Rat Renal Tubules Research Articles

Cell inward transport of L-DOPA and 3-O-methyl-L-DOPA in rat renal tubules.

1. The present study has determined the kinetics of the uptake of L-3,4-dihydroxyphenylalanine (L-DOPA) and 3-O-methyl-L-DOPA (3-OMDOPA) in rat renal tubules and examined the effect of 3-OMDOPA on the inward transport of L-DOPA and on its conversion into dopamine in kidney homogenates. 2. The accumulation of both L-DOPA and 3-OMDOPA in renal tubules was found to occur through non-saturable and saturable mechanisms. The kinetics of the saturable component of L-DOPA and 3-OMDOPA uptake in renal tubules were as follows: L-DOPA, Vmax = 11.1 nmol mg-1 protein h-1 and Km = 216 microM (n = 6); 3-OMDOPA, Vmax = 8.1 nmol mg-1 protein h-1 and Km = 231 microM (n = 5). The diffusion constant of the non-saturable component for the accumulation of L-DOPA and 3-OMDOPA was 0.0010 and 0.0014 mumol-1, respectively. 3. 3-OMDOPA (100 to 2000 microM) was found to produce a concentration-dependent decrease (29% to 81% reduction) of the saturable component of the tubular uptake of L-DOPA; the Ki value of 3-OMDOPA for inhibition of L-DOPA uptake was found to be 181 microM (n = 5). The accumulation of L-DOPA obtained in experiments conducted at 4 degrees C was not affected by 3-OMDOPA. 4. In experiments conducted in kidney homogenates only L-DOPA (10 to 5000 microM) was found to be decarboxylated. The Vmax and Km values for aromatic L-amino acid decarboxylase determined in the absence of 3-OMDOPA (Vmax = 14.1 nmol mg-1 protein h-1; Km =62 MicroM) were not significantly different from those observed when the decarboxylation of L-DOPA was carried out in the presence of 1000 MicroM 3-OMDOPA (Vmax = 15.7 nmol mg-1 protein h-1; Km = 68 MicroM).5. It is concluded that the tubular uptake of both L-DOPA and 3-OMDOPA occur through nonsaturable and saturable mechanisms; only the saturable tubular uptake of L-DOPA was found to be inhibited by 3-OMDOPA. It is further shown that 3-OMDOPA neither undergoes decarboxylation into 3-MT nor affects the decarboxylation of L-DOPA.

Role of lindane in membranes. Effects on membrane fluidity and activity of membrane-bound proteins.

The influence of lindane (gamma-hexachlorocyclohexane) on fluidity of plasma membranes from rat renal cortical tubules has been investigated. Preincubation with lindane increased membrane fluidity. This effect was accompanied by (i) a decrease in the transport of glucose with regard to the controls and (ii) an inhibition of the beta-adrenergic stimulatory activity upon cyclic AMP accumulation. However, a significant decrease of the membrane fluidity was found when rats were injected with lindane for 12 days. The injection of lindane exerted the opposite effect on the membrane proteins, the glucose transporter and the beta-adrenergic receptor, enhancing the glucose uptake and increasing the isoproterenol-stimulated cycle AMP accumulation. A possible explanation of the difference could involve a resistance to membrane disordering by lindane through a regulatory mechanism that would balance the activity of many lindane-sensitive proteins in insecticide-injected rats.

Distribution of the adrenocortical inner zone antigen.

Previous studies using a mouse monoclonal antibody (IZAb) have identified inner zone-specific antigens (IZAg 1 and 2) in the rat adrenal cortex. The expression of IZAg1 is enhanced by dibutyryl-cyclic AMP or ACTH in vitro and by in vivo ACTH treatment. These studies also suggest that IZAg may be involved in steriogenesis in rat adrenals. Using sensitive detection methods, the distribution of IZAg in adrenals from other species and in other rat tissues was studied. Pig and rabbit adrenals expressed an IZAb-immunoreactive protein with molecular mass identical with that of IZAg1, but no immunoreactivity was detected in bovine, guinea-pig or mouse adrenals. In the rabbit adrenal, as in the rat, IZAb bound only to zonae fasciculata/reticularis cells. However, the whole adrenal cortex in the pig immunoreacted with IZAb. Although immunofluorescence was observed in human adrenal sections, predominantly in the zona reticularis, no significant immunoreactive protein band was detected by immunoblot analysis. Apart from the zonae fasciculata/reticularis in the adrenal gland, an IZAb-immunoreactive protein with a molecular mass of 26,000 Da, corresponding to IZAg1, was also found in rat hepatocytes, renal tubules, ovary (corpus luteum, interstitial cells, theca interna of mature follicles) and Leydig cells. The observations suggest that IZAg may be involved in the processes of biosynthesis, metabolism and/or excretion of steroid hormones.

