Regulation of renal parathyroid hormone receptor expression by 1, 25-dihydroxyvitamin D3 and retinoic acid.

The renal distal convoluted tubule (DCT) is the major site of parathyroid hormone (PTH) and 1alpha,25-dihydroxyvitamin D3 [1, 25(OH)2D3]-regulated calcium absorption. 1,25(OH)2D3 augments PTH-stimulated calcium transport by DCT cells, while having no effect of its own. 1,25(OH)2D3 mediates its effects on gene expression by binding to a nuclear vitamin-D receptor (VDR), which then associates with the retinoid-X receptor (RXR) as a heterodimer. We studied the effects of 1,25(OH)2D3, 9-cis- and all-trans-retinoic acid on PTH/PTHrP receptor expression. mRNAs for the PTH/PTHrP, VDR, and RXR receptors were detected in immortalized DCT cells by reverse transcriptase-polymerase chain reaction. Changes in PTH/PTHrP receptor mRNA expression were quantified by slot blot hybridization. 1,25(OH)2D3 maximally increased PTH/PTHrP receptor mRNA levels by 70%. The stimulation was specific since 1,25(OH)2D3 treatment had no effect on the expression of adrenergic receptor or Na+/H+ exchanger mRNA levels. Likewise, the inactive form, 25(OH)2D3 had no effect on PTH/PTHrP receptor mRNA expression. In combination with the putative RXR ligand, 9-cis-retinoic acid, 1,25(OH)2D3 increased PTH/PTHrP receptor mRNA levels 4-fold. 9-cis-Retinoic acid had no effect of its own on steady-state PTH/PTHrP receptor mRNA expression. The putative ligand for the retinoic acid receptor, all-trans-retinoic acid, increased PTH/PTHrP receptor mRNA expression alone and in combination with 1,25(OH)2D3. 9-cis-Retinoic acid alone, and in combination with 1,25(OH)2D3, also increased specific PTH/PTHrP receptor binding to plasma membranes isolated from DCT cells. These results indicate that 1,25(OH)2D3 upregulated PTH/PTHrP receptor expression at both mRNA and protein levels in a manner consistent with VDR/RXR heterodimers transactivating the PTH/PTHrP receptor gene by binding a vitamin D response element in the PTH/PTHrP gene.

Na+ -phosphate cotransport in mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression.

While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+-dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+-dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 +/- 3% of control), a competitive inhibitor of Na+-Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi-free medium for 21 h increased Na+-Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12-myristate 13-acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+-Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+-Pi cotransport, suggesting that other Na+-Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphoteric murine retrovirus (Ram-1) that also function as Na+-Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+-Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+-Pi cotransport in the proximal and distal segments of the nephron.

Parathyroid hormone stimulation of calcium transport is mediated by dual signaling mechanisms involving protein kinase A and protein kinase C.

PTH stimulates calcium absorption by renal distal convoluted tubules. The PTH receptor is capable of coupling to adenylyl cyclase and phospholipase C. However, it is not known whether the actions of PTH require activation of both pathways. Three approaches were taken to identify the signaling pathways responsible for stimulating calcium entry in distal convoluted tubule cells: second messengers formed in response to PTH were identified, the effects on calcium uptake of inhibiting protein kinase A (PKA) or protein kinase C (PKC) with chemical or peptide blockers were determined, and calcium transport was reconstituted by the addition of exogenous second messengers. PTH increased cAMP formation in primary cultures of mouse distal and proximal tubule cells. However, PTH stimulated inositol trisphosphate formation only in proximal tubule cells. Blocking PKA with Rp-cAMPS or the cAMP-dependent protein kinase inhibitor inhibited PTH-stimulated Ca uptake. Likewise, the PKC inhibitors, calphostin C and PKC pseudosubstrate, inhibited PTH-induced calcium uptake. Addition of forskolin (30 nM) or phorbol 12-myristate 13-acetate (10 nM) alone had no effect on Ca uptake. However, when added in combination, Ca uptake was stimulated to nearly the same extent as with concentrations of PTH that maximally stimulate calcium transport. We conclude that stimulation of calcium uptake by distal convoluted tubule cells requires activation of both PKA and PKC.

Immunomagnetic separation, primary culture, and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney.

Renal cortical thick ascending limbs of Henle's loop (CAL) and distal convoluted tubules (DCT) represent sites at which much of the final regulation of urinary ionic composition, particularly that of calcium, is accomplished in both humans and in rodents. We sought in the present work to develop an efficient means for isolating parathyroid hormone (PTH)-sensitive cells from these nephron segments and to grow them in primary culture. [CAL+DCT] cells were isolated from mouse kidney using an antiserum against the Tamm-Horsfall glycoprotein which, in the renal cortex, is produced exclusively by these cells. A second antibody conjugated to coated ferrous particles permitted magnetic separation of [CAL+DCT] cells from Tamm-Horsfall negative renal cortical cells. Approximately 3 X 10(6) cells per kidney with a trypan blue exclusion greater than 94% were isolated by these procedures. Experiments were performed to characterize the cells after 7 to 10 days in primary culture. PTH and isoproterenol, but neither calcitonin nor vasopressin, stimulated cyclic AMP (cAMP) formation in [CAL+DCT] cells, consistent with the pattern of hormone-activated cAMP synthesis found in freshly isolated CAL and DCT segments. Alkaline phosphatase, an enzyme present dominantly in proximal tubule brush border membranes, was virtually absent from [CAL+DCT] cells but was present in Tamm-Horsfall negative cells. Similarly, Na-glucose cotransport was absent in [CAL+DCT] cells but present in Tamm-Horsfall negative renal cortical cells. Finally, transport-related oxygen consumption in [CAL+DCT] cells was blocked by bumetanide and by chlorothiazide, diuretics that inhibit sodium transport in CAL and DCT nephron segments. These results demonstrate that PTH-sensitive [CAL+DCT] cells can be isolated in relatively high yield and viability and grown in cell culture. Primary cultures of these cells exhibit a phenotype appropriate to their site of origin in the nephron.

Amiloride, protein synthesis, and activation of quiescent cells.

Amiloride is known to inhibit both influx of sodium ions and activation of quiescent cells by growth factors. The coincidence of these effects has been cited to support the proposal that influx of sodium ions acts as a mitogenic signal. Although it was noted that amiloride inhibited protein synthesis, this was attributed to an action on transport of amino acids, particularly those coupled to sodium fluxes. We find, however, that amiloride directly inhibits polypeptide synthesis in a reticulocyte lysate. In Swiss 3T3 cells, concentrations of amiloride and of cycloheximide that are nearly matched in their degree of inhibition of protein synthesis, produce about the same degree of inhibition of transit of cells from G0 to S. Inhibition of protein synthesis is sufficient to explain the effect of amiloride on mitogenesis; the drug, therefore, is not suitable for testing the hypothesis that sodium influx is a mitogenic signal.