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PubMed1986

Effect of osmolarity on cation fluxes in medullary thick ascending limb cells.

The effects of a hypertonic extracellular medium on furosemide-sensitive Na and K fluxes were studied in isolated cells from the rabbit medullary thick ascending limb of Henle's loop (mTALH). In the control incubation medium, the furosemide-sensitive 22Na uptake was 379.1 +/- 24.4 pmol . mg protein-1 . min-1 and the furosemide-sensitive 86Rb uptake was 30.5 +/- 16.9. The furosemide-sensitive 22Na flux was not stimulated by K gradients directed into the cells, and, conversely, the furosemide-sensitive 86Rb flux was not stimulated by Na gradients directed into the cells. These findings are consistent with a Na-Cl cotransport system. In the presence of 200 mM mannitol, the furosemide-sensitive 22Na and 86Rb fluxes were increased dramatically to 919.4 +/- 76.6 and 106.1 +/- 29.2 pmol . mg protein-1 . min-1, respectively. When the osmolarity of the incubation medium was increased, not only were the furosemide-sensitive fluxes increased but these fluxes became inter-dependent, i.e., removing Na or K prevented the increase in the furosemide-sensitive flux of the other cation. This finding is consistent with a Na-K-2Cl cotransport system in the mTALH cells. The data suggest that the Na-Cl and the Na-K-2Cl cotransport systems may be distinct functions of the same furosemide-sensitive cotransport system and that their expression may be regulated by changes in cell volume.

Animals↗
PubMed1986

Enhanced renal tubular ouabain-sensitive ATPase in streptozotocin diabetes mellitus.

The effect of streptozotocin-induced diabetes mellitus on rat renal ouabain-sensitive ATPase in six distinct nephron segments was studied. Twenty-four hours after administration of streptozotocin, blood glucose increased threefold (P less than 0.001), and glucosuria was evident. Aldosterone levels increased almost twofold (P less than 0.001). Ouabain-sensitive ATPase increased in the proximal segments PC (proximal convoluted tubule) and PS (proximal straight tubule) by 43 and 62%, respectively, (P less than 0.001) and CD (cortical collecting duct) ouabain-sensitive ATPase increased 77% (P less than 0.001). Ouabain-sensitive ATPase in the cortical (CTAL) and medullary (MTAL) thick ascending limbs of Henle's loop and in the DC (distal convoluted tubule) remained unchanged after 24 h of streptozotocin administration. Eight days after streptozotocin administration, when glomerular filtration rate (GFR) was already markedly elevated, ouabain-sensitive ATPase remained increased in the PC, PS, and CD but was significantly less compared with the activity after 24 h (P less than 0.05), whereas in the CTAL and MTAL a marked increase in ouabain-sensitive ATPase occurred by 54% in the CTAL and 65% in the MTAL (P less than 0.001). Aldosterone levels remained elevated compared with control but less than after 24 h. Pretreatment with deoxycorticosterone acetate abolished the increase in ouabain-sensitive ATPase in the CD. These findings show that streptozotocin-induced diabetes mellitus in the rat is associated with a substantial increase in ouabain-sensitive ATPase activity along most of the nephron. This increase in enzyme activity may represent a mechanism of physiological adaptation of the nephron to maintain electrolyte homeostasis in diabetes in face of the increased GFR and osmotic diuresis.

Aldosterone↗
PubMed1986

In vitro stimulation of Na-K-ATPase in rat thick ascending limb by dexamethasone.

Dexamethasone has been reported to stimulate Na-K-ATPase activity in the medullary thick ascending limb of adrenalectomized animals within a few hours. The present study was aimed at characterizing the mechanism of this action by investigating the stimulatory effect of the hormone in vitro. Dexamethasone (10(-8) M) added in vitro to segments of the medullary thick ascending limb of Henle's loop, which were microdissected from adrenalectomized rats, restored in a dose-dependent manner the depressed Na-K-ATPase activity within one h of incubation. This stimulation of Na-K-ATPase was inhibited by cycloheximide and actinomycin D. Dexamethasone also stimulated the component of oxidative metabolism coupled to sodium transport. These results, which confirm previous in vivo observations, demonstrate that dexamethasone-induced stimulation of Na-K-ATPase is a direct tubular action of the hormone mediated by protein synthesis. They suggest that this short-term effect of dexamethasone corresponds to the stimulation of sodium reabsorption by the dilution segment.

