Summary
Sex-dependent protein handling in the rat renal tubular system was studied both qualitatively and quantitatively using the method of direct fluorescent protein tracing. The protein tracer, fluorescent ovalbumin, was synthesized by conjugating hen ovalbumin with fluorescein isothiocyanate (FITC), and the fluorescence characteristics of fluores-ceinthiocarbamyl (FTC)-ovalbumin conjugates with different degrees of labelling were studied. Heavily labelled tracer was intravenously injected into male and female rats, and both kidneys were perfused; the right kidney was then homogenized and used for quantitative fluorometric measurements, while the left kidney was perfusion fixed and prepared for fluorescence mciroscopy. The tubular reabsorption of fluorescent ovalbumin was studied 4 min and 10 min after the injection of different doses (1.4, 7.0 and 14.0 mg/kg body weight) of the tracer, and the tubular catabolism was investigated in animals killed 60 and 120 min after the injection. Fluorescence microscopy demonstrated that, in both sexes and regardless of the dose administered and the time after injection, specifically fluorescent protein or its degradation products was only present in the epithelial cells of the proximal tubule. With regard to sex-dependent differences in protein handling, fluorometry indicated that at 4 min (7.0 mg) and at 10 min (all doses) after injection, female animals had reabsorbed more fluorescent protein than males. With regard to the catabolic phase, both the fluorescence microscopy and the fluorometric results showed that the female rats had degraded the fluorescent tracer at a significantly higher rate than males. The results are discussed in connection with sex-dependent proteinuria in rats.
Similar content being viewed by others
References
Asan E, Kugler P (1985) Maleate effects on kidney peptidases and proteinuria of male and female rats. Histochemistry 82:81–92
Bode F, Ottosen PD, Madsen KM, Maunsbach AB (1980) Does transtubular transport of intact protein occur in the kidney? In: Maunsbach AB, Olson TS, Christensen EI (eds) Functional ultrastructure of the kidney. Academic Press, London New York, pp 389–395
Carone FA, Peterson DR, Oparil S, Pullman TN (1979) Renal tubular transport and catabolism of proteins and peptides. Kidney Int 16:271–278
Christensen EI (1976) Rapid protein uptake and digestion in proximal tubule lysosomes. Kidney Int 10:301–310
Christensen EI, Maunsbach AB (1974) Intralysosomal digestion of lysozyme in renal proximal tubule cells. Kidney Int 6:396–407
Curtain CC (1961) The chromatographic purification of fluorescein-antibody. J Histochem 9:484–486
Falk N (1984) Nachweis exogener Meerrettichperoxidase im Hauptstück der Niere männlicher und weíblicher Ratten. Z mikrosk-anat Forsch 98:451–464
Fothergill LA, Fothergill JE (1970) Structural comparison of ovalbumins from nine different species. Eur J Biochem 17:529–532
Geisow MJ, Evans WH (1984) pH in the endosome. Measurements during pinocytosis and receptor-mediated endocytosis. Exp Cell Res 150:36–46
Goldstein G, Slizys IS, Chase MW (1961) Studies on fluorescent antibody staining. I. Non-specific fluorescence with fluorescein-coupled sheep anti-rabbit globulins. J exp Med 114:89–110
Herbert M (1983) Elektronenmikroskopisch-morphometrische Untersuchungen am Nierenhauptstück männlicher und weiblicher Ratten nach Kastration und Testosteronsubstitution. Z mikrosk-anat Forsch 97:189–239
Holt S (1959) Factors governing the validity of staining methods for enzymes, and their bearing upon the Gomori acid phosphatase technique. Exp Cell Res (Suppl) 7:1–27
Jedrzejewski K, Kugler P (1982) Peptidases in the kidney and urine of rats after castration. Histochemistry 74:63–84
Kenny AJ (1979) Proteinases associated with cell membranes. In: Barret AJ (ed) Proteinases in mammalian cells and tissue. North Holland, Amsterdam New York Oxford, pp 393–444
