Staged in vitro reconstitution and implantation of engineered rat kidney tissue.

A major hurdle for current xenogenic-based and other approaches aimed at engineering kidney tissues is reproducing the complex three-dimensional structure of the kidney. Here, a stepwise, in vitro method of engineering rat kidney-like tissue capable of being implanted is described. Based on the fact that the stages of kidney development are separable into in vitro modules, an approach was devised that sequentially induces an epithelial tubule (the Wolffian duct) to undergo in vitro budding, followed by branching of a single isolated bud and its recombination with metanephric mesenchyme. Implantation of the recombined tissue results in apparent early vascularization. Thus, in principle, an unbranched epithelial tubular structure (potentially constructed from cultured cells) can be induced to form kidney tissue such that this in vitro engineered tissue is capable of being implanted in host rats and developing glomeruli with evidence of early vascularization. Optimization studies (of growth factor and matrix) indicate multiple suitable combinations and suggest both a most robust and a minimal system. A whole-genome microarray analysis suggested that recombined tissue recapitulated gene expression changes that occur in vivo during later stages of kidney development, and a functional assay demonstrated that the recombined tissue was capable of transport characteristic of the differentiating nephron. The approach includes several points where tissue can be propagated. The data also show how functional, 3D kidney tissue can assemble by means of interactions of independent modules separable in vitro, potentially facilitating systems-level analyses of kidney development.

Organic anion and cation transporter expression and function during embryonic kidney development and in organ culture models.

Organic anion and cation transporters (OATs, OCTs, and OCTNs) mediate the proximal tubular secretion of numerous clinically important compounds, including various commonly prescribed pharmaceuticals. Here, we report determination of the ontogeny of these transporters and of NaP(i)2 and SGLT1, using quantitative polymerase chain reaction (QPCR) to determine expression levels of transporter genes in rat embryonic kidneys on each day of gestation from embryonic day (ed) 13 to ed18, in cultures of induced and uninduced metanephric mesenchyme (MM), and on each day of 1 week of whole embryonic kidney (WEK) culture. We also examined ontogeny of Oat1 protein expression in rat embryonic kidney by immunohistochemistry. Finally, we used uptake of fluorescein (FL) as a novel in vitro functional assay of OAT expression in WEK and MM. Developmental induction of OAT and OCT genes does not occur uniformly: some genes are induced early (e.g., Oat1 and Oat3, potential early markers of proximal tubulogenesis), and others after kidney development is relatively advanced (e.g., Oct1, a potential marker of terminal differentiation). The ontogeny of transporter genes in WEK and MM is similar to that observed in vivo, indicating that these organ culture systems may represent convenient in vitro models to study the developmental induction of OATs, OCTs, and OCTNs. Functional transport was evidenced by accumulation of FL in the developing tubule in WEK and MM organ cultures. Our findings on the renal ontogeny of OATs and OCTs could carry implications both for the development of more rational therapeutics for premature infants, as well as for our understanding of proximal tubule differentiation.

Dietary magnesium, not calcium, regulates renal thiazide receptor.

This study reports for the first time a relationship between dietary Mg and the renal thiazide-sensitive Na-Cl cotransporter (TZR, measured by saturation binding with 3H-metolazone). Ion-selective electrodes measured plasma ionized magnesium (PMg++), calcium (PCa++), and potassium (PK+). Restricting dietary Mg for 1 wk decreased PMg++ 18%, TZR 25%, and renal excretion of magnesium (UMg) and calcium (UCa) more than 50% without changing PCa++, PK+, or plasma aldosterone. A low Mg diet for 1 d significantly decreased PMg++, TZR, UMg and UCa. Return of dietary Mg after 5 d of Mg restriction restored PMg++ and TZR toward normal. In the control, Mg-deficient, and Mg-repleting animals, TZR correlated with PMg++ (r = 0.86) and with UMg (r = 0.87) but not UCa (r = 0.09). Increasing oral intake of Mg for 1 wk increased PMg++ 14%, TZR 32%, UMg 74%, and UCa more than fourfold without changing PCa++ or PK+. In contrast, increasing dietary Ca content from 0.02% to 1.91% did not change TZR, but increased UCa fivefold without changing PCa++. Hormonal mediators (if any) involved in the relationship between dietary Mg and TZR remain to be elucidated, as does the relationship between TZR and tubular reabsorption of Mg.