Adrenalectomy↗
PubMed1987

Enhanced glomerular filtration and Na+-K+-ATPase with furosemide administration.

To evaluate the effect of furosemide on kidney function, glomerular filtration rate (GFR), urinary Na excretion (UNaV), Na reabsorption (NAR), and Na+-K+-ATPase in isolated nephron segments were measured in 1) rats treated with furosemide 10 mg X 100 g-1 X 24 h-1 ip for 7 days, and 2) rats receiving an oral Na load for 12 days. In furosemide-treated rats, GFR rose from 0.61 +/- 0.03 (mean +/- SD) to 0.83 +/- 0.06 ml/min (P less than 0.01), UNaV rose from 904 +/- 71 to 1,402 +/- 85 mueq/day (P less than 0.001), and net NAR rose from 87.5 +/- 3.7 to 116.7 +/- 9.0 mueq/min (P less than 0.01). Na+-K+-ATPase remained unchanged in the proximal convoluted tubule (PCT), proximal straight tubule (PST), cortical thick ascending limb of Henle's loop (cTALH), and medullary thick ascending limb of Henle's loop (mTALH), but was increased in the distal convoluted tubule (DCT) and in cortical collecting duct (CCD) from 48.5 +/- 1.2 to 75.3 +/- 0.7 (P less than 0.001) and from 18.6 +/- 0.7 to 27.1 +/- 2.7 (P less than 0.02) X 10(-11) mol X mm-1 X min-1, respectively. In Na-loaded rats GFR rose from 0.61 +/- 0.04 to 0.86 +/- 0.03 ml/min (P less than 0.001), UNaV rose from 1,064 +/- 118 to 18,532 +/- 2,045 mueq/day (P less than 0.001), net NAR from 88.1 +/- 3.0 to 107.8 +/- 3.9 mueq/min and Na-K-ATPase in the mTALH rose from 40.3 +/- 1.4 to 56.2 +/- 2.11 (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Effectiveness of a salt transport cascade in the renal medulla: computer simulations.

Based on morphological observations, it has been concluded that the upper parts of the long descending limbs of Henle's loops should be able to secrete salts into the tubular fluid (Kriz, W. Federation Proc. 42:2379-2385, 1983). In the same article, a hypothesis is given depicting how this active transport in concert with certain characteristics of the medullary architecture might produce a transport cascade toward the papillary tip, thus supporting the accumulation of salts in this region. The effectiveness of the proposed mechanism can be judged by a mathematical model only. The computer simulations of the present study demonstrate that this mechanism indeed leads to an increase of the concentrating capability of the renal countercurrent system.

Biological Transport, Active↗
PubMed1988

Basal and hormone-activated calcium absorption in mouse renal thick ascending limbs.

These studies examined transepithelial routes of basal and hormone-activated calcium absorption in thick ascending limbs of Henle's loop perfused in vitro. Single cortical (cTAL) or medullary (mTAL) thick ascending limbs were dissected from kidneys of normal mice. Tubules were perfused at 37 degrees C at pH 7.4 in bicarbonate-buffered media for cTALs and in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions for mTALs. Calcium was determined by electrothermal atomic absorption spectrophotometry. External driving forces for calcium were eliminated by perfusing and bathing tubules with symmetric solutions containing identical calcium concentrations and by abolishing the transepithelial voltage (VT), by adding furosemide or bumetanide to the luminal perfusate. Basal calcium absorption averaged 60 X 10(-12) mol.s-1.cm-2 in cTAL. After addition of 1 U/ml parathyroid hormone (PTH)-(1-34), net calcium absorption increased to 162 X 10(-12) mol.s-1.cm-2 (P less than 0.05) with no change in VT. Similarly, addition of 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) to external bathing solutions increased net calcium absorption to 201 from 70 X 10(-12) mol.s-1.cm-2 in control conditions (P less than 0.025). These results demonstrate that PTH, acting at least in part through cAMP, activates a cellular pathway for calcium absorption in cTAL. In segments of mTAL, calcium absorption was strictly voltage dependent: when VT was abolished, net calcium absorption fell to values indistinguishable from zero and was not altered in the presence of dibutyryl cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1988

cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH.

Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-[32P]-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-[32P]cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-[32P]cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1988

Hypotonic cell volume regulation in mouse medullary thick ascending limb: effects of ADH.

Differential interference contrast microscopy was used in combination with standard electrophysiological techniques in the in vitro perfused mouse medullary thick ascending limb of Henle's loop (MAL) to evaluate the cell volume responses of this nephron segment during and following exposure to hypotonic media and to assess the role of antidiuretic hormone (ADH) and net salt absorption on the associated volume regulatory processes. Reductions in extracellular osmolality by 50 mosmol resulted in rapid increases in cell volume of approximately 20% with or without exposure to ADH. Cell volume recovery (volume-regulatory decrease, VRD) was much slower in the presence, than in the absence, of ADH. This hormone-mediated impairment of the VRD response could be overcome by the abolishment of net salt absorption with luminal 10(-4) M furosemide. An inverse linear relationship was observed between the rates of net salt absorption and VRD, indicating a finite ability of this nephron segment to enhance solute exit mechanisms whether induced by increases in transcellular traffic or by hypotonic cell swelling. Finally, returning to the isotonic media resulted in cell shrinkage under all conditions [+/- ADH and +(ADH and furosemide)] consistent with cell solute loss mediating VRD. However, recovery of cell volume back to the initial isotonic control value [post-VRD volume regulatory increase (VRI)] was only observed in ADH-treated tubules and was independent of net salt absorption. The post-VRD VRI response could be abolished by isohydric CO2-HCO3- removal or by addition of 10(-4) M amiloride to the peritubular medium. The latter results suggest that parallel Na+-H+ and Cl- -HCO3- exchangers located in basolateral membranes mediate the post-VRD VRI response.

Absorption↗
PubMed1988

Substrate specificity to maintain cellular ATP along the mouse nephron.

To evaluate substrate utilization along the mouse nephron, cellular ATP content was measured under various conditions by the luciferin-luciferase technique. Individual micro-dissected nephron segments were incubated in a modified Hanks' solution (pH 7.4) with or without each of the following substrates: D-glucose, DL-lactate, beta-hydroxybutyrate (HBA), and L-glutamine. ATP production from glucose was minimal in the early proximal tubule (S1), but was substantial in the late proximal tubule (S3). Glutamine and lactate were the preferred substrates in proximal tubules. In contrast, ATP production from glutamine was less than that from the other substrates in distal nephron segments, including medullary and cortical thick ascending limbs of Henle's loop (MTAL and CTAL), distal tubule including the connecting tubule (DT), and cortical and medullary collecting tubules (CCT and MCT). Lactate and HBA were preferred substrates for ATP maintenance in CTAL, MTAL, and DT. Glucose was the best substrate in CCT. In addition, the specific contribution of anaerobic metabolism to maintaining cellular ATP was low in MTAL and CTAL. On the other hand, the glycolytic capacity of CCT and MCT was high. The above results demonstrate the substrate requirements for maintaining cellular ATP content within specific nephron segments.

3-Hydroxybutyric Acid↗
PubMed1989

Effects of Cl- transport inhibitors on Cl- permeability across hamster ascending thin limb.