Stimulatory effect of ethanol on weak organic acid uptake in rat renal tubules

Ethanol at relatively low concentrations (10–40 mM) significantly stimulated the uphill uptake of a weak organic acid, fluorescein, in the superficial proximal tubules of rat renal cortex slices, but it did not affect the rate of glucose production from lactate or pyruvate in rat renal cortex fragment suspension. In a low Na + medium, ethanol failed to stimulate fluorescein uptake, although under the conditions employed in the present study, the baseline weak organic acid uptake was not dependent on external Na +. The stimulation of fluorescein uptake by ethanol (20 mM) was abolished by an inhibitor of alcohol dehydrogenase (EC 1.1.1.1), pyrazole (1 mM), or an inhibitor of aldehyde dehydrogenase (EC 1.2.1.3), cyanamide (0.3 mM), suggesting that oxidation of ethanol mediated its effect on the uptake. Among gluconeogenesis inhibitors tested, only d-malate (2 mM) abolished the stimulatory effect of ethanol, while the rest either did not affect (quinolinate) or even slightly augmented (α-cyano-4-hydroxycinnamate and phenylpyruvate) it. The effect of ethanol was markedly increased by an inhibitor of the tricarboxylic acid cycle, fluoroacetate. It was concluded that the stimulation by ethanol of weak organic acid uptake in rat renal tubules was mediated by the production of acetate.

Stimulation of weak organic acid uptake in rat renal tubules by cadmium and nystatin

The uphill uptake of a weak organic acid, fluorescein, in superficial proximal tubules of the rat kidney was stimulated by CdCl 2 (0.1 mM) or nystatin (20 μM) in the absence of metabolic substrates in the incubation medium. The stimulation could be observed during the initial period of incubation (up to 30 min) only and was prevented completely by ouabain (0.1 mM), fluoroacetate (1 mM), malonate (10 mM), α-cyano-4-hydroxycinnamate (0.1 mM), phenylpyruvate (1 mM), d-malate (2 mM) or phenazine methosulfate (20 μM). In the renal cortex fragment suspension, both Cd 2+ and nystatin increased the ouabain-sensitive, basal oxygen consumption and inhibited the rate of glucose production from pyruvate, but not from lactate. In the presence of lactate (0.5–5 mM) in the incubation medium, Cd 2+ and nystatin rather inhibited fluorescein uptake, while externally added pyruvate did not influence their stimulatory effects. Taken together, these data suggest that both activation of the tricarboxylic acid cycle and export of reducing equivalents from the mitochondria to the cytosol are necessary for the stimulatory effects of Cd 2+ and nystatin on the weak organic acid uptake to develop.