Genetic control of renal thiazide receptor response to dietary NaCl and hypertension.

Excess NaCl increases blood pressure in some strains of animals but not others. An 8% NaCl diet did not change renal thiazide receptor (TZR) density in two salt-resistant normotensive rat strains (Wistar-Kyoto and Sprague-Dawley) [Fanestil, D. D., D. A. Vaughn, and P. Blakely. Am. J. Physiol. 273 (Regulatory Integrative Comp. Physiol. 42): R1241-R1245, 1997]. However, the renal response to salt differs in normal and hypertensive kidneys [Rettig, R., N. Bandelow, O. Patschan, B. Kuttler, B. Frey, and A. Uber. J. Hum. Hypertens. 10: 641-644, 1996]. Therefore, we examined two strains with salt-aggravated hypertension. Renal TZR did not change when Dahl-S (salt sensitive) animals became hypertensive with 8% dietary NaCl. In contrast, renal TZR decreased 34%, whereas blood pressure increased further, in SHR with 8% dietary NaCl. Blood pressure increased after NG-nitro-L-arginine in SHR, but renal TZR did not change, indicating the salt-induced decrease in TZR in SHR cannot be attributed nonspecifically to elevated arterial pressure. We conclude that the renal response to NaCl-induced increases in blood pressure can be genetically modulated independently of the genes that mediate either the primary hypertension or the salt sensitivity of the hypertension. This finding may be of use in future studies directed at identifying genotypes associated with salt-dependent hypertension.

Dietary NaCl and KCl do not regulate renal density of the thiazide diuretic receptor.

We tested the postulate that the renal density of the thiazide-inhibitable Na-Cl cotransporter or thiazide receptor (TZR) is modulated as part of the renal homeostatic response to changes in dietary intake of NaCl or KCl. Renal excretion of NaCl or KCl varied > 10-fold in response to alterations in oral intake. Renal TZR density was quantitated by binding of [3H]metolazone to renal membranes. Renal TZR density was not altered by sodium deficit (with increased plasma aldosterone concentration), by sodium surfeit (8% NaCl content of diet), by potassium deficit (with hypokalemia), or by potassium surfeit (drinking 1% KCl solution). Unexpectedly, we conclude that regulation of the renal density of TZR is not part of the renal homeostatic responses that adjust excretion of NaCl and KCl to changes in dietary intake of NaCl or KCl.

Metabolic acid-base influences on renal thiazide receptor density.

The renal responses to metabolic acidosis/alkalosis involve changes in the proximal tubule, loop of Henle, and collecting ducts. We tested for acid- or base-induced changes in the distal convoluted tubule (DCT) by examining the renal density of the DCT's receptor for thiazide-type diuretics (TZR), as estimated by the binding of [3H]metolazone in Wistar-Kyoto rats. TZR density significantly decreased by 17% in rats ingesting NH4Cl for 3.5 days and by nearly 30% after 7 days; TZR increased up to 40% in rats ingesting NaHCO3 for 2-4 days but was no longer significantly increased after 7 days. Urinary excretion of chloride increased as renal density of the TZR decreased, a finding consistent with the interpretation that acidosis/alkalosis not only altered TZR density but coordinately altered reabsorption of NaCl by the thiazidesensitive Na-Cl cotransporter. The result is that delivery of Na from DCT is enhanced during acidosis and decreased during alkalosis, assisting in compensatory changes in distal nephron secretion of hydrogen ion. The integrated renal response to metabolic acidosis/alkalosis involves a decrease in renal TZR with acidosis and an increase in TZR with alkalosis.

Thiazide diuretics normalize urinary calcium in spontaneously hypertensive male rats.

