Localization of thyroxine 5'-monodeiodinase activity in the renal proximal tubules in rabbits and rats

To determine the localization of T4 5'-monodeiodinase activity in rabbit and rat nephron segments, the formation of tri-iodothyronine (T3) from thyroxine (T4) was measured in kidney homogenate and in isolated nephron segments obtained by the microdissection method. In order of decreasing activity, homogenates of rabbit renal cortex, outer medulla and inner medulla were capable of converting T4 to T3. In the isolated nephron segments of the rabbit cortex, the activities were noted in both proximal convoluted and proximal straight tubules. On the other hand, the activities were not detected in segments including the cortical thick ascending limb of Henle's loop, the distal convoluted tubule, the connecting tubule, and the cortical collecting tubule. It is concluded that both the convoluted and the straight tubules are the sites of T3 production in the kidney.     

Immunolocalization of C1-tetrahydrofolate synthase in the rat kidney

The distribution of the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) was examined in the rat kidney by immunolocalization with anti-C1-THF synthase serum using the peroxidase-antiperoxidase method. C1-THF synthase immunoreactivity was detected in both distal and proximal epithelial cells. Staining of the distal tubule epithelia was more intense and granular whereas staining of the proximal tubule epithelia was diffuse. All cells of the cortical collecting duct showed positive granular staining. In the outer medullary collecting duct, the intercalated cells showed intense granular cytoplasmic staining and the principal cells were either negative or weakly positive. The ascending thick limb of Henle's loop was also positive. Glomeruli and the inner medulla showed no staining for C1-THF synthase.     

A cytophotometric analysis of the activity of oxidative enzymes and Na, K-ATPASE in vertebrate nephrons at different levels of sodium transport in the kidney.]

Using quantitative cytochemistry, activities of Na, K-ATPase, succinate dehydrogenase (SDH) and alpha-keto-glutarate dehydrogenase (alpha-KDH) was investigated in cells of renal tubules at different levels of sodium reabsorption in the kidney. The activity of these enzymes in mammals and birds renal tubule cells was found to be higher than in the cells of corresponding renal tubules of cold-blooded vertebrates. This corresponds to the increased total amount of reabsorbed sodium in the kidney of warm-blooded animals. The summer frogs, as compared to the winter ones, exhibit higher activities of SDH and Na,K-ATPase in the proximal tubule cells where changes in sodium reabsorption are also noted. In the kidney of marine teleosts, a negative correlation between U/PNa and the activity of SDH and Na,K-ATPase in the cells of proximal and distal tubule was observed. Aldosterone was found to stimulate sodium reabsorption and to activate Na,K-ATPase.SDH and alpha-KDH mainly in the distal convoluted tubule. Furosemide was observed to inhibit sodium reabsorption and to reduce SDH and Na,K-ATPase activities in cells of the proximal tubule and Henle's loop. In the kidney of adrenalectomized rats, both sodium reabsorption and activities of Na,K-ATPase, SDH, alpha-KDH decreased in all the segments of the nephron. The data obtained suggest that changes in sodium reabsorption may be coupled with those in the activities of the investigated enzymes.     

Guanylyl cyclase-C receptor mRNA distribution along the rat nephron

Guanylin (GN) and uroguanylin (UGN) are two recently identified peptides that have been shown to affect water and electrolyte transport in both the intestine and the kidney. Mechanistically, the effects of both peptides are thought to be mediated by intracellular cGMP which results from ligand binding to a plasma membrane guanylyl cyclase-C (GC-C) receptor. To date, the specific intrarenal site(s) of GN and UGN action have not been established. To begin to address this issue, the present studies utilized semi-quantitative RT-PCR to assess the distribution of GC-C mRNA in specific microdissected segments of the rat nephron. GC-C mRNA expression was highest in the cortical collecting tubule, followed by the proximal convoluted tubule, medullary thick ascending limb and collecting tubule, and thin limbs of Henle's loop. Expression levels were significantly lower in all other segments tested, including the glomerulus. The renal tubular expression pattern for cGMP-dependent protein kinase II (cGK-II) mRNA, which is activated in response to GN/UGN-dependent cGMP accumulation, was similar to that for GC-C. Notably, both GN and UGN mRNAs were also expressed along the nephron. The highest levels of expression for both peptides were detected in the medullary collecting tubule. Lower, but comparable levels of GN and UGN expression also occurred in the cortical collecting tubule, cortical and medullary thick ascending limb, and thin limbs of Henles loop. In the proximal convoluted tubule, GN mRNA expression was also quite high, while UGN mRNA was almost undetectable. The presence of renal GC-C and cGK-II in the kidney are consistent with a proposed endocrine function for GN and UGN. In addition however, the present data suggest that intrarenally synthesized GN and UGN may also contribute to the regulation of renal tubular transport.     