The highly conductive Cl- transport pathway exists in the ascending thin limb (ATL) of Henle's loop. To characterize the mechanism of the Cl- conductance across the ATL, we examined effects on Cl- permeability across hamster ATL of Cl- transport inhibitors, including 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), diphenylamine carboxylate (DPC), and anthracene-9-carboxylic acid (9-AC), by the in vitro microperfusion technique. NPPB added to the bath caused reversible suppression of the relative permeability of Cl- to Na+ (PCl/PNa), as estimated from the NaCl diffusion voltage in a dose-dependent manner in a range from 3 x 10(-6) to 10(-3) M. The concentration of NPPB that inhibited PCl/PNa by 50% (ID50) was approximately 3 X 10(-5) M. When 3 X 10(-5) M NPPB was added to the bath, the lumen-to-bath flux coefficient for 36Cl (Kl----b,Cl- 10(-7) cm2/s) was decreased from 130.7 +/- 7.3 to 52.2 +/- 11.6 (n = 7, P less than 0.01). Application of NPPB in the lumen also caused reversible suppression of PCl/PNa, but this effect was less potent compared with the application of the drug via the bath. Whereas 10(-3) M 9-AC or 10(-3) M DPC decreased PCl/PNa by 6.5 +/- 1.2 and 10.2 +/- 2.6%, respectively, 3 X 10(-4) M NPPB decreased PCl/PNa by 69.7 +/- 3.8%. In maleimide-treated tubules, addition of 10(-3) M N-ethylmaleimide (NEM) increased PCl/PNa from 1.0 +/- 0.1 to 1.8 +/- 0.1 (n = 7, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

K+ alters cytochrome P-450-dependent arachidonate metabolism by rabbit renomedullary cells.

We studied the effects of K+ on cytochrome P-450-dependent arachidonic acid (P450-AA) metabolism by cells isolated from the rabbit medullary thick ascending limb of Henle's loop (MTAL) by varying K+ from 0 to 7.5 mM in the incubating medium because of the known effects of K+ on AA metabolism. Rabbit MTAL cells convert AA to metabolites that segregate into two peaks (P1 and P2) on reverse-phase high-performance liquid chromatography; P1 contains vasodilator material and P2 an inhibitor(s) of Na(+)-K(+)-ATPase activity. Formation of P450-AA metabolites by MTAL was enhanced by reducing external K+ (P less than 0.01) and was not affected by changes in external Cl- but was dependent on the presence of intact MTAL cells, suggesting that P450-AA metabolism was related to altering ion fluxes and/or cell volume changes. The effects of altered external K+ on MTAL P450-AA metabolism could be nullified by increasing K+ intake before killing the rabbits. Evidence for the absence of a direct effect of zero K+ on Na(+)-K(+)-ATPase was provided by the demonstration that ouabain failed to affect AA metabolism in MTAL cells. We conclude that P450-AA metabolism by MTAL cells can be influenced either directly by altering external K+ in the incubate or indirectly by changing dietary K+ before killing the rabbits. Furthermore, MTAL P450-AA metabolism was independent of changes in external Cl- and Na(+)-K(+)-ATPase activity.

Animals↗
PubMed1990

Isoproterenol increases Ca, Mg, and NaCl reabsorption in mouse thick ascending limb.

The effect of isoproterenol (Iso) on tubular transport in the thick ascending limb of Henle's loop (TAL) was investigated by in vitro microperfusion of MTAL (medullary) and CTAL (cortical) from White Swiss mouse kidney. The pattern of activation of adenylate cyclase along the distal tubule was investigated in this strain: results indicated that Iso stimulated adenylate cyclase fivefold in MTAL and ninefold in CTAL. Data from microperfusion experiments showed that Iso (10(-7) M in the bath) significantly and reversibly increased Ca and Mg reabsorption in CTAL. No net transport of Ca and Mg was observed in MTAL whether Iso was present or not. With regard to Na and Cl, Iso significantly stimulated their reabsorption in both segments and increased the transepithelial voltage in MTAL. Iso abolished K reabsorption in MTAL and induced a net K secretion in CTAL, the latter effect being also observed with 10(-9) M of Iso. When applied on CTAL, propranolol (10(-6) M in the bath) inhibited all these effects. These data indicate that beta-adrenergic agonists are involved in the multihormonal modulation of the TAL function.