The role of the thiazide-sensitive distal convoluted tubule (DCT) in the hypercalciuria of the spontaneously hypertensive rat (SHR) strain was examined by determining (a) the renal density of the thiazide diuretic receptor with 3H-metolazone, and (b) the renal response to a maximal dose of bendroflumethiazide (BFTZ). We confirm that the renal thiazide receptor density was greater in SHR than WKY (0.936 +/- 0.026 vs. 0.797 +/- 0.045 pmol/mg protein; P = 0.02). Prior to BFTZ the urinary excretion of calcium (0.525 +/- 0.061 vs. 0.274 +/- 0.049 micromol per micromol creatinine, P < 0.01) and sodium (12.6 +/- 1.27 vs. 7.89 +/- 0.926 micromol per micromol creatinine; P < 0.01) were greater in SHR versus WKY. BFTZ decreased the excretion of calcium only in SHR and to a level (0.250 +/- 0.032) not significantly different (P = 0.519) from WKY (0.225 +/- 0.032). Surprisingly, BFTZ increased chloride excretion to a greater extent in WKY than in SHR (P = 0.008). We postulate that hypercalciuria in SHR is a manifestation of incomplete uptake of calcium from the tubule lumen across the apical cell membrane in the DCT of the SHR nephron.

Amylin, calcitonin gene-related peptide, and adrenomedullin: effects on thiazide receptor and calcium.

We previously reported that salmon calcitonin, but not rat calcitonin, increased renal thiazide receptor (TZR) density and decreased renal calcium [urinary calcium excretion (U(Caex))] in the rat. Since calcitonins, islet amyloid polypeptide (amylin), calcitonin-gene related peptide (CGRP), and adrenomedullin interact with a family of calcitonin-related receptors, we examined the effects of these peptides on 1) TZR density, as quantitated by binding of [3H]metolazone to renal membranes; 2) plasma ionic composition; and 3) urinary electrolyte excretion. Subcutaneous amylin both increased TZR density nearly twofold and decreased U(Caex), with maximal effects by 24 h. The decreased U(Caex) occurred with plasma amylin levels in the physiological range, whereas the increased TZR did not reach maximum even with plasma amylin >100 times above normal. Similar doses of adrenomedullin increased TZR density modestly but without effect on U(Caex), whereas CGRP did not alter TZR density and tended to increase U(Caex). We propose that U(Caex) and TZR density in the rat kidney are regulated by rat amylin but not by rat calcitonin.

Effects of calcium-modulating hormones on thiazide receptor density.

Thiazide diuretic drugs act in the distal convoluted tubule (DCT) to inhibit a Na+Cl- cotransporter and enhance reabsorption of luminal calcium. The density of receptors for thiazides in the rat DCT is known to be increased by adrenocortical steroids, furosemide, and bendroflumethiazide, but decreased by ischemia. Because the DCT is a physiologic site of action by calcitonin and parathyroid hormone, this study examined the effects of these calcitropic hormones in thyroparathyroidectomized Sprague-Dawley rats on (1) the density of the rat thiazide receptor (TZR), as quantitated by binding of (3H)metolazone to renal membranes, and (2) urinary electrolyte excretion rate. Salmon calcitonin (sCT) (20 to 100 ng/h) (1) increased the density of the renal TZR twofold, an effect that is maximal by 6 h after sCT administration, and (2) decreased urinary calcium excretion rate. Adequate dietary calcium must be provided for the effects of sCT to be observed. Regression analysis demonstrated that renal TZR density correlated negatively with total urinary calcium excretion rate but not with plasma calcium ion concentration. In addition, neither rat calcitonin (rCT), at doses that cause hypocalcemia, nor parathyroid hormone, at doses that cause hypercalcemia, produce direct effects on TZR density in the DCT of the thyroparathyroidectomized rat. Our findings indicate that upregulation of TZR by sCT, which occurs independently of plasma calcium-ion concentration, is likely via a calcitonin-like receptor other than that for rat calcitonin itself.

Adrenocortical steroids increase renal thiazide diuretic receptor density and response.