Kidwai SA, Ansari AA, Salahuddin A (1976) Effect of succinylation (3-Carboxypropionylation) on the conformation and immunological activity of ovalbumin. Biochem J 155:171–180
Kierszenbaum F, Levison SA, Dandliker WB (1969) Fractionation of fluorescent-labeled proteins according to the degree of labeling. Anal Biochem 28:563–572
Kiesewetter F, Kugler P (1985a) Sex different cytochrome-c uptake in the proximal tubule of the rat kidney. Histochemistry 82:557–564
Kiesewetter F, Kugler P (1985b) Geschlechtsdifferenter lysosomaler Abbau von exogenem Cytochrom C im Nierenhauptstück der Ratte. Z mikrosk-anat Forsch 99:855–865
Kugler P (1982a) On angiotensin-degrading aminopeptidases in the rat kidney. Adv Anat Embryol Cell Biol, vol 76
Kugler P (1982b) Quantitative histochemistry of the lysosomal dipeptidyl aminopeptidase II in the proximal tubule of the rat kidney. Histochemistry 76:557–566
Kugler P, Vornberger G (1986) Renal cathepsin-B activities in rats after castration and treatment with sex hormones. Histochemistry (in press)
Loida Z, Gossrau R, Schiebler TH (1979) Enzyme histochemistry. Springer, Berlin Heidelberg New York
Maack T, Johnson V, Kau ST, Figueiredo J, Sigulem D (1979) Renal filtration, transport, and metabolism of low-molecular-weight proteins: A review. Kidney Int 16:251–270
Maunsbach AB (1966) Observations on the segmentation of the proximal tubule in the kidney. Comparison of results from phase contrast, fluorescence and electron microscopy. J Ultrastruct Res 16:239–258
McKinney RM, Spillane JT, Pearce GW (1966) A simple method for determining the labeling efficiency of fluorescein isothiocyanate products. Anal Biochem 14:421–428
Nairn RC (1976) Fluorescent protein tracing. Churchill Livingstone, Edinburgh London New York
Nisbet AD, Saundry RH, Moir AJG, Fothergill LA, Fothergill JE (1981) The complete amino-acid sequence of hen ovalbumin. Eur J Biochem 115:335–345
Nguyen NY, Chrambach A (1978) A test of the resolving power of cascade electrofocusing and cascade stacking: separation of BSA from ovalbumin. Anal Biochem 87:576–585
Ottosen PD, Bode F, Madsen KM, Maunsbach AB (1979) Renal handling of lysozyme in the rat. Kidney Int 15:246–254
Perlman GE (1955) Nature of phosphorus linkages in phosphoproteins. Adv Prot Chem 10:1–30
Riggs JL, Loh PC, Eveland WC (1960) A simple fractionation method for preparation of fluorescein-labeled gamma globulin. Proc Soc Exp Biol 105:655–658
Rühmke PH, Breekveldt-Kielich JC, van den Broecke-Siddré A (1970) Sex-associated urinary protein in the rat. Biochim Biophys Acta 200:275–283
Schiller AA, Schayer RW, Hess EL (1952) Fluorescein-conjugated bovine albumin-physical and biological properties. J Gen Physiol 36:489–506
Sellers AL, Goodman MC, Marmorston J, Smith M (1950) Sex differences in proteinuria in the rat. Am J Physiol 163:662–667
Straus W (1967) Changes in intracellular location of small phagosomes (micropinocytotic vesicles) in kidney and liver cells in relation to time after injection and dose of horseradish peroxidase. J Histochem Cytochem 15:381–393
Sumpio BE, Maack T (1982) Kinetics, competition, and selectivity of tubular absorption of proteins. Am J Physiol 243:379–392
Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecylsulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412
Wells AF, Miller CE, Nadel MK (1966) Rapid fluorescein and protein assay method for fluorescent-antibody conjugates. Appl Microbiol 14:271–275
Author information
Authors and Affiliations
Additional information
In honour of Prof. P. van Duijn
Supported by the Deutsche Forschungsgemeinschaft (SFB 105)
Rights and permissions
About this article
Cite this article
Asan, E., Kugler, P. & Schiebler, T.H. Sex-related differences in the handling of fluorescent ovalbumin by the proximal tubule of the rat kidney. Histochemistry 84, 408–417 (1986). https://doi.org/10.1007/BF00482971
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00482971