Monoclonal antibodies against luminal membranes of renal proximal tubules which are kidney-specific

After immunization with porcine brush-border membrane proteins, 11 monoclonal antibodies were generated which react with proximal tubules. Their antigenic polypeptides were characterized with respect to apparent molecular weight, histochemical localization in porcine and human kidney, and tissue distribution in pig. In porcine kidney, six antibodies bind selectively to the proximal tubule whereas the others also react with other nephron segments. With the exception of one antibody which reacts with the luminal and the basolateral membrane of the porcine proximal tubule, the other antibodies specific for the proximal tubule only stain the brush-border membrane. Four of them react along the entire length of the porcine proximal tubule, whereas one (R1A2) binds to the S3-segment in pig and to the entire length of the proximal tubule in man. This indicates that segment-specific expression may be species-dependent. Testing the antibodies in 21 different extrarenal tissues it was found that three of the antibodies, specific for the brush-border membrane in renal proximal tubules, only react in kidney. Two of these are specific for pig kidney whereas one also reacts with human kidney. This antibody (N4A4) is directed against a polypeptide with an apparent molecular weight of 400,000. Electron microscopic immunohistochemistry showed that N4A4 binds to the intervillus region of the brush-border membrane and to subapical vesicles

Analysis of Na⁺-D-glucose cotransporter and other renal brush border proteins in human urine

Analysis of Na+-D-glucose cotransporter and other renal brush border proteins in human urine. A sensitive quantitative radioimmunoassay is described by which different antigens in the urine can be assayed simultaneously. Urinary excretion of three proteins from proximal tubules was compared: 1) the Na+-D-glucose cotransporter from brush border membranes and subapical vesicles; 2) a kidney-specific hydrophobic Mr 400,000 polypeptide from intermicroviUar invaginations and subapical vesicles; and 3) villin from microvilli cores. In the normal urine about 50% of the excreted Na+-D-glucose cotransporter and villin, and about 25% of the Mr 400,000 polypeptide was associated with brush border membrane vesicles, whereas trie remaining fractions of the three proteins formed small sedimentable aggregates which contained some cholesterol and fatty acids but no phospholipids. The normal urinary excretion of the Na+-D-glucose cotransporter was correlated with that of villin and the Mr 400,000 polypeptide. The data show that membrane proteins from the proximal tubule are excreted by the shedding of different brush border membrane areas. They suggest that some microvilli are released in total, and that a large fraction of the brush border membrane proteins is excreted without being associated with a phospholipid bilayer. In an attempt to define protein excretion patterns during kidney malfunctions, the excretion of brush border membrane proteins was analyzed after one intravenous injection of the X-ray contrast medium, iopamidol. No change in villin excretion was observed, but a reversible increase in the excretion of brush border membrane proteins was found in patients without diabetes. With diabetes a more pronounced iopamidol effect on the excretion of brush border membrane proteins and a significant increase in the excretion of villin was observed