The density of the rat renal pharmacologic receptor for thiazide-type diuretics, as quantitated by the maximal specific binding of (3H)metolazone, decreased to one-third normal after adrenalectomy. Selective glucocorticoid (dexamethasone or RU-28362) replacement increased thiazide receptor density to or above the normal level over the dose range of steroid that decreased thymus weight, which served as a bioassay for glucocorticoid activity. Mineralocorticoid (fludrocortisone or aldosterone), in doses that did not decrease thymus weight, also increased thiazide diuretic receptor density to or above normal. The addition of glucocorticoid (RU-28362) to maximal aldosterone increased thiazide receptor above that produced by aldosterone alone and to threefold normal. Similarly, the addition of aldosterone to high-dose RU-28362 also increased thiazide receptor density above that produced by the glucocorticoid alone and to threefold normal. Hence, the effects of glucocorticoids and mineralocorticoids appeared to be additive. The increase in renal thiazide receptor density produced by fludrocortisone, at a dose that elicited both mineralocorticoid and glucocorticoid effects, was unrelated to the basal (prethiazide) renal excretion of sodium, potassium, chloride, or calcium. However, fludrocortisone-pretreated animals responded to bendroflumethiazide with a greater natriuresis than did controls. In addition, the magnitudes of the thiazide-elicited natriuresis and chloriuresis correlated significantly with thiazide receptor. It was concluded that both the density of the renal thiazide receptor and the quantity of sodium and chloride reabsorbed by the thiazide-sensitive Na-Cl cotransporter in the kidney are under adrenocortical regulation.

Influence of gender on renal thiazide diuretic receptor density and response.

The influence of gender and gonadectomy on (1) the density of the renal thiazide-sensitive ion transporter, as quantitated by the ability of renal membranes to bind (3H)metolazone, and (2) the changes in the urinary excretion of electrolytes caused by maximal bendroflumethiazide (BFTZ) in Sprague-Dawley rats was determined. The density of the thiazide receptor was twofold higher (P < 0.001) in females than in males. Orchiectomy increased thiazide receptor significantly in one of two studies (P < 0.01). Ovariectomy decreased thiazide receptor by more than 20% (P < 0.01) in both studies. The rates of the urinary excretion of sodium and chloride after BFTZ and the increases in the urinary excretion of sodium, chloride, and ammonium caused by BFTZ were greater in intact females than in intact males; BFTZ decreased the urinary excretion of calcium 50% in intact females, but not in intact males. Regression analysis of the thiazide receptor (in intact and gonadectomized animals) versus the urinary excretion of electrolytes before and after BFTZ yielded a model in which one-third of the variation in thiazide receptor could be related to the change in the excretion of calcium and ammonium produced by BFTZ, raising the possibility that the density of the thiazide receptor might be related to calcium or acid-base homeostasis. It was concluded that the renal excretion of sodium, chloride, calcium, and ammonium are, in part, controlled by gender and sex hormones via their regulation of the renal density of the thiazide diuretic receptor.

Effects of diuretic treatment and of dietary sodium on renal binding of 3H-metolazone.

We report a series of experiments designed to determine if agents and conditions that have been reported to alter sodium reabsorption, Na-K-ATPase activity or cellular structure in the rat distal nephron might also regulate the density or affinity of binding of 3H-metolazone to the putative thiazide receptor in the distal nephron. Experimental conditions selected for study were acute (60-min) and chronic hydrochlorothiazide (HCTZ), acute acetazolamide, acute and chronic furosemide, and 14 days of varied intake of dietary sodium. The density of the binding of 3H-metolazone was increased 47% by acute HCTZ (P less than 0.001) and 39% (P less than 0.001) by acute furosemide. In contrast, acute acetazolamide produced no change in binding despite eliciting a dramatic diuresis. Chronic HCTZ (5 days) and chronic furosemide (7 days) increased binding of 3H-metolazone by 46% (P less than 0.001) and by 101% (P less than 0.001), respectively. Variation of dietary sodium intake over a range that allowed normal growth of the animal and that produced urinary excretion of Na varying from 0.28 to 2.62 mEq/100 g/day failed to alter the density of binding of 3H-metolazone. These studies are the first indication that the density of the thiazide receptor is regulated by a variety of both acute and chronic conditions that have previously been associated with changes in transport, ultrastructure or Na-K-ATPase activity in the distal nephron.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization of thiazide diuretic receptors from rat kidney membranes.

