Sertoli cells, proximal convoluted tubules in the kidney, and neurons in the brain contain cyclic protein-2.

We analyze by immunocytochemistry the in vivo distribution in rat Sertoli cells of Cyclic Protein-2 (CP-2), which is maximally synthesized and secreted in vitro at stages VI and VII of the cycle of the seminiferous epithelium. This analysis demonstrates that CP-2 staining is strongest in Sertoli cells in stage VI and VII tubules. Additionally, we demonstrate that the staining for CP-2 within a stage VII tubule differs from the staining of another Sertoli cell secretory product, androgen-binding protein. CP-2 is not detected by immunocytochemistry in any other tissues of the reproductive tract, though immunoblot analysis demonstrates the presence of CP-2 in rete testis and epididymal fluids. CP-2 was immunocytochemically detected in only three other organs: the kidney, the brain (with greatest concentration in the supraoptic and paraventricular nuclei), and the posterior pituitary. The presence of CP-2 in the kidney was confirmed by metabolic radiolabeling, immunoprecipitation, and peptide analysis. The presence of CP-2 in the brain was confirmed by immunoblot analysis of radioinert protein immunoprecipitated from the anterior hypothalamus.

Detection of specific mRNAs in single nephron segments by use of the polymerase chain reaction.

We have developed a procedure to detect specific mRNAs in single renal nephron segments. This approach combines microdissection, reverse transcription (RT) of the target mRNA, and amplification of the resulting cDNA using the polymerase chain reaction (PCR). After microdissection, the sample is placed in a tube where it is permeabilized and where all reactions are performed directly without the need for isolation of the RNA. Our model target was the mRNA for aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol. Its expression is modulated by changes in extracellular osmolality in the renal medulla. RT-PCR of inner medullary collecting duct (1 mm) and glomeruli (6-10) yielded a product of the predicted length (670 base pairs) defined by the PCR primers. Its identity was confirmed by a specific oligonucleotide probe that differed from the primers. RT-PCR of proximal tubules (1 mm) resulted in no aldose reductase-specific amplification product. RT-PCR is generally applicable for measuring specific gene expression in single nephron segments or small numbers of cultured cells. Utility, limitations, and refinements of this approach are discussed.

In situ hybridization evidence for angiotensinogen messenger RNA in the rat proximal tubule. An hypothesis for the intrarenal renin angiotensin system.

We examined angiotensinogen gene expression in rat kidney by in situ hybridization histochemistry. Using a rat cRNA probe to angiotensinogen, we demonstrated angiotensinogen mRNA to be localized predominantly in the proximal renal tubule, with considerably lesser amounts in distal tubular segments and glomerular tufts. Previous studies have localized renin immunoreactivity to the juxtaglomerular cells, glomerular tufts, and proximal tubules. Such findings provide further evidence for a local tissue renin angiotensin system within the kidney which may influence regional function. Based on our data, we hypothesize that a major site of angiotensin production is the proximal tubule. We postulate that angiotensin synthesized in and/or around the proximal tubule may directly modulate tubular transport of sodium, bicarbonate, and water. In addition to the proximal tubule, the specific localization of the renin angiotensin components elsewhere in the kidney would also support the other proposed regional functions of the intrarenal system, including modulation of tubuloglomerular balance.

Imidazoline-guanidinium receptive site in renal proximal tubule: asymmetric distribution, regulation by cations and interaction with an endogenous clonidine displacing substance.

In the present report we have used [3H]idazoxan to characterize the rabbit renal imidazoline preferring site by defining its plasmalemma distribution, its regulation by cations and the type of interaction with the clonidine displacing substance (CDS), a putative endogenous ligand for the imidazoline receptor. The density of [3H]idazoxan binding sites was 12-fold higher in purified basolateral membranes than in brush-border membranes (maximal binding activity, 566 +/- 118 vs. 46 +/- 2 fmol/mg of protein). In basolateral membranes, [3H]idazoxan binding was inhibited not only by imidazoline compounds but also by guanidinium analogs such as guanabenz, amiloride, 5-(M-ethyl-N-isopropyl)amiloride and phenamylamiloride. Amiloride had no effect on the dissociation rate of [3H]idazoxan, suggesting a direct interaction of this molecule with the ligand binding site. [3H]Idazoxan binding was 80% inhibited by 150 mM K+ or Rb+. The effect of K+ appeared to occur through the interaction with an allosteric site in as much as both the apparent dissociation constant and the dissociation rate of [3H]idazoxan were increased in the presence of 75 mM K+. CDS inhibited [3H]idazoxan binding with a half-maximal effective concentration of 2 U/250 microliters. The competitive nature of CDS effect was indicated by the increase in the apparent dissociation constant of [3H]idazoxan (Kd from 3 +/- 0.3 to 8.5 +/- 0.2 nM, P less than .01) in the presence of CDS. In conclusion, our findings showed that the imidazoline-guanidinium receptive site is located mainly in the basolateral side of the tubular cell, recognizes CDS and is regulated by K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical study of a rat membrane protein which induces a selective potassium permeation: its localization in the apical membrane portion of epithelial cells.