Absorption↗
PubMed1995

Hypertonic NaCl enhances adenosine release and hormonal cAMP production in mouse thick ascending limb.

Adenosine 3',5'-cyclic monophosphate (cAMP), accumulated in the presence of adenosine, was measured in medullary portions of mouse thick ascending limbs of Henle's loop, suspended either in classic extracellular buffer or in the presence of added NaCl. Under control conditions (140 mmol/l NaCl), adenosine (< 10(-5) mol/l) and N6-cyclohexyladenosine, an A1 adenosine receptor agonist, inhibit the cAMP accumulation induced by arginine vasopressin (AVP). On the other hand, high concentrations of adenosine and CGS-21680, an A2 adenosine receptor agonist, stimulate cAMP formation. Addition of NaCl (+300 mmol/l) to extracellular buffer stimulates the release of endogenous adenosine. It also enhances A2 receptor-induced cAMP accumulation but suppresses A1 receptor-mediated inhibition of adenylyl cyclase. This hypertonic NaCl medium also potentiates the stimulatory action of AVP on adenylyl cyclase. The modifications of tubular responses to both AVP and A1 and A2 agonists, brought about by hypertonic NaCl, were all inhibited by adenosine deaminase, thereby demonstrating the involvement of endogenous adenosine. Adenosine, the release and the effects of which are modulated by hypertonic NaCl, thus appears to act as an endogenous physiological modulator of kidney medulla function.

Adenosine↗
PubMed1995

Role of NF-kappa B in the regulation of inducible nitric oxide synthase in an MTAL cell line.

The effects of cytokines, lipopolysaccharide (LPS), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), and pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappa B) activation, on inducible nitric oxide synthase (iNOS) expression were studied in the medullary thick ascending limb of Henle's loop cell line ST-1. LPS + interferon-gamma (IF-gamma) promoted a time-dependent increase in nitrite (a NO metabolite) and iNOS mRNA and the appearance of NF-kappa B p50 and p65 in nuclear protein extracts. Actinomycin D but not cycloheximide prevented the LPS + IF-gamma induction of iNOS mRNA and NO synthesis, indicating that iNOS transcriptional activation by LPS + IF-gamma does not require newly synthesized proteins. PDTC inhibited the LPS + IF-gamma induction of NO, iNOS mRNA, and the appearance of NF-kappa B in nuclear protein extracts, suggesting that NF-kappa B mobilization and trans-activation of the iNOS gene mediates this induction. In contrast to other cell types, cycloheximide did not alter iNOS mRNA stability, and 8-BrcAMP did not alter basal or LPS+IF-gamma induced NO production in ST-1 cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Cloning and characterization of maxi K+ channel alpha-subunit in rabbit kidney.

We have identified in rabbit renal cells two alternatively spliced transcripts of the alpha-subunit rbslo1 and rbslo2. Rbslo1 has a novel "in-frame" 174-bp insertion immediately after the predicted S8 transmembrane segment, whereas rbslo2 has a 104-bp deletion between S9 and S10, creating a frameshift and a premature termination codon. Amino acid identity between mouse maxi K- channel alpha-subunit (mslo) and rbslol was 99%. Two transcript sizes of 4.2 and 7.5 kb were detected in brain, kidney, stomach, testis, and lung. Rbslo is expressed in glomeruli, thin limbs of Henle's loop, medullary and cortical thick ascending limbs of Henle's loop, and cortical, outer, and inner medullary collecting ducts; however, it was rarely detected in proximal convoluted tubules. Rbslo1 is most abundant in inner medulla. Expressed in Xenopus oocytes, rbslo1 generates depolarization-activated, outwardly rectifying K+ currents. Rbslo1 expressed in Chinese hamster ovary cells could be activated by depolarization and Ca2+. These data suggest that rbslo transcripts are expressed in multiple nephron segments and that the magnitude of mRNA expression varies among different nephron segments.

Amino Acid Sequence↗
PubMed1997

Perfusion pressure dependency of in vivo renal tubular dynamics.