The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule

Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues.     

Carbonic anhydrase in the monkey kidney

The distribution of carbonic anhydrase in the kidney of the cynomolgus monkey was studied by the histochemical method of Hansson. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed high enzyme activity at the brush border and the basolateral membranes and the cytoplasm. Straight proximal tubules were less intensely stained. In nephrons with long loops of Henle, the descending thin limb contained weak enzyme activity, whereas the ascending thin limb was inactive. The thick limb of Henle's loop displayed most enzyme activity at the luminal cell border. In distal convoluted tubules enzyme activity was restricted to the basal part of the cells. In the late distal tubule, intercalated cells appeared among the "ordinary" distal cells and contained abundant cytoplasmic enzyme. Many intensely stained intercalated cells were also found in the cortical and outer medullary segments of the collecting duct, intermingled with more weakly stained chief cells. In the inner medullary segment of the collecting duct, enzyme activity gradually disappeared. Many capillaries were clearly stained for enzyme activity. The capillary staining apparently varied with that of the kidney tubules; virtually all capillaries in the cortex, but very few in the inner medulla, were stained. The distribution of carbonic anhydrase in the kidney tubules of the monkey is very similar to that in man and in the rat, but the primate kidney differs from the rat kidney by the presence of capillary enzyme activity. The functional importance of this difference is not clear at present.     

Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney

Summary Tamm-Horsfall protein (THP) has been previously detected in cells of the thick ascending limb of Henle's loop (TAL) of different mammalian species using immunocytochemical methods. A nearly complete identity between THP and uromodulin, an immunosuppressive glycoprotein present in the urine of pregnant females, has been established recently. This paper describes the cellular location of THP mRNA by high-resolution in situ hybridization using a [³⁵S]-labeled human uromodulin cRNA (antisense-) probe of a length of 665 base pairs. Control experiments were performed using an mRNA (sense-) probe of the same length. The probe was hybridized to frozen sections of the rat kidney. THP mRNA distribution in the kidney was found to be homologous to the immunocytochemical labeling pattern: Autoradiographic signal was present along the entire length of the TAL including the post-macula segment which leads to the distal convoluted tubule. Tubular cells of the macula densa were negative. Labeling intensity of the TAL epithelium was found to increase from the origin of the TAL at the transition between inner and outer medulla to its end beyond the macula densa. Labeling of the medullary segment in the inner stripe was weak, whereas outer medullary and cortical segments very strongly expressed THP mRNA. The glomerulus, the portions of the nephron proximal to the TAL, the distal convoluted tubule as well as the collecting duct system were negative.     

Renal synthesis of epidermal growth factor is unchanged by vitamin D deficiency.

In mammalian kidney, epidermal growth factor (EGF) is produced as a small internal domain of an abundant high molecular weight peptide associated with the luminal membrane of the thick ascending limb of Henle's loop and distal convoluted tubule. At present, there is no evidence to indicate a mitogenic function for the EGF-containing molecule in kidney; consideration of its molecular structure suggests the possibility of a membrane-associated physiologic role. In this study, we examine regulation of renal EGF synthesis during induction of vitamin D deficiency in mice. Despite evidence of marked hyperparathyroidism, urinary excretion of EGF was equivalent in control (2.54 +/- 0.72 micrograms/mg creatinine) and vitamin D deficient (2.13 +/- 0.97 micrograms/mg creatinine) animals. Similarly, EGF mRNA levels in kidney were comparable in the two groups. These data indicate that parathyroid hormone has no effect on renal EGF regulation, although it is known to stimulate calcium reabsorption in distal nephron segments.     