We previously reported a novel rat membrane protein that exhibits a voltage-dependent potassium channel activity on the basis of molecular cloning combined with an electrophysiological assay. This protein, termed IsK protein, is small and different from the conventional potassium channel proteins but induces selective permeation of potassium ions on its expression in Xenopus oocytes. In this investigation, we examined cellular localization of rat IsK protein by preparing three different types of antibody that specifically reacts with a distinct part of rat IsK protein. Immunohistochemical analysis using these antibody preparations demonstrated that rat IsK protein is confined to the apical membrane portion of epithelial cells in the proximal tubule of the kidney, the submandibular duct and the uterine endometrium. The observed tissue distribution of rat IsK protein was consistent with that of the IsK protein mRNA determined by blot hybridization analysis. In epithelial cells, the sodium, potassium-ATPase pump in the basolateral membrane generates a sodium gradient across the epithelial cell and allows sodium ions to enter the cell through the apical membrane. Thus, taking into account the cellular localization of the IsK protein, together with its electrophysiological properties, we discussed a possible function of the IsK protein, namely that this protein is involved in potassium permeation in the apical membrane of epithelial cells through the depolarizing effect of sodium entry.

Effect of dietary taurine on renal taurine transport by proximal tubule brush border membrane vesicles in the kitten.

Renal adaptation of the kitten to altered dietary taurine intake was assessed using proximal tubule brush border membrane (BBM) vesicles. Three groups of kittens were adapted to purified diets containing 43.5% soy protein that were either taurine-free (OT) or contained 0.15% taurine (NT) or 1.0% taurine (HT). The plasma taurine concentration of the kittens fed OT decreased from 104 +/- 25 microM to 16 +/- 5 microM and 1.7 +/- 0.5 microM in 1 and 6 wk, respectively. Feeding HT increased plasma taurine concentration to 350 +/- 116 microM in 1 wk. Compared to NT, taurine accumulation by BBM vesicles was significantly elevated after 4 wk of feeding OT and decreased after 2 wk or less of feeding HT (P less than 0.05). Maximum renal adaptation occurred by 6 wk of feeding OT (206% increase in taurine uptake/15 s compared to NT) and by 2 wk or less of feeding HT (43% decrease in taurine uptake/15 s compared to NT). Evaluation of transport kinetics using renal cortex from groups of four kittens (16 determinations) fed NT, OT (12 wk) or HT (10 wk) revealed a Vmax of 55 +/- 10, 123 +/- 24 or 39 +/- 7 pmol.mg protein-1.10 s-1 and a Km of 32 +/- 7, 16 +/- 2 or 37 +/- 8 microM, respectively. The differences in Vmax and Km were significant between NT and OT (P less than 0.05), but not significant between NT and HT (P greater than 0.05). Our results suggest that renal adaptation of the kitten to changes in dietary taurine occurs with modifications of both Vmax and Km of the taurine transport system.

Silver enhancement of tissue mercury: demonstration of mercury in autometallographic silver grains from rat kidneys.

The autometallographic silver enhancement method has been applied increasingly to detect trace amounts of mercury in preparations of biological tissue. It has, however, been difficult to establish the presence of a core of mercury within the silver grain by direct methods such as energy dispersive X-ray analysis. In the present work, a sample of autometallographic silver grains was prepared from kidneys of rats exposed to mercury in the drinking water. Frozen sections from the kidneys were silver-enhanced and subsequently all organic material was removed by enzymatic digestion. The remaining pellet of silver grains was analyzed by proton-induced X-ray emission (PIXE) and mercury was demonstrated in an amount of 0.1-0.5% compared to silver. In addition, it was demonstrated that two pools of catalytic mercury compounds exist, probably corresponding to sulfide- and selenium-bound mercury.

A new double-barrelled, ionophore-based microelectrode for chloride ions.