To examine whether changes in renal perfusion pressure (RPP) within the range of autoregulation induce detectable changes in tubular dynamics in an entire nephron population of the intact kidney, we measured, using electron beam computed tomography (EBCT), transit times (TT, s) and intratubular concentration (%) of filterable contrast media in various nephron segments simultaneously with renal regional perfusion. In seven dogs (group A) this was performed at the upper and lower limits of autoregulation (RPP = 130 and 95 mmHg, respectively) while group B (n = 5) served as control. In group A alone, a decrease in RPP led to an increase in TT by 40%, 68%, and 32% in the proximal tubules, ascending limb of Henle's loop, and distal tubules, respectively, in association with an increase in intratubular concentration (+ 50%, 80%, and 42%, respectively). Papillary perfusion decreased, whereas perfusion of the adjacent, outlying inner medulla increased. The decrease in papillary perfusion correlated positively with the concurrent change in sodium excretion (R = 0.81). This study demonstrates that changes in RPP within the autoregulatory range elicit changes of tubular sodium reabsorption mainly in proximal, distal, and ascending tubules, in which most of the nephrons participate. These tubular changes are associated with an alteration of perfusion circumscribed to two areas of the inner renal medulla.

Animals↗
PubMed1998

Nitric oxide increases the activity of the apical 70-pS K+ channel in TAL of rat kidney.

We have previously shown that nitric oxide (NO) mediates the stimulatory effect of angiotensin II on the apical 70-pS K+ channel in the thick ascending limb (TAL) of Henle's loop of the rat kidney (12). In the present study, we used the patch-clamp technique to examine the effects of NO on the 70-pS K+ channel. Addition of 10 microM S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, increased the channel activity in cell-attached patches. In contrast, application of 100 microM N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), reduced the channel activity by 75 +/- 7%. The effect of L-NAME was the result of inhibiting NOS, since D-NAME, which does not block NOS activity, had no effect on the channel activity. In addition, the effect of L-NAME was abolished in the presence of 1 mM L-arginine or by addition of 10 microM SNAP, further supporting the role of NO. Finally, the L-NAME-induced inhibition was also reversed by adding 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). That the effect of NO is mediated by the cGMP-dependent pathway is also suggested by experiments in which inhibition of guanylate cyclase abolished the effect of SNAP. Finally, 10 microM SNAP significantly increased cGMP concentration of the medullary TAL from 12.4 fM/microgram protein to 38.9 fM/microgram protein, as measured with ELISA. We conclude that NO is involved in regulating the activity of the apical 70-pS K+ channel in the TAL of the rat kidney.

Animals↗
PubMed1998

Ultrastructural localization of Na-K-2Cl cotransporter in thick ascending limb and macula densa of rat kidney.

A bumetanide-sensitive Na-K-2Cl cotransporter, BSC-1, is believed to mediate the apical component of transcellular NaCl absorption in the thick ascending limb (TAL) of Henle's loop. To study its ultrastructural localization in kidney, we used an affinity-purified, peptide-derived polyclonal antibody against rat BSC-1. Immunoblots from rat kidney cortex and outer medulla revealed a solitary 161-kDa band in membrane fractions. Immunocytochemistry of 1-micrometer cryosections demonstrated strong BSC-1 labeling of the apical and subapical regions of medullary and cortical TAL cells. Notably, macula densa cells also exhibited distinct labeling. Distal convoluted tubules and other renal tubule segments were unlabeled. Immunoelectron microscopy demonstrated that BSC-1 labeling was associated with the apical plasma membrane and with subapical intracellular vesicles in medullary and cortical TAL and in macula densa cells. Smooth-surfaced TAL cells, in particular, had extensive BSC-1 labeling of intracellular vesicles. These results support the view that BSC-1 provides the apical pathway for NaCl transport across the TAL and that an extensive intracellular reservoir of BSC-1 is present in a subpopulation of TAL cells. Furthermore, the BSC-1 localization in the apical plasma membrane of macula densa cells is consistent with its proposed role in tubuloglomerular feedback.

Animals↗