Thiazide-type diuretics act at receptors to inhibit NaCl transport in the renal distal tubule. We solubilized high-affinity [3H]metolazone binding sites from rat kidney membranes with Triton X-100, which was more effective than several other detergents. Phosphatidylcholine and a mixture of proteinase inhibitors were needed to stabilize binding so that 57% of solubilized binding remained after 72 h at 4 degrees C. The affinities of solubilized (Kd = 11.4 +/- 0.5 nM) and membrane-bound receptors (Kd = 12.0 +/- 1.7 nM) were similar. The maximal number of binding sites/mg protein of solubilized receptors was 46 +/- 3% (n = 5) of membrane-bound receptors. Diuretics with a wide range of affinities had similar affinities for the solubilized and membrane-bound sites. Chloride inhibited and sodium stimulated the binding of [3H]metolazone to solubilized receptors, as they do with membrane-bound receptors. These studies demonstrate that, as judged by ligand binding, thiazide receptors can be solubilized in an active conformation and provide the basis for future purification and reconstitution.

Thiazide diuretic receptors in spontaneously hypertensive rats and 2-kidney 1-clip hypertensive rats.

Thiazide diuretic receptor density was assessed in kidneys from spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats by measuring hydroflumethiazide-displaceable 3H-metolazone binding to renal membranes in vitro. Renal thiazide receptor density was not significantly different in 4 week old SHR and WKY rats, but was significantly increased by 20%-40% in 14-49 week old SHRs compared to WKY rats. Affinity of receptors for 3H-metolazone did not differ between SHRs and WKY rats at any age. In WKY rats with 2 kidney-1 clip (2K-1C) hypertension, thiazide receptor density was not significantly different in either clipped or unclipped kidneys from sham-operated controls. Thus, increased renal thiazide receptor density occurs in SHRs along with the development of hypertension and does not appear to be secondary to increased renal perfusion pressure. This increase may reflect altered hormonal or ionic input to the distal tubule and may contribute to elevated sodium reabsorption in this segment in the SHR.

Thiazide diuretic receptors: autoradiographic localization in rat kidney with [3H]metolazone.

The localization of binding sites for [3H]metolazone, a quinazolinesulfonamide diuretic with thiazide-like actions, was determined by in vitro autoradiography. [3H]Metolazone bound saturably to rat kidney sections incubated in vitro with a dissociation constant (Kd) = 3.4 nM and binding site density = 0.14 pmol/mg of protein. Incubation conditions were used that excluded binding to low affinity sites and carbonic anhydrase. Pharmacological specificity of binding was consistent with labeling of physiologically relevant thiazide diuretic receptors, as identified in previous studies of [3H]metolazone binding to renal membranes. Autoradiographs obtained with tritium-sensitive film demonstrated that binding sites were limited to the renal cortex and were relatively sparsely distributed. Higher resolution autoradiography indicated that [3H] metolazone binding sites were localized in a highly specific manner over short lengths of tubular segments, which by their morphology and distribution most likely represented distal convoluted tubules. In the short sections of tubule that contained receptors, labeling was very dense and appeared to be more prevalent over luminal than peritubular surfaces. The intrarenal distribution of [3H]metolazone binding sites provides further evidence for their identity as thiazide diuretic receptors. These results are consistent with physiological studies demonstrating that the early distal tubule is the location of thiazide-sensitive sodium chloride cotransport.

Reversible downregulation of thiazide diuretic receptors by acute renal ischemia.