REABSORPTION OF PROTEINS IN RENAL TUBULES

In order to investigate the mechanism of the reabsorption of a protein in renal tubules of rats, we employed OCT-7000, which is a recombinant variant of natural human interleukin-1α with a molecular mass of approximately 18, 000. In this study, OCT-7000 uptake in renal tubules was examined using immunoelectron microscopic technique with immunogold staining. The effects of various proteins or synthetic polypeptides on the urinary excretion of OCT-7000 were also investigated. Immunoelectron microscopic observations showed that OCT-7000 was taken up into the endocytic vesicle close to the brush border membrane located in segment 2 of the proximal tubules, followed by accumulation of secondary lysosomes. Urinary excretion of OCT-7000 after systemic administration was extremely low, accounting for 0.014% of the dose. Human serum albumin had no effect on the excretion of OCT-7000, while increases in the urinary excretion of OCT-7000 were found in rats treated with a trypsin inhibitor, myoglobin and trypsinogen, in a dose-dependent manner. The order of potency for urinary excretion of OCT-7000 was trypsinogen > myoglobin > trypsin inhibitor. Poly-L-lysine, a synthetic polypeptide dose-dependently increased the urinary excretion of OCT-7000, whereas poly-L-glutamic acid had no effect on excretion. Specifically, the data reveal that reabsorption of OCT-7000 in the proximal tubules was inhibited by trypsinogen, myoglobin, trypsin inhibitor or poly-L-lysine, resulting in an increase of urinary excretion of OCT-7000. Furthermore, it was considered that negative charges on the brush border membrane in the proximal tubules were involved in the reabsorption of OCT-7000 because the inhibitory potency of proteins or synthetic polypeptides on the reabsorption of OCT-7000 was increased with a high isoelectric point. From the above, the mechanisms of reabsorption of protein in renal tubule are speculated as follows. (1) The reabsorption site of proteins is located in segment 1 and/or 2 of the proximal tubules in the kidneys. (2) Binders with negative charges likely exist on the surface of the brush border membrane, glomerular filtrated proteins are bound to the binder. The binder has an unusually broad binding specificity for filtered proteins, and the binding affinity between them is involved in only molecular charges. (3) After binding of filtered proteins to the binder on the membrane, the membrane invaginates into the cell, and then the endocytic vesicle including filtered proteins is incorporated into the cell. (4) Primary lysosome and endocytic vesicle are fused to constitute a secondary lysosome.

Light- and electronmicroscopic distribution of taurine, an organic osmolyte, in rat renal tubule cells

Several lines of evidence suggest that taurine acts as an organic osmolyte in the kidney. We investigated the cellular and subcellular distribution of this amino acid in rat renal tubule cells. Semi- and ultrathin sections of plastic-embedded rat kidney were incubated with an antiserum against conjugated taurine, using peroxidase-antiperoxidase and immunogold procedures, respectively. Extensive control tests confirmed the selectivity of the antiserum. Our immunocytochemical preparations revealed a highly differentiated labeling pattern. Strong labeling (judged visually or by computer-aided calculation of gold particle densities) was found in collecting duct cells throughout cortex and medulla, in proximal straight tubule cells, and in cells of the descending thin limbs of Henle's loop. Intermediate gold particle densities occurred in proximal convoluted tubule cells and intercalated cells of the collecting ducts (the gold particle in the latter being 30% of that in the collecting duct cells). The distal convoluted tubules, and thick and thin ascending limbs were almost immunonegative. It cannot be excluded that the proportion of free taurine that is retained by the fixative varies somewhat among the different cell types. Yet the highly differentiated labeling pattern that was obtained suggests that taurine is heterogeneously distributed among different populations of tubule cells, and that its level varies substantially even among cells that are exposed to the same osmotic stress.

Interpretation of male rat renal tubule tumors.

Based on an analysis of recent scientific studies, a Technical Panel of the U.S. Environmental Protection Agency's (EPA) Risk Assessment Forum recently advised EPA risk assessors against using information on certain male rat renal tubule tumors to assess human risk under conditions specified in a new Forum report. Risk assessment approaches generally assume that chemicals producing tumors in laboratory animals are a potential cancer hazard to humans. For most chemicals, including classical rodent kidney carcinogens such as N-ethyl-N-hydroxyethylnitrosamine, this extrapolation remains appropriate. Some chemicals, however, induce accumulation of alpha 2u-globulin (alpha 2u-g), a low molecular weight protein, in the male rat kidney. The alpha 2u-g accumulation initiates a sequence of events that appears to lead to renal tubule tumor formation. Female rats and other laboratory mammals administered the same chemicals do not accumulate low molecular weight protein in the kidney, and they do not develop renal tubule tumors. Because humans appear to be more like other laboratory animals than like the male rat, in this special situation, the male rat is not a good model for assessing human risk. The Forum report stresses the need for full scrutiny of a substantial set of data to determine when it is reasonable to presume that renal tumors in male rats are linked to a process involving alpha 2u-g accumulation and to select appropriate procedures for estimating human risks under such circumstances.