Intrarenal localization of degradation of atrial natriuretic peptide in isolated glomeruli and cortical nephron segments

Using isolated glomeruli and nephron segments obtained from collagenase treated rabbit kidneys, we examined the in vitro degradation of alpha-human atrial natriuretic polypeptide (alpha-hANP). The ANP-degrading activity was measured by the amount of immunoreactive ANP remaining after incubation of about 50 fmoles alpha-hANP with each tissue preparation for 7.5 min. The sequence of degrading activity among isolated nephron segments was as follows: proximal straight tubule greater than proximal convoluted tubule greater than cortical collecting tubule greater than distal convoluted tubule greater than cortical thick ascending limb. A single glomerulus exhibited the degrading activity which was comparable to approximately 50% of the activity of 1 mm proximal convoluted tubule. Phosphoramidon, an inhibitor of endopeptidase, prevented the degradation of ANP in proximal convoluted tubule and glomerulus by 68% and 89%, respectively, but not in cortical thick ascending limb and cortical collecting tubule. From these results, we conclude that the degradation of ANP by endopeptidase occurs mainly in the proximal tubule and glomerulus.     

Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney

Tamm-Horsfall protein (THP) has been previously detected in cells of the thick ascending limb of Henle's loop (TAL) of different mammalian species using immunocytochemical methods. A nearly complete identity between THP and uromodulin, an immunosuppressive glycoprotein present in the urine of pregnant females, has been established recently. This paper describes the cellular location of THP mRNA by high-resolution in situ hybridization using a [35S]-labeled human uromodulin cRNA (antisense-) probe of a length of 665 base pairs. Control experiments were performed using an mRNA (sense-) probe of the same length. The probe was hybridized to frozen sections of the rat kidney. THP mRNA distribution in the kidney was found to be homologous to the immunocytochemical labeling pattern: Autoradiographic signal was present along the entire length of the TAL including the post-macula segment which leads to the distal convoluted tubule. Tubular cells of the macula densa were negative. Labeling intensity of the TAL epithelium was found to increase from the origin of the TAL at the transition between inner and outer medulla to its end beyond the macula densa. Labeling of the medullary segment in the inner stripe was weak, whereas outer medullary and cortical segments very strongly expressed THP mRNA. The glomerulus, the portions of the nephron proximal to the TAL, the distal convoluted tubule as well as the collecting duct system were negative.     

Benzodiazepine receptors along the nephron: [3H]PK 11195 binding in rat tubules

Binding of [3H]PK 11195, an isoquinoline carboxamide derivative, was measured in microdissected tubule segments of rat nephron. High specific binding capacities (1.1-1.8 fmol X mm-1) were found in the thick ascending limb of the Henle's loop and in the collecting tubule, whereas specific binding could not be detected in the proximal tubule. In the medullary collecting tubule, the association and dissociation rate constants at 4 degrees C were k1 = 3.0 X 10(6) M-1 X min-1 and k-1 = 0.021 min -1; the ratio k-1/k1 = 7.0 nM was in agreement with the estimated equilibrium dissociation constant (Kd = 2.4 nM). [3H]PK 11195 binding sites from medullary ascending limb and medullary collecting tubule revealed the following sequence of specificity: PK 11195 = Ro 5-4864 much greater than clonazepam, indicating that tubule binding sites might be the peripheral benzodiazepine receptors of the rat kidney.     

Polyol determination along the rat nephron

The polyols sorbitol and inositol were determined in single freshly microdissected tubule segments of rat kidney. Twenty different structures were separated from six different kidney zones reaching from cortex to papillary tip. Picomol amounts of sorbitol and inositol were quantitated by use of an enzymatic bioluminescence procedure. Experimental conditions (700 mosmol/kg, 4 degrees C) were chosen to assure constant polyol concentrations over 3 h dissection period. Sorbitol exhibited a concentration gradient in the collecting duct system from the outer/inner medullary border (3.9 +/- 0.5 pmol/mm) to the papillary tip (78.8 +/- 6.9 pmol/mm). In the same region descending and ascending limbs of Henle's loop contained 1.5 +/- 0.5 to 5.3 +/- 1.6 pmol/mm and 2.5 +/- 0.8 to 8.35 +/- 1.5 pmol/mm, respectively. In contrast, all outer medullary and cortical structures had lower sorbitol concentrations. Inositol amounts increased continuously in the collecting duct from cortex (5.3 +/- 0.5 pmol/mm) to inner medulla (30.7 +/- 3.8 pmol/mm). This polyol was also found in thick ascending limb of Henle's loop (6.2 +/- 1.1 pmol/mm in cortex to 11.2 +/- 1.4 pmol/mm in outer medulla) and in proximal tubules (5.6 +/- 1.2 pmol/mm in S1 and 4.5 +/- 1.5 pmol/mm in S3). When related to cellular volume measured by planimetry, intracellular sorbitol concentration was calculated to be 51 mmol/l in papillary collecting duct and inositol 28 mmol/l in outer medullary thick ascending limb cells. These data confirm the role of sorbitol in the renal concentrating process in papilla. Inositol seems to have additional function in thick ascending limb of Henle's loop and the proximal tubule.     

