes were determined by TUNEL staining. The average number of podocytes per glomerulus was quantified by immunohistochemistry. Expressions of p-Smad2/3, p-Smad1/5/8, Fas, Fas-Associated protein with Death Domain (FADD), Bax, and Bcl-2 proteins were examined by Western blot assay.Results: Compared with control group, proteinuria, FPW, apoptotic podocytes, caspase-3 activity, the protein expressions of p-Smad2/3, Fas, FADD, and Bax were significantly increased, podocyte density, p-Smad1/5/8 and Bcl-2 expression were decreased in ADR group. 1,25(OH)₂D₃ significantly reduced proteinuria, FPW, caspase-3 activity, expressions of p-Smad2/3, Fas, FADD, and Bax and apoptosis of podocytes, but increased serum albumin, number of viable podocytes , p-Smad1/5/8 and Bcl-2 expression in ADR treated rats.Conclusion: ADR-induced podocyte apoptosis was associated with the imbalance of p-Smad2/3, p-Smad1/5/8 the activity of caspase-3 and aberrant expressions of, Fas, FADD, Bax and Bcl-2. The beneficial effects of 1,25(OH)₂D₃ on podocytes may be attributable to inhibit podocyte apoptosis and the amelioration of podocytopenia.