Functional endothelin ET B receptors on renal papillary tubules

Addition of endothelin-1 or endothelin-3 to rat renal papillary tubules produced a dose-dependent inhibition of the cAMP response to vasopressin stimulation. The average EC 50 values were 1.1 ± 0.6 and 2.6 ± 1.1 nM, respectively, indicating mediation by an endothelin ET B receptor. Phorbol myristate acetate (1 μM) also inhibited the vasopressin-cAMP response and this inhibition was not additive with that to endothelin, indicating that the endothelin inhibition is mediated by activation of protein kinase C. These findings demonstrate functionally relevant endothelin ET B receptors on renal papillary tubules. Such receptors are a possible target for endothelin-3 produced within the kidney.

The Effect of Dopamine on Adenylate Cyclase and Na+, K+-ATPase Activity in the Developing Rat Renal Cortical and Medullary Tubule Cells

Dopamine has an age-dependent natriuretic and diuretic effect. We have investigated the ontogeny of the dopamine response on adenylate cyclase activity and Na+,K(+)-ATPase activity in two different cell populations in the infant (10-d-old) and the adult (40-d-old) rat kidney. Basal- and forskolin-stimulated adenylate cyclase activity in tubular suspensions of renal cortex was 5.4-fold (p < 0.05) higher in the 10-d-old rats than in the 40-d-old rats but unchanged between the ages in a suspension of medullary tubules. The dopamine-1-specific agonist fenoldopam did not stimulate adenylate cyclase activity in the cortical cells from 10-d-old rats but did stimulate activity 51 +/- 16% (p < 0.05) in the 40-d-old rats. In the medullary suspension, fenoldopam stimulated adenylate cyclase activity by 43.5 +/- 5% (p < 0.001) in the 10-d-old rats and by 32.0 +/- 7% (p < 0.01) in the 40-d-old rats. In the isolated proximal convoluted tubule, dopamine inhibited Na+,K(+)-ATPase activity in both the 10-d-old (34 +/- 3%, p < 0.001) and 40-d-old rats (44 +/- 7%, p < 0.001). In contrast, in the medullary thick ascending limb of Henle, inhibition of Na+,K(+)-ATPase activity by fenoldopam was more pronounced in the 10-d-old (56 +/- 6%, p < 0.001) than in the 40-d-old rat (33 +/- 6%, p < 0.001). In summary, the renal tubular effects of dopamine on adenylate cyclase and Na+,K(+)-ATPase activity change during postnatal development in a cell-specific manner.

Lyophilization can generate artifacts in chromatographic profiles of inositol phosphates

Lyophilized extracts of [ 3H]inositol-labelled rat heart or renal tubule preparations were found to contain unidentified 3H-labelled compounds in addition to the inositol phosphates. The appearance of these labelled substances was caused by the presence in the extracts of compounds which bound [ 3H]inositol when lyophilized together with it. These studies demonstrate a previously undescribed source of [ 3H]inositol-labelled compounds which can complicate chromatographic profiles of inositol phosphates. These problems can be overcome either by not lyophilizing the samples or by lyophilizing in the presence of 0.3 M urea, which prevents the association with [ 3H]inositol and does not interfere with the chromatography.

Cytokine-induced expression of a nitric oxide synthase in rat renal tubule cells.

Nitric oxide (NO.) has been implicated in the regulation of renal vascular tone and tubular sodium transport. While the endothelial cell is a well known source of NO(.), recent studies suggest that tubular epithelial cells may constitutively generate NO(.). An inducible isoform of nitric oxide synthase which produces far greater quantities of NO. exists in some cell types. We sought to determine whether kidney epithelial cells exposed to cytokines could express an inducible nitric oxide synthase. Primary cultures of rat proximal tubule and inner medullary collecting duct cells generated NO. on exposure to TNF-alpha and IFN-gamma. NO. production by both cell types was inhibited by NG-monomethyl-L-arginine; this inhibition was partially reversed by the addition of excess L-arginine. Stimulation of kidney epithelial cells with TNF-alpha and IFN-gamma dramatically increased the level of inducible nitric oxide synthase mRNA. In summary, renal proximal tubule and inner medullary collecting duct cells can produce NO. via expression of an inducible isoform of nitric oxide synthase.

