Characterization of Aldose Reductase from the Thick Ascending Limb of Henle’s Loop of Rabbit Kidney

Functional and immunohistological studies were performed to identify basolateral chloride/bicarbonate exchange in macula densa cells. Using the isolated, perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, macula densa intracellular pH (pHi) was measured with fluorescence microscopy and BCECF. For these experiments, basolateral chloride was reduced, resulting in reversible macula densa cell alkalinization. Anion exchange activity was assessed by measuring the maximal net base efflux on readdition of bath chloride. Anion exchange activity required the presence of bicarbonate, was independent of changes in membrane potential, did not require the presence of sodium, and was inhibited by high concentrations of DIDS. Inhibition of macula densa anion exchange activity by basolateral DIDS increased luminal NaCl concentration-induced elevations in pHi. Immunohistochemical studies using antibodies against AE2 demonstrated expression of AE2 along the basolateral membrane of macula densa cells of rabbit kidney. These results suggest that macula densa cells functionally and immunologically express a chloride/bicarbonate exchanger at the basolateral membrane. This transporter likely participates in the regulation of pHi and might be involved in macula densa signaling.

Download Full-text

A potassium channel in the apical membrane of rabbit thick ascending limb of Henle's loop

AJP Renal Physiology ◽

10.1152/ajprenal.1990.258.2.f244 ◽

1990 ◽

Vol 258 (2) ◽

pp. F244-F253 ◽

Cited By ~ 19

Author(s):

W. H. Wang ◽

S. White ◽

J. Geibel ◽

G. Giebisch

Keyword(s):

Apical Membrane ◽

Rabbit Kidney ◽

K Channel ◽

Thick Ascending Limb ◽

Dependent Manner ◽

Patch Clamp Technique ◽

Bath Solution ◽

Henle's Loop ◽

Voltage Dependent ◽

Inside Out

We used the patch-clamp technique to study the activity of single potassium channels in the apical membrane of isolated thick ascending limbs of Henle's loop (TAL) of rabbit kidneys. In cell-attached patches with NaCl Ringer or high-K+ solution in the bath and 140 mM K+ in the pipette, an inwardly rectifying K+ channel was observed with an inward slope conductance of 22.0 +/- 0.5 pS and outward slope conductance of 10.2 +/- 0.3 pS at 22 degrees C (n = 15). The channel was highly selective for K+, with a calculated permeability ratio for K(+)-to-Na+ of 20:1 (n = 4). The open probability (Po) of the channel was 0.89 +/- 0.03 (n = 15) and was not voltage dependent. In inside-out patches with 140 mM K+ in both the bath and the pipette solutions, both Po and conductance of the channel were similar to that in cell-attached patches. Addition of 0.1 mM Ba2+ to the pipette solution reduced Po of the channel in a voltage-dependent manner. Lowering the pH of the bath solution from 7.4 to 6.9 or increasing Ca2+ concentration from 0 to 0.5 mM in inside-out patches did not alter either Po or conductance of the channel. Addition of 2 mM ATP to the bath solution completely inhibited channel activity. This ATP-induced inhibition was fully reversible and was found to be dependent on the ratio of ATP to ADP, since adding 1 mM ADP to the bath solution relieved the ATP-induced blockade. The property of this small-conductance K+ channel make it a likely candidate for recycling of K+ across the apical membrane of TAL of the rabbit kidney. ATP and ADP are possible intracellular regulators of the channel's activity.

Download Full-text

Evidence for electroneutral sodium chloride cotransport in the cortical thick ascending limb of Henle's loop of rabbit kidney

Pflügers Archiv - European Journal of Physiology ◽

10.1007/bf01063936 ◽

1983 ◽

Vol 396 (4) ◽

pp. 308-314 ◽

Cited By ~ 164

Author(s):

Rainer Greger ◽

Eberhard Schlatter ◽

Florian Lang

Keyword(s):

Sodium Chloride ◽

Rabbit Kidney ◽

Thick Ascending Limb ◽

Henle's Loop

Download Full-text

Immunocytochemical characterization of Na(+)-H+ exchanger isoform NHE-1 in rabbit kidney

AJP Renal Physiology ◽

10.1152/ajprenal.1992.263.5.f833 ◽

1992 ◽

Vol 263 (5) ◽

pp. F833-F840 ◽

Cited By ~ 33

Author(s):

D. Biemesderfer ◽

R. F. Reilly ◽

M. Exner ◽

P. Igarashi ◽

P. S. Aronson

Keyword(s):

Fusion Protein ◽

Renal Cortex ◽

Apical Membrane ◽

Basolateral Membrane ◽

Rabbit Kidney ◽

Thick Ascending Limb ◽

Nephron Segments ◽

Basolateral Plasma Membrane ◽

Tubule Cells

We have recently isolated cDNAs encoding a Na(+)-H+ exchanger isoform, referred to as NHE-1, from rabbit kidney and LLC-PK1 cells. To identify the NHE-1 protein and to establish its cellular and subcellular localization in the rabbit kidney, we prepared antibodies to a NHE-1 fusion protein. cDNA encoding the COOH-terminal 41 amino acids of NHE-1 was subcloned into a maltose-binding protein vector and the purified fusion protein (FP347A) used to immunize guinea pigs. To identify the NHE-1 protein, we performed Western blot analysis against membrane fractions prepared from rabbit renal cortex. Anti-FP347A antibody specifically reacted with a polypeptide with an apparent molecular mass of 100–110 kDa that was enriched in basolateral membrane fractions. When indirect immunofluorescence was performed on semithin (0.5 micron) cryosections of paraformaldehyde-lysine-periodate-fixed rabbit kidney, anti-FP347A specifically stained the basolateral plasma membrane of cells of the proximal tubule, thick ascending limb, and distal convoluted tubule. Anti-FP347A similarly stained connecting tubule cells and principal cells. No staining was detected on the apical membrane of any cells of the rabbit nephron. We conclude that NHE-1 is a 100- to 110-kDa protein expressed on the basolateral membrane of multiple nephron segments.

Download Full-text