A new Cl- selective microelectrode based on the ionophore 5,10,15,20-tetraphenyl-21H,23H-porphin manganese(III) chloride is presented which discriminates better against HCO3- and several organic anions than electrodes containing the Corning 477913 ion-exchanger. Using a redesigned construction procedure, fine-tip double-barrelled microelectrodes were produced which had slopes of -52.4 +/- 0.6 mV (SE, n = 24), resistances of about 7.10(11) omega and a selectivity coefficient log KpotCLHCO3 of -1.40 +/- 0.03. Some electrodes showed a small unexplained sensitivity to pH greater than 7.6. When used to puncture cells of isolated S3 segments of rabbit renal proximal tubule during perfusion with HCO3- Ringer solution, the electrodes gave a membrane potential of -69.8 +/- 1.5 mV and an intracellular Cl- activity, [Cl-]i, of 35.3 +/- 2.6 mmol/l. Upon switching bath and lumen perfusions to Cl- -free solutions the "residual" [Cl-]i dropped to 1.20 +/- 0.03 mmol/l, while in similar measurements with ion-exchanger electrodes the "residual" [Cl-]i dropped only to 10.9 +/- 0.5 mmol/l. These observations demonstrate the superiority of the new electrode and prove that previously determined high [Cl-]i values in Cl- -free ambient solutions reflect interference problems rather than non-exchangeable intracellular chloride.

Primary cultures of rabbit renal proximal tubule cells: I. Growth and biochemical characteristics.

Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular dome formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme, N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture, respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytotoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/micrograms DNA in preconfluent cultures to 0.059 mg protein/micrograms DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology.

Relationship between cell adenosine triphosphate and glutathione content and protection by glycine against hypoxic proximal tubule cell injury.

We designed studies to characterize metabolic aspects of the protective effects of glycine and glutathione against hypoxic proximal tubule cell injury b clarifying the relationship between protection and preservation of tubule cell adenosine triphosphate (ATP) and glutathione levels. The tubule preparation was glutatione depleted as isolated although some recovery occurred during incubation at 37 degrees C, and this recovery was enhanced by treatment with glutatione precursors. Increasing the duration of hypoxia from 30 minutes to 60 minutes produced increasingly extensive lethal tubule cell injury that was almost completely prevented, even at the 60-minute duration, by inclusion of either 2 mmol/L glutathione or 2 mmol/L glycine in the tubule incubation medium. Cell ATP levels decreased to the same extent and at the same rate in protected and unprotected hypoxic tubules. Glycine- and glutathione-protected tubules maintained higher cell glutathione levels than unprotected tubules at all durations of hypoxia studied. However, completely eliminating this increment of glutathione with either the gamma-glutamylcysteine synthetase inhibitor, buthionine sulfoximine, or the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1-nitrosourea, did not prevent protection. The data indicate that the striking protection against hypoxic injury to the isolated tubules provided by treatment with glycine or glutathione is independent of preservation of tubule cell ATP and glutathione levels, to the extent that difference of these levels can be discriminated in intact cells with present methods.

B-cell markers in the human kidney.

This report describes the reactivity of a panel of 89 monoclonal antibodies with the normal human kidney. The antibodies were all submitted to the B-cell panel of the Third Leukocyte Differentiation Workshop. Several different patterns of staining of renal structures were identified. Most antibodies clustered in defined CD groups showed the same pattern of reactivity in the kidney, but some, notably CD19 antibodies, showed heterogeneity of binding to the kidney. These findings are important for the use of monoclonal antibodies to define phenotype in cell culture and immunohistology experiments.

Intact teratogenic immunoglobulins may reach the rat embryo.

It is known that heterologous antiserum against rat kidney homogenate may induce congenital malformations when injected into pregnant rats during the period of organogenesis. Teratogenic rabbit antibodies against a purified rat renal tubular glycoprotein were isolated, labelled with 125I and injected into pregnant rats on the 10th day of gestation. Extracts of visceral yolk-sacs (VYS) and embryos were obtained 16 h later and chromatographed separately on a Sephacryl S-300 gel filtration column. The resultant chromatograms showed several radioactive peaks, one of which coincided with the eluate of intact rabbit immunoglobulins G (IgG). We interpret the result as an indication that some undigested intact teratogenic IgG were present in VYS and the embryo.

Free cytosolic calcium in renal proximal tubules from the spontaneously hypertensive rat.