Endothelin-1 and -3 binding to ETB receptors in rat renal tubules.

To increase understanding of endothelin (ET) function in the kidney, we investigated binding of the radioligand of endothelin isopeptides to microdissected rat nephron segments. Specific ET-1 binding was highest in the inner medullary collecting duct, whereas the cortical and outer medullary collecting ducts showed moderate binding, as did the glomeruli. There was slight ET-1 binding to the early portion of the proximal tubule. Other nephron segments displayed little ET-1 binding. The binding profile of ET-3 along the nephron markedly resembled that of ET-1. Scatchard analyses of ET-1 and ET-3 binding to cortical collecting ducts revealed a single class of receptor for both ET-1 and ET-3. Displacement of [125I]-ET-1 binding by unlabeled ET-3 was similar to that produced by unlabeled ET-1. Moreover, a specific ETB agonist, BQ-3020, almost completely inhibited [125I]-ET-1 binding in cortical collecting ducts, whereas a specific ETA antagonist, BQ-123, had little effect. These data indicate that cortical collecting ducts express ETB receptors, to which both ET-1 and ET-3 bind equally.

Effects of inhibitors of gluconeogenesis on weak organic acid uptake in rat renal tubules

Using inhibitors of gluconeogenesis (phenylpyruvate, α-cyano-4-hydroxycinnamate, quinolinate, d-malate, aminooxyacetate), we analysed mechanisms by which the gluconeogenic substrates, lactate and pyruvate, as well as a short-chain fatty acid, acetate, stimulate the uptake of a weak organic acid, fluorescein, in the rat kidney. We have shown that these inhibitors modified both the rate of glucose production from lactate and pyruvate in the renal cortex fragment suspension and the stimulatory effects of the metabolic substrates on fluorescein uptake in superficial proximal tubules in the renal cortex slices. The peculiarities of the effects of lactate and pyruvate on the uptake were correlated with the partial divergence of the pathways of gluconeogenesis from these precursors. The linkage of the weak organic acid uptake with gluconeogenesis is interpreted in terms of the hypothesis that the uptake is controlled by the cytoplasmic pyridine nucleotide redox potential, which is maintained with the participation of certain processes involved in glucose synthesis.

Detection of endothelin-1 mRNA by RT-PCR in isolated rat renal tubules

A combination of reverse transcription and polymerase chain reaction was utilized to detect baseline endothelin-1 (ET-1) mRNA expressed in renal tubules dissected from rats. 5′ and 3′ primers were constructed according to human ET-1 genomic DNA. Rat ET-1 mRNA was found to be expressed only in cortical through medullary collecting ducts in addition to glomeruli. Sites for tubular synthesis of ET-1 corroborate well with major ET-1 binding sites along the nephron, indicating autocrine/paracrine role of ET-1 in the renal tubules and supporting a prevailing concept on such function of ET-1 in many differing tissues.

Physiological basis for an animal model of the renal Fanconi syndrome: use of succinylacetone in the rat.

1. The biochemical basis for the human renal Fanconi syndrome, including glucosuria, phosphaturia and aminoaciduria, remains enigmatic. This is due, in part, to the lack of an appropriate animal model. Since there is an association between the human genetic disease hereditary tyrosinaemia, for which urinary excretion of the compound succinylacetone constitutes a biochemical marker, and a renal Fanconi syndrome, we have examined the relationship between succinylacetone and renal tubular function in the rat. 2. Intraperitoneal injection of succinylacetone for 3 consecutive days into adult male Sprague-Dawley rats resulted in succinylacetone plasma concentration of 3 mmol/l. This concentration was associated with glucosuria, aminoaciduria, polyuria, reduced renal phosphate reabsorption and normal creatinine clearance. In addition, urinary porphobilinogen and total porphyrin excretions were markedly reduced. In animals permitted to recover for 7 days after succinylacetone administration, these renal functional changes remitted partially or completely. Ultrastructural examination of the kidneys after the 3 days of treatment showed no fine structural changes. 3. We conclude that the physiological alterations produced in normal rat renal tubules by succinylacetone provide the basis for the study of the biochemical changes underlying the human renal Fanconi syndrome.

