In normal rabbit, immunolabeling of intercalated cells in the outer medullary collecting duct (OMCD) demonstrates band 3-like protein in the basolateral plasma membrane (15) and H(+)-adenosinetriphosphatase (H(+)-ATPase) in the apical plasma membrane and cytoplasmic vesicles (30). However, in type A intercalated cells in the cortical collecting duct (CCD), band 3-like protein is located primarily in multivesicular bodies and cytoplasmic vesicles (15), whereas H(+)-ATPase is present in cytoplasmic vesicles only in most intercalated cells (30). In this study, we observed the effect of chronic acid loading on immunolocalization of these transporters in the collecting duct. Adult New Zealand White rabbits received either normal tap water (controls) or 75 mM NH4Cl for 12 days plus eight daily gavages of 2-6 meq NH4Cl/kg body wt. At time of death, mean urine pH of acid-loaded animals was 5.96 (SD = 0.69), vs. 8.47 (SD = 0.07) in controls. Kidneys were fixed by in vivo perfusion and processed for light and electron microscopic immunoperoxidase localization of band 3-like protein and immunogold localization of H(+)-ATPase. In controls, band 3-like protein was largely confined to multivesicular bodies in the majority of positive-staining intercalated cells in the CCD and to the basolateral plasma membrane of intercalated cells in the OMCD. In acid-loaded rabbits, band 3 protein-positive intercalated cells in the inner CCD and the in the outer stripe of the OMCD (OMCDo) were strikingly stellate in form. Basolateral plasma membrane label was intensified, while the number of labeled multivesicular bodies was diminished. Morphometric analysis demonstrated an increase in the amount of basolateral plasma membrane in these intercalated cells. In control rabbits, H(+)-ATPase immunoreactivity in intercalated cells in the CCD was located predominantly over cytoplasmic vesicles. A minority of intercalated cells exhibited basolateral plasma membrane label, and only an occasional cell displayed apical plasma membrane label. In acid-loaded rabbits, H(+)-ATPase immunoreactivity was enhanced along the apical plasma membrane of intercalated cells in the inner CCD, and morphometric analysis demonstrated increased apical plasma membrane in band 3-positive intercalated cells in this segment. These results suggest that rabbits respond to acid loading via enhancement of both electrogenic proton secretion and Cl-/HCO3- exchange in intercalated cells in the inner CCD and the OMCDo.

The intralysosomal proteinases cathepsins B and L were measured in microdissected segments of rat nephron. Z-Arg-Arg-NMec served as the substrate for cathepsin B and Z-Phe-Arg-NMec for cathepsin B and L together. Individual S1, S2, and S3 segments of proximal tubules, TDL, MTAL, CTAL, DCT, CCD, and OMCD were dissected from young female rats weighing 130 +/- 11 g with low protein excretion (0.68 +/- 0.1 mg/24 h), from older female rats weighing 289 +/- 9 g with protein excretion of 10 +/- 6.3 mg/24 h, from older male rats weighing 404 +/- 11 g with protein excretion of 22 +/- 6 mg/24 h, from female rats weighing 198 +/- 10 g with albumin-induced proteinuria (411 +/- 134 mg/24 h), and from female rats weighing 203 +/- 11 g with low protein excretion (2.7 +/- 0.4 mg/24 h). The distributions of cathepsin activities along the nephron were similar. In all five groups, S1 and S2 segments had enzyme activities three times higher than in all remaining segments. In S2 and S3, enzyme activities were two to three times higher in proteinuric animals. These findings suggest that in proteinuric animals the increase in the protein load delivered to the proximal tubules selectively stimulated cathepsin activities in the S2 and S3 segments, presumably because of an increase in protein uptake, and that cathepsins B and L participate in lysosomal digestion of protein reabsorbed from the glomerular filtrate via endocytosis.

The intralysosomal proteinases, cathepsins B and L, were measured in microdissected segments of rat nephrons following a single injection of puromycin aminonucleoside (PAN). Z-Phenylalanyl-arginine-7-amido-4-methylcoumarin served as substrate. Enzyme activities, proteinuria, creatinine clearance and renal morphology were determined at specific time intervals following induction of PAN nephrosis. During the first three days following PAN injection, enzyme activities in S2 and S3 segments, protein excretion, creatinine clearance and appearance of the renal parenchyma resembled control animals. The enzyme activity in S1 segments was slightly decreased, but returned to control levels at day six after injection. Days four through eight post-PAN injection were characterized by a dramatic increase in protein excretion and an increase in cathepsin B and L activity in S2 and S3 segments of the proximal tubule. During days 9 through 15 enzyme activity decreased significantly in S2 segments despite continued proteinuria. Overt necrosis and cell injury were seen in the proximal tubule and probably account for the decrease in proteolytic activity. After day 15 following PAN injection, the level of proteinuria decreased, restoration of cathepsin activities occurred and a histopathologic picture of healing was present. The data suggest a positive relationship exists between stimulation of cathepsin B and L activity in S2 and S3 segments of the proximal tubule and increased protein filtration in PAN nephrosis. The increased enzyme activity reflects enhancement of the proteolytic capacity of the lysosomal system that is necessary for increased protein catabolism.