Frank A. Gesek

IMMUNOMAGNETIC SEPARATION, PRIMARY CULTURE, AND CHARACTERIZATION OF CORTICAL THICK ASCENDING LIMB PLUS DISTAL CONVOLUTED TUBULE CELLS FROM MOUSE KIDNEY

SummaryRenal 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 × 106 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.

Cell-specific signaling and structure-activity relations of parathyroid hormone analogs in mouse kidney cells.

PTH is an 84-amino acid protein. Occupancy of its cognate receptor generally results in activation of adenylyl cyclase and/or phosphoinositide-specific phospholipase Cβ (PLCβ). In the kidney, PTH receptors are present on proximal and distal tubule cells. In proximal tubules, PTH induces calcium signaling, typified by a transient rise in intracellular calcium ([Ca2+]i) and inositol trisphosphate formation, but does not affect calcium absorption. By contrast, in distal tubules, PTH increases calcium absorption that is associated with a slow and sustained rise in [Ca2+]i, but does not stimulate phospholipase C (PLC) or cause inositol trisphosphate accumulation. Nonetheless, stimulation of distal calcium transport requires activation of protein kinase C (PKC) and protein kinase A. We now characterize the origin of the differential effects of ligand occupancy by using synthetic human PTH analogs that preferentially activate adenylyl cyclase and/or PLCβ. We further tested the hypothesis that phospholipase D is ...

Pentoxifylline Ameliorates Renal Tumor Necrosis Factor Expression, Sodium Retention, and Renal Hypertrophy in Diabetic Rats

Background/Aim: Diabetic nephropathy contributes substantially to cardiovascular morbidity and mortality associated with diabetes. Urinary tumor necrosis factor (TNF) excretion is increased during diabetes and serves as an important mediator of pathological changes during the initial stages of diabetic nephropathy, including sodium retention and renal hypertrophy. We tested the hypothesis that pentoxifylline (PTF), an agent that inhibits TNF synthesis, could prevent sodium retention and renal hypertrophy during diabetes. Methods: Proximal and distal tubule TNF expression, urinary TNF excretion, sodium retention, and renal hypertrophy were examined in control, diabetic, and PTF-treated diabetic rats. Results: TNF mRNA and protein levels were increased in proximal tubule cells isolated from diabetic rats compared to control rats. In contrast, TNF expression in distal tubule cells was not increased during diabetes. PTF prevented the increase in TNF mRNA and protein in proximal tubule cells during diabetes and reduced urinary TNF excretion. PTF therapy decreased whole animal sodium retention by enhancing urinary sodium excretion in diabetic rats. In addition, PTF reduced renal hypertrophy in diabetic rats. Conclusions: The proximal tubule is an important site of TNF production during diabetes and PTF is an effective therapy for preventing the pathological changes accompanying early diabetic nephropathy.

Distinct Calcium Channel Isoforms Mediate Parathyroid Hormone and Chlorothiazide-stimulated Calcium Entry in Transporting Epithelial Cells

Abstract. Some cells express multiple calcium channel isoforms that are likely to have distinct functions. The present study used molecular cloning and antisense techniques to identify calcium channel isoforms mediating calcium entry in mouse distal convoluted tubule (DCT) cells. The DCT is the major site of hormone- and diuretic-regulated calcium transport in the kidney. Cellular calcium absorption involves entry through apical membrane calcium channels that are sensitive to dihydropyridine-type calcium channel antagonists. Partial cDNA clones corresponding to one isoform of the calcium channel α1 pore-forming subunit, α1C, and one isoform of the calcium channel β accessory subunit, β3, were isolated by RT-PCR. Full-length transcripts were detected by Northern blot analysis in immortalized DCT cells. Antisense oligonucleotides complementary to the α1C sequence inhibited the rise of intracellular calcium ([Ca2+]i) induced by the thiazide diuretic, chlorothiazide (CTZ), but not that induced by parathyroid hormone (PTH). However, antisense oligonucleotides complementary to the β3 sequence inhibited both CTZ- and PTH-induced rises of [Ca2+]i.β3 antisense oligonucleotides also inhibited the membrane hyperpolarization induced by CTZ but not that triggered by PTH. Thus, members of the voltage-gated calcium channel family are expressed in DCT cells, where they are responsible for hormone- and drug-induced calcium uptake. The results suggest that DCT cells contain multiple calcium channels with distinct roles in the regulation of cellular calcium.

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.

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 1α,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.

α1- and α2-Adrenoceptor Control of Sodium Transport Reverses in Developing Hypertension

Abstract —α-Adrenergic receptor (AR) activation enhances sodium retention in certain forms of hypertension. The objective of the present study was to understand the role of α-ARs in regulating sodium transport by distal tubules (DT). DT cells were isolated from kidneys of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at 6 weeks, when hypertension is developing, or at 12 weeks, when hypertension is established. The α 1 -AR agonist phenylephrine increased 22 Na uptake by 50% into DT cells of 6-week SHR; no effect was observed with WKY cells. The α 2 -AR agonist B-HT 933 increased uptake by only 10%. At 12 weeks, the pattern of α-AR regulation was reversed: α 1 -AR–induced sodium uptake was only 15%, whereas α 2 -AR activation increased sodium uptake by 35% in SHR and WKY cells. α 1 -AR–induced sodium uptake in 6-week SHR cells was abolished by prazosin; α 2 -AR–stimulated sodium uptake was blocked by yohimbine in 12-week SHR and WKY. Competitive binding studies were performed with [ 3 H]prazosin and α 1A -, α 1B -, and α 1D -selective antagonists with DT cell membranes from 6- and 12-week SHR and WKY. α 2 -AR subtypes were determined with [ 3 H]rauwolscine and α 2A - and α 2B -selective antagonists. Expression of α 1B -ARs was increased 4-fold in DT cells during the developing phase of hypertension in SHR. No change was detected in α 2 -AR expression. DT cells transiently increase [Ca 2+ ] i in response to α 1 -AR agonists from 6-week but not 12-week SHR. Conversely, α 2 -AR agonists increase [Ca 2+ ] i at 12 weeks. In summary, during developing hypertension, α 1 -ARs increase sodium uptake and [Ca 2+ ] i in SHR cells. Expression of α 1B -ARs is selectively upregulated during developing hypertension. In established hypertension (and normotension), α 2 -ARs regulate sodium transport and [Ca 2+ ] i in DT cells. We conclude that a molecular switch of α 1 -AR and α 2 -AR signaling occurs in DT cells during the development of hypertension.

Identification and Antisense Inhibition of Na-Ca Exchange in Renal Epithelial Cellsa

In summary, DCT cells express multiple isoforms of the Na-Ca exchanger and exhibit functional exchange, and antisense oligonucleotides to a downstream region of the exchanger transcript inhibit activity. These experiments provide direct evidence for Na-Ca exchange in DCT cells mediated by NACA2, NACA3, or NACA6.