Age-related alterations in calbindin-D28K induction by 1,25-dihydroxyvitamin D3 in primary cultures of rat renal tubule cells.

In vivo studies have indicated that renal calbindin-D28K protein and mRNA levels decrease in adult and old rats, and this decrease parallels the age-associated decline in serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels. However, diminished renal responsiveness to 1,25-(OH)2D3 with advancing age could also contribute to decreased calbindin-D28K expression. To study renal responsiveness with age, primary cell cultures were established from the kidney cortices of young (1 month old), adult (10-12 months old), and old (20-24 months old) rats. Cells were incubated in medium K-1 containing 2% fetal calf serum. Calbindin-D28K protein levels were determined by Western blot and enzyme-linked immunosorbent assay. In young animals, the levels of calbindin-D28K declined from 12.1 +/- 1.3 micrograms/mg protein in the intact kidney to 1.6 +/- 0.07 micrograms/mg protein in cells that had been cultured for 3 days in the absence of 1,25-(OH)2D3. This sharp decline in calbindin-D28K protein concentration moderated by days 6-8. The continuous presence of 10(-7) M 1,25-(OH)2D3 in the medium did not abolish the decline. The low levels of calbindin-D28K in the cells cultured in the absence of 1,25-(OH)2D3 provided an excellent experimental system in which to compare the response of the cells to 1,25-(OH)2D3 between age groups. In cultured cells treated with 1,25-(OH)2D3 for 72 h, calbindin-D28K induction was greater in cells from adult and old animals compared to cells from young animals. The ratios of calbindin-D28K content (with vitamin D/without vitamin D) were 2.2 +/- 0.2, 4.7 +/- 0.5, and 7.1 +/- 1.5 for young, adult, and old cells, respectively. These studies suggested that the observed in vivo decrease in renal calbindin-D28K with age is primarily due to the lowered circulating 1,25-(OH)2D3.

Age-related alterations in calbindin-D28K induction by 1,25- dihydroxyvitamin D3 in primary cultures of rat renal tubule cells

Age-related alterations in calbindin-D28K induction by 1,25- dihydroxyvitamin D3 in primary cultures of rat renal tubule cells

Activation of protein kinase C modulates dihydroxycholecalciferol synthesis in rat renal tubules

1,25(OH) 2D 3, the biologically active metabolite of vitamin D, is produced from 25(OH)D 3, by the renal mitochondrial 25(OH)D 3 1α-hydroxylase. Several studies have implicated reversible phosphorylation and a possible role for protein kinase C (PKC) in acute regulation of 1,25(OH) 2D 3 production. In the experiments described here, we studied 1,25(OH) 2D 3 production in freshly isolated rat renal tubules treated with activators and inhibitors of PKC. In this mammalian system, TPA, but not its inactive analogue 4αPDD, inhibited 1,25(OH) 2D 3 production in a dose-dependent fashion within 20 min. The acute inhibition of 1,25(OH) 2D 3 production by TPA exposure was preceded by an increase in membrane associated PKC activity, which was paralleled by a decrease in cytosolic PKC activity. Pre-incubation of tubules with staurosporine, a PKC inhibitor, abolished the inhibitory effect of TPA on 1,25(OH) 2D 3 production. Chronic (18 h) exposure of tubules to high dose TPA resulted in down regulation of both membrane and cytosolic PKC activity and re-exposure to TPA did not affect PKC translocation or 1,25(OH) 2D 3 production in down regulated tubules. Our data strongly suggest that modulation of renal PKC activity may be an important mechanism for acute regulation of 1,25(OH) 2D 3 production.

Interaction between vasopressin and endothelin in renal papillary tubules: uncoupling following cell isolation and culture.

1. In freshly prepared rat renal papillary tubules, endothelin-related peptides inhibited the vasopressin-stimulated accumulation of cAMP. No inhibition was observed when tubules were cultured overnight ex vivo. 2. Endothelin-1, endothelin-3 and sarafatoxin S6b had similar potencies as inhibitors of cAMP accumulation, indicating an endothelin (ETb) receptor. 3. These results demonstrate an interaction between ETb receptors and vasopressin receptors at the level of their signal transduction pathways, and show that this relationship is lost following cell culture.