The transition of the thick ascending limb of Henle's loop into the distal convoluted tubule in the nephron of the rat kidney

Summary Examination of serial semithin sections of rat kidney cortex and a subsequent electron microscopic study of selected areas revealed that the characteristic epithelium of the cortical part of the thick ascending limb of Henle extends for a varying distance beyond the macula densa. The transition from the relatively thin epithelium of the thick ascending limb at this site to the three -or even four-fold thicker epithelium of the convoluted part of the distal tubule is sharply defined and occurs without the interposition of an intermediate cell type.The position of the macula densa at the end but still clearly within the ascending limb of Henle's loop is functionally interpreted to guarantee the separation of the sensor point macula densa from disturbing influences which might arise from the secretory activity of the subsequent tubular portion.Investigations supported by the Deutsche Forschungsgemeinschaft. The skillful technical assistance of Mrs. Saliha Sabanovic is gratefully acknowledged     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

Aminoglycosides inhibit hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells

The clinical use of aminoglycosides often leads to renal magnesium wasting and hypomagnesemia. Of the nephron segments, both the thick ascending limb of Henle's loop and the distal tubule play significant roles in renal magnesium conservation but the distal convoluted tubule exerts the final control of urinary excretion. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. Peptide hormones, such as parathyroid hormone (PTH), glucagon, calcitonin, and arginine vasopressin (AVP) stimulate Mg2+ uptake in MDCT cells that is modulated by extracellular polyvalent cations, Ca2+ and Mg2+. The present studies determined the effect of aminoglycosides on parathyroid hormone (PTH)-mediated cAMP formation and Mg2+ uptake in MDCT cells. Gentamicin, a prototypic aminoglycoside, elicited transient increases in intracellular Ca2+ from basal levels of 102 +/- 13 nM to 713 +/- 125 nM, suggesting a receptor-mediated response. In order to determine Mg2+ transport, MDCT cells were Mg(2+)-depleted by culturing in Mg(2+)-free media for 16 h and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.0 mM MgCl2. The mean rate of Mg2+ uptake, d([Mg2+]i)/dt, was 138 +/- 24 nM/s in control MDCT cells. Gentamicin (50 microM) did not affect basal Mg2+ uptake (105 +/- 29 nM/s), but inhibited PTH stimulated Mg2+ entry, decreasing it from 257 +/- 36 nM/s to 108 +/- 42 nM/s. This was associated with diminished PTH-stimulated cAMP formation, from 80 +/- 2.5 to 23 +/- 1 pmol/mg protein x 5 min. Other aminoglycosides such as tobramycin, streptomycin, and neomycin also inhibited PTH-stimulated Mg2+ entry and cAMP formation. As these antibiotics are positively charged, the data suggest that aminoglycosides act through an extracellular polyvalent cation-sensing receptor present in distal convoluted tubule cells. We infer from these studies that aminoglycosides inhibit hormone-stimulated Mg2+ absorption in the distal convoluted tubule that may contribute to the renal magnesium wasting frequently observed with the clinical use of these antibiotics.     

Proteinase-activated receptor 2 stimulates Na,K-ATPase and sodium reabsorption in native kidney epithelium