Free intracellular calcium was measured in renal proximal tubules obtained from spontaneously hypertensive rats (SHR) and from age-matched Wistar-Kyoto rats (WKY) ingesting a normal diet. Experiments were performed on renal proximal tubule suspensions using fura-2 to monitor cytosolic calcium. In 4-week-old rats, when systolic blood pressure was not significantly different between the two groups, renal proximal tubule cytosolic calcium was similar (143 +/- 28 and 144 +/- 15 nM, respectively). By the age of 5 weeks, cytosolic calcium increased significantly in both SHR and WKY (214 +/- 24 and 262 +/- 34 nM, respectively, p less than 0.05). Calcium, however, was not significantly different between the two groups, even though at this age blood pressure was higher in SHR than in WKY. As compared with values in 4-week-old rats, cytosolic calcium was also found increased in tubules from both SHR and WKY aged 10 to 12 weeks (261 +/- 42 and 279 +/- 30 nM, respectively) and 20 to 24 weeks (263 +/- 42 and 308 +/- 28 nM, respectively). However, no significant differences in cytosolic calcium were found between SHR and WKY even though at these ages systolic blood pressure increased markedly in the SHR. Moreover, regression analysis failed to reveal a correlation between cytosolic calcium and blood pressure when data from either group of rats of all ages studied were pooled. Exposure to ouabain (10(-3) M) to inhibit Na+,K+-adenosine triphosphatase and increase intracellular sodium had no significant effect on cytosolic calcium in tubules from either SHR or WKY (260 +/- 69 and 250 +/- 45 nM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and characteristics of a novel human osteosarcoma-associated cell surface antigen.

The characteristics of a new osteosarcoma-associated cell surface antigen were studied by means of two murine monoclonal antibodies, TP-1 and TP-3, which were found to bind to two different epitopes on the same antigen, a monomeric polypeptide with a molecular weight of approximately 80,000. Immunohistochemical studies showed that the antigen was present in all osteogenic sarcomas tested, in most cases of malignant fibrous histiocytoma, in two malignant hemangiopericytomas and in a few synovial sarcomas, but not in other main groups of sarcomas and nonsarcomatous malignancies. In normal tissues it was detected only in clusters of cells in the adrenal medulla and in proximal kidney tubules. Also endothelial cells in proliferating capillaries in placenta and in most tumors were stained. The antigen was absent in resting but present in actively proliferating osteoblastic cells. The epitopes were resistant to proteolytic and sugar-cleaving enzymes but sensitive to high temperatures and could not be detected in paraffin-embedded specimens. The tissue distribution and properties of the antigen show that it is different from the sarcoma-associated antigens previously studied. In contrast to previous findings with three other anti-sarcoma monoclonal antibodies, no correlation was found between serum alkaline phosphatase activity and the amount of TP-binding substances in the same sera. Nevertheless, an apparently complex association between alkaline phosphatase and the TP-binding antigen seems to exist. Thus, the Mr 80,000 antigen extracted from an osteosarcoma cell line showed enzyme activity, whereas TP-binding molecules precipitated from patient sera contained alkaline phosphatase activity only in a few of the cases studied. Altogether our data suggest that the antigen defined by the TP antibodies may be a marker of osteoblastic differentiation. The pattern of antigen expression in malignant tumors is unique, inasmuch as the antigen is found selectively in sarcomas and in all 31 osteosarcomas tested.

Comparison of the toxicity of the radiocontrast agents, iopamidol and diatrizoate, to rabbit renal proximal tubule cells in vitro.

Radiographic contrast agent-induced acute renal failure is an increasingly recognized clinical event. Multiple factors have been implicated in its development. Recent experiments have demonstrated that sodium diatrizoate, a common ionic radiocontrast agent, is moderately toxic to proximal tubule cells in vitro, and that this toxicity is enhanced by hypoxia. In this study, we compare toxicities of the nonionic radiocontrast agent, iopamidol, and the commonly used ionic contrast agent, diatrizoate. Suspensions enriched in proximal tubule segments were exposed for 82.5 min to 10 or 25 mM diatrizoate or 10 or 25 mM iopamidol with or without 22.5 min or 30 min of hypoxia. Cell viability parameters, including basal and uncoupled respiratory rates, tubule cell potassium and calcium levels and cell ATP content were measured. No consistent differences in tubule viability parameters were observed between tubule suspensions exposed to 10 mM concentrations of the radiocontrast agents during either oxygenated or hypoxic conditions. Under oxygenated conditions, both 25 mM iopamidol and diatrizoate exposure produced greater metabolic alterations in renal tubules than control conditions, but the effects were not statistically significant. With concomitant hypoxia, the alterations after 25 mM diatrizoate exposure were significantly greater than those seen after exposure to 25 mM iopamidol. Iopamidol had less of a detrimental effect on renal tubule potassium content and both basal and uncoupled respiratory rates than that of diatrizoate under these conditions. Thus, diatrizoate is more toxic to rabbit renal proximal tubule cells than iopamidol in vitro, and this difference in toxicity is enhanced by hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical detection of cytochrome P450C-M/F and NADPH-cytochrome P450 reductase in rat liver and kidney.