Receptors for thiazide diuretic drugs in the rat renal cortex have recently been identified through the binding of [3H]metolazone, a potent diuretic with a thiazide-like mechanism of action. The present studies describe the rapid and reversible alterations that occur in thiazide receptors following acute renal ischemia in the rat. The apparent density of thiazide receptors in kidney membranes as measured by the binding of [3H]metolazone was reduced by 90% following 10 min of renal ischemia produced by clamping the renal pedicle. With release of the clamp and subsequent reperfusion for 10 min, thiazide receptor density returned to within 40% of control levels. Ischemia did not alter apparent affinity of receptors for [3H]-metolazone. Sections prepared from renal cortex and incubated in oxygenated media in vitro displayed similar rapid changes in thiazide receptors. Hypoxia of 10- to 30-min duration produced by incubating sections in vitro in nitrogen-saturated media caused a significant decrease in [3H]metolazone binding that was reversible with return to oxygenated media. Similar decreases were obtained in oxygenated sections that were incubated with mitochondrial inhibitors, dinitrophenol and rotenone, but not in sections incubated with ouabain. These results indicate that renal thiazide receptors undergo a rapid and reversible form of regulation and that controlling mechanisms are dependent on metabolic energy.

Thiazide diuretic drug receptors in rat kidney: identification with [3H]metolazone.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. We report here that [3H]metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. [3H]Metolazone bound with high affinity (Kd = 4.27 nM) to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or no binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated with their clinical potency. Halide anions (Cl-, Br-, and I-) specifically inhibited high-affinity binding of [3H]metolazone to this site. [3H]Metolazone also bound with lower affinity (Kd = 289 nM) to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had Ki values of 0.6-10 microM for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity [3H]metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.

Interactions of lipids with peripheral-type benzodiazepine receptors.

Peripheral-type benzodiazepine receptors (PBRs) are present at high densities in the rat kidney distal tubule. [3H]RO 5-4864 binding to PBRs in kidney membranes is inhibited by several unidentified low molecular weight hydrophobic compounds in urine and serum. We tested representative hydrophobic compounds from several lipid classes for ability to inhibit binding to rat kidney PBRs of two high affinity ligands, [3H]RO 5-4864 and [3H]PK 11195. Unsaturated fatty acids and alcohols inhibited [3H]RO 5-4864 binding with half-maximal inhibition occurring at 3 X 10(-6) M to 10(-4) M. Inhibitory potency increased with the degree of unsaturation. Phospholipids inhibited [3H]RO 5-4864 in the same concentration range, with inhibitory potency in this case dependent both upon an unsaturated fatty acid moiety and upon the polar head group. Phosphatidylethanolamine was the most potent phospholipid tested (IC50 = 2 X 10(-6) M), whereas phosphatidylcholine was not inhibitory. Although phospholipids inhibited both [3H]RO 5-4864 and [3H]PK 11195 binding equally, unsaturated fatty acids had a much greater inhibitory effect upon [3H]RO 5-4864 than upon [3H]PK 11195 binding. Similar effects were obtained with digitonin-solubilized PBRs. These data demonstrate that in our experiments PBR binding was inhibited by specific lipids and that binding of proposed agonist (RO 5-4864) and antagonist (PK 11195) ligands was differentially affected by unsaturated fatty acids.

Binding of calcium to the proteolipid phosphorin.

Phosphorin is a phosphate-binding proteolipid isolated from rabbit kidney brush border membrane vesicles that binds inorganic phosphate with high affinity and specificity. This binding of phosphate has a Hill coefficient of 1.92 and an absolute requirement for the presence of a divalent metal. We now describe the binding of Ca2+ to phosphorin that had been depleted of endogenous divalent metal. The dependence of the binding of Ca2+ over the concentration range of 5-100 microM produced a sigmoidal curve, yielding a Hill coefficient of 2.46. (Ruthenium red and La3+ were also potent inhibitors of Ca2+ binding). The divalent metals Mn2+ and Mg2+ were able to inhibit binding of Ca2+ and produced Hill coefficients of 1.75 and 1.98; however, Ba2+ and Sr2+ were less effective in their ability to inhibit binding. In addition, the amine-reactive reagent, 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS), which had previously been found to inhibit binding of phosphate to phosphorin, also completely inhibited binding of Ca2+. We propose that a phosphorin-Me2+ complex forms the molecular species that binds phosphate.