Proteinase-activated receptors 2 (PAR2) are expressed in kidney, but their function is mostly unknown. Since PAR2 control ion transport in several epithelia, we searched for an effect on sodium transport in the cortical thick ascending limb of Henle's loop, a nephron segment that avidly reabsorbs NaCl, and for its signaling. Activation of PAR2, by either trypsin or a specific agonist peptide, increased the maximal activity of Na,K-ATPase, its apparent affinity for sodium, the sodium permeability of the paracellular pathway, and the lumen-positive transepithelial voltage, featuring increased NaCl reabsorption. PAR2 activation induced calcium signaling and phosphorylation of ERK1,2. PAR2-induced stimulation of Na,K-ATPase Vmax was fully prevented by inhibition of phospholipase C, of changes in intracellular concentration of calcium, of classical protein kinases C, and of ERK1,2 phosphorylation. PAR2-induced increase in paracellular sodium permeability was mediated by the same signaling cascade. In contrast, increase in the apparent affinity of Na,K-ATPase for sodium, although dependent on phospholipase C, was independent of calcium signaling, was insensitive to inhibitors of classical protein kinases C and of ERK1,2 phosphorylation, but was fully prevented by the nonspecific protein kinase inhibitor staurosporine, as was the increase in transepithelial voltage. In conclusion, PAR2 increases sodium reabsorption in rat thick ascending limb of Henle's loop along both the transcellular and the paracellular pathway. PAR2 effects are mediated in part by a phospholipase C/protein kinase C/ERK1,2 cascade, which increases Na,K-ATPase maximal activity and the paracellular sodium permeability, and by a different phospholipase C-dependent, staurosporine-sensitive cascade that controls the sodium affinity of Na,K-ATPase.     

NAD+-15-hydroxyprostaglandin dehydrogenase distribution in rat kidney.

Rat kidney NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was measured in zones and substructure of the rat kidney nephron. This was accomplished utilizing an assay procedure based upon determining the amount of prostaglandin E1 present before and after the reaction with the 15-hydroxyprostaglandin dehydrogenase contained in the tissue sample. The enzyme activity was assayed in freeze dried, quick frozen rat kidney sections and its distribution within the rat kidney was determined. In kidney zones, it was localized to medullary rays and inner cortex. In kidney substructure, activity was highest in collecting tubule, pars recti tubule, distal convoluted tubule and the ascending limb of Henle (14.2, 11.5, 6.4 and 9.2 mM kg-1hr-1, respectively). Activity in glomeruli, proximal convoluted tubule and small arteries was lower (2.1, 2.8 and 2.1 mM kg-1hr-1, respectively). The assay procedure was verified by established assays (spectrophotometric, fluorometric and radiometric TLC) which are often used in homogenate and purified PGDH preparations.     

The age-related characteristics of the hormonal regulation of Na+, K(+)-ATPase activity in the kidney cortex of rats]

The aldosterone binding in isolated distal convoluted and cortical collecting tubules of renal nephrons and the influence of hormonal induction on the Na, K-ATPase activity in membrane fraction of kidney cortex were studied in 10-day- and 2-month-old rats. No reliable difference in aldosterone-specific binding was revealed (0.26 +/- 0.04 and 0.22 +/- 0.03 fmol/mm of tubule length, respectively, at the age of 10 days and 2 months). It was found that Na, K-ATPase activity increased with age from 0.39 +/- 0.06 to 0.72 +/- 0.10 mumol Pi/mg of protein.1 hour.100 microliters. Aldosterone induction caused approximately a 3-fold increase of the enzyme activity in both age groups comparing to the control level. Co-induction of aldosterone and spironolactone resulted in a 50% decrease of Na, K-ATPase activity in adult rats, but did not influence that in young rats. The revealed age-related differences in the mechanism of hormonal Na, K-ATPase regulation are supposed to underlie the absence of physiological reaction of the kidney to aldosterone in early postnatal ontogenesis.     

Glycocalyx heterogeneity of rat kidney urinary tubule: demonstration with a lectin-gold technique specific for sialic acid

The distribution of sialic acid residues in rat kidney urinary tubule was investigated by light and electron microscopy with a lectin-gold technique. The application of the sialic acid specific Limax flavus lectin resulted in intense plasma membrane labeling of the epithelium of the entire proximal tubule and thin limbs of loop of Henle. In contrast, the plasma membrane of the epithelium lining the medullary portion of the thick ascending limb of Henle was not labeled. In the cortical portion, however, microvilli-bearing positive and smooth-surfaced negative cells were present. Moreover, all cells of the convoluted distal tubules were labeled along their plasma membrane. These data demonstrate the existence of a gross difference in glycocalyx composition between proximal tubules and thin limbs of loop of Henle on one hand and thick ascending limbs on the other. In addition, fine heterogeneity in glycocalyx composition between medullary and cortical portion of thick ascending limb exists. It is concluded that the differences in sialic acid content of the glycocalyx may be related to the functional diversity exhibited by these tubular regions.