The localization and distribution of NADPH-cytochrome P450 reductase and cytochrome P450C-M/F were investigated immunohistochemically in the liver and the kidney of untreated rats employing both an unlabelled antibody peroxidase-antiperoxidase method and a peroxidase labelled primary antibody technique. In both immunohistochemical procedures, the reductase and P450C-M/F were detected in hepatocytes throughout the liver. In contrast, the reductase and P450C-M/F in the kidney were only detectable in the proximal tubule cells.

Epinephrine and norepinephrine enhance p-aminohippurate transport into basolateral membrane vesicles.

The effect of exogenous l-norepinephrine (NE) and l-epinephrine (EP) on transmembrane transport of p-aminohippurate (PAH) was studied in rat proximal tubular basolateral membrane vesicles. A gradient of 50 mM Na+ (out greater than in) and preloading of vesicles with unlabeled PAH were utilized to promote the influx of [3H]PAH into the vesicles. At final concentrations of 1 microM, NE and EP each produced significant elevations in vesicle uptake of [3H]PAH. The enhancement of PAH transport by NE or EP was inhibited by either phentolamine (100 microM) or yohimbine (100 microM). Prazosin (100 microM) or atenolol (100 microM) were unable to inhibit the response to NE. Similarly, prazosin or propranolol (100 microM) were unable to inhibit the response to EP. Clonidine (1 microM) also produced a significant elevation of PAH uptake, an effect inhibited by both phentolamine and yohimbine. Basolateral Na+-K+-adenosine triphosphatase activity also was increased significantly by either NE or EP (1 microM). Both agonists produced significant elevations of PAH uptake into vesicles preloaded with ATP. However, in the absence of NE or EP, PAH uptake into ATP-loaded vesicles was not significantly greater than into control vesicles. It was concluded that NE and EP enhance Na+-coupled PAH transport and that this effect may be mediated by alpha-2 adrenergic receptors. Activation of Na+-K+-adenosine triphosphatase is a possible mechanism whereby adrenergic agonists may exert effects on Na+-coupled transport across the basolateral membrane.

Selective intracellular beryllium localization in rat tissue by mass-resolved ion microprobe imaging.

Beryllium absorption sites in the kidney and liver of rats have been located and imaged at approximately 70 nm lateral resolution with a scanning ion microprobe utilizing secondary ion mass spectrometry. Embedded sections and lyophilized cryosections of these organs were prepared after in vivo administration of beryllium in soluble form. Beryllium distribution images were correlated with the histological microstructure revealed by CN- images. In the kidney, beryllium concentrates selectively within the nuclei of proximal tubule cells and occasionally within modified podocytes or mesangial cells in the glomerulus. In the liver, beryllium is seen to localize within severely altered lysosomal structures as well as within hepatocyte nuclei. These observations are relevant to understanding aspects of the toxic and carcinogenic properties of absorbed beryllium compounds.

Luminal secretion of ammonia in the mouse proximal tubule perfused in vitro.

A major portion of the total ammonia (tNH3 = NH3 + NH+4) produced by the isolated perfused mouse proximal tubule is secreted into the luminal fluid. To assess the role of Na+-H+ exchange in net tNH3 secretion, rates of net tNH3 secretion and tNH3 production were measured in proximal tubule segments perfused with control pH 7.4 Krebs-Ringer bicarbonate (KRB) buffer or with modified KRB buffers containing 10 mM sodium and 0.1 mM amiloride. Net tNH3 secretion was inhibited by 90% in proximal tubule segments perfused with the pH 7.4 modified KRB buffer while tNH3 production remained unaffected. The inhibition of net tNH3 secretion by perfusion with the modified KRB buffer was only partially reversed by acidifying the modified KRB luminal perfusate from 7.4 to as low as 6.2. These data indicate that the Na+-H+ exchanger facilitates a major portion of net tNH3 secretion by the proximal tubule and that luminal acidification may play only a partial role in the mechanism by which the Na+-H+ exchanger mediates net tNH3 secretion.