Digital three-dimensional (3-D) functional reconstructions of inner medullary nephrons were performed. Antibodies against aquaporins (AQP)-1 and -2 and the chloride channel ClC-K1 identified descending thin limbs (DTLs), collecting ducts (CDs), and ascending thin limbs (ATLs), respectively, through indirect immunofluorescence. Tubules were labeled in transverse sections and assembled into 3-D arrays, permitting individual tubule or combined surface representations to depths of 3.3 mm to be viewed in an interactive digital model. Surface representations of 75 tubules positioned near the central region of the inner medulla were reconstructed. In most DTL segments that form loops below 1 mm from the inner medullary base, AQP1 expression begins at the base, becomes intermittent for variable lengths, and continues nearly midway to the loop. The terminal DTL segment exhibiting undetectable AQP1 represents nearly 60% of the distance from the medullary base to the tip of the loop. AQP1 expression was entirely undetectable in shorter long-looped DTLs. ClC-K1 is expressed continuously along the terminal portion of all DTLs reconstructed here, beginning with a prebend region approximately 164 microm before the bend in all tubules and continuing through the entire ascent of the ATLs to the base of the inner medulla. CDs express AQP2 continuously and extensive branching patterns are illustrated. 3-D functional reconstruction of inner medullary nephrons is capable of showing axial distribution of membrane proteins in tubules of the inner medulla and can contribute to further development and refinement of models that attempt to elucidate the concentrating mechanism.

Intracellular pH (pHi) was measured in isolated, nonperfused and perfused rat papillary thin limbs of Henle's loops in N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES)- or HEPES/bicarbonate-buffered medium at pH 7.4 using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Resting pHi was about 6.7 in descending thin limbs (DTL) and about 6.9 in ascending thin limbs (ATL), even with a medium pH of 7.4. These values appeared to reflect the acid pH of the blood in the neighboring vasa recta found in vivo. The resting pHi did not differ whether or not the medium contained bicarbonate although the total buffering capacity of the tubule cells was increased in the presence of bicarbonate. In nonperfused DTL and ATL, pHi was further acidified following an NH4Cl pulse. The rate of recovery of pHi from this level to the resting pHi was reduced by Na+ removal from the bath in both DTL and ATL and by the addition of ethylisopropylamiloride (EIPA) to the bath in the presence of Na+ in DTL. The rate of recovery was not affected by Cl- removal from the bath or K+ (75 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) addition to the bath in either DTL or ATL. These results suggest that the common, amiloride-sensitive, basolateral Na+/H+ exchanger plays a role in the regulation of pHi in rat papillary DTL but that a different basolateral Na+/H+ exchanger or a luminal Na+/H+ exchanger is important in rat papillary ATL.

In nonperfused proximal tubules isolated from chicken long-looped mammalian-type nephrons, intracellular pH (pHi), measured with the pH-sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, was ∼7.3 under control conditions (HEPES-buffered medium with pH 7.4 at 37°C) and was reduced to ∼7.0 in response to NH4Cl pulse. The rate of recovery of pHi from this level to the resting level was 1) significantly reduced by the removal of Na+ from the bath, 2) significantly increased by the removal of Cl− from the bath, 3) unchanged by the removal of both Na+ and Cl− from the bath, 4) significantly reduced by the addition of either ethylisopropylamiloride or DIDS to the bath, 5) significantly increased by a high bath K+ concentration, and 6) unchanged by the addition of Ba2+ to the bath. These data suggest that both Na+-coupled and Cl−-coupled basolateral acid-base fluxes are involved in determining the rate of recovery of pHi after acidification. The most likely ones to be important in regulating pHi are a Na+/H+exchanger and a Na+-coupled Cl−/[Formula: see text]exchanger. In birds, long-looped mammalian-type nephrons resemble short-looped transitional nephrons but differ markedly from superficial loopless reptilian-type nephrons.

No abstract

In proximal tubules isolated from chicken superficial loopless reptilian-type nephrons, intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein, was ∼7.1–7.2 under control conditions ( N-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid-buffered medium with pH 7.4 at 37°C), and was reduced to ∼6.9 in response to NH4Cl pulse. The rate of recovery of pHi(control value ≅ 5 × 10−3 pH U/s) from this acid level was 1) significantly decreased by removal of Na+ or both Na+ and Cl− from the bath or addition of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (0.25 mM) to the bath, 2) significantly increased by high bath K+ (75 mM), and 3) unchanged by removal of Cl− alone from the bath or addition of ethylisopropylamiloride (1 mM) or Ba2+ (5 mM) to the bath. Resting pHi was 1) significantly decreased by Na+ or simultaneous Na+ and Cl− removal, 2) significantly increased by high K+, and 3) unchanged by Cl− removal alone or addition of Ba2+. The data do not fit the concept of pHi regulation by the most commonly suggested basolateral transporters (Na+/H+exchanger, Na+-dependent and Na+-independent Cl−/[Formula: see text]exchangers, or Na+-[Formula: see text]-[Formula: see text]cotransporter).