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Filtration of Protein in the Anti-Glomerular Basement Membrane Nephritic Rat: a Micropuncture Study

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Filtration of Protein in the Anti-Glomerular Basement Membrane Nephritic Rat: a Micropuncture Study View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, Vol. 10 (1976) p. 425—437 Filtration of protein in the anti-glomerular basement membrane nephritic rat: A micropuncture study HANS VON BAEYER, JUDITH B. VAN LIEW, JOHN KLASSEN and JOHN W. BOYLAN with the technical assistance of NANCY MANZ and PATRICIA MUIR Departments of Medicine, Physiology and Microbiology, State University of New York at Buffalo and Veterans Administration Hospital, Buffalo, New York Filtration of protein in the anti-glomerular basement membrane (GBM). We have used this animal model to examine, nephritic rat: A micropuncture study. Production on an anti-gb- with micropuncture techniques, changes in the filtra- merular basement membrane (anti-GBM) nephritis in the rat re- sults in a 30-fold increase in glomerular membrane permeability to tion and reabsorption of protein during the first to albumin. The concentration of albumin in glomerular filtrate, esti- third weeks of the disease (days 2 to 17) and to mated from proximal tubular fluid samples, is ten times the normal correlate some of the renal functional deficits ob- value. Tubular reabsorption of albumin is not enhanced so that essentially the filtered load is excreted. A nephrotic syndrome served with pathological findings. The period of develops rapidly. Total kidney gbomerular filtration rate (GFR) is study therefore complements the work of Baldamus reduced to 40% of normal with a proportional reduction in filtra- et al [1] on changes in protein excretion during the tion fraction. Glomerulo-tubular balance is maintained since prox- imal fractional reabsorption remains constant near control levels. first 24 hr and the recent paper of Allison, Wilson and Calculated efferent arteriolar plasma colloid osmotic pressure Gottschalk [2] whose functional data for the anti- (COP) is about one-third normal. Sodium excretion, sharply cur- GBM nephritic rat span the 10th to 30th day. The tailed in the first days of anti-GBM nephritis, returns to control values after the fourth postinjection day. Restoration of sodium consequence of proteinuria, decreased colloid os- balance despite reduced filtered load and constant proximal frac- motic pressure of plasma, has been examined in rela- tional reabsorption must be accomplished by adjustments at a tion to glomerular filtration, proximal reabsorption distal site in the nephron. and sodium retention. Filtration de protéine chez le rat atteint de néphrite anti-GBM: Etude par microfonction. La creation d'une néphrite anti-GBM chez le rat a pour consequence une augmentation de 30 fois de Ia Methods perméablité gbomérulaire a l'albumine. La concentration d'albu- mine dans le filtrat glomerulaire, evaluée a partirdéchantillons A total of 87 male Wistar rats (200 to 350 g body tubaluires proximaux, est dix fois Ia valeur normale. La réabsorp- tion tubulaire de l'albumine n'est pas augmentée de telle sorte que wt) was used. Sixty-two were utilized for micro- Ia quantité filtrée est pratiguement excrétée. Un syndrome néphro- puncture studies and 25 for evaluation of filtration tique se developpe rapidement. Le debit de filtration glomérulaire fraction and sodium balance. Animals were main- est réduit a 40% de La valeur normale avec une reduction propor- tionnelle de Ia fraction filtrée. La balance glomérulo-tubulaire est tained on a regular laboratory diet containing 1% maintenue du fait que Ia reabsorption fractionnelle proximale reste NaCl. constante, au voisinage des valeurs contrôles. La pression colloido Production of the lesion. Nephrotoxic nephritis [3] osmotique calculée dans l'arteriole efférente est environ 1/3 de le normale. L'excretion du sodium, brusquement diminuée dans les was produced by administration of heterologous se- premiers jours de Ia néphrite anti-GBM, revient aux valeurs con- rum containing GBM antibodies. The serum was troles apres le 4ejour.La restauration du bilan du sodium malgré raised in sheep by repeated intradermal injections of une charge filtrée diminuée et avec une reabsorption fractionnelle proximale constante doit etre assurée par des ajustements, dans 10 mg of isolated GBM, prepared according to the des regions distales du néphron. method of Krakower and Greenspon [4] and in- corporated in complete Freund's adjuvant. Injections A typical nephrotic syndrome develops in rats fol- were given at two-week intervals and serum was lowing the injection of serum containing antibodies tested for potency according to its ability to induce to homologous glomerular basement membrane proteinuria (> 100 mg/24 hr) in a young rat. A single batch of serum of demonstrated potency was har- Received for publication March 10, 1976; vested, divided into 1 .5-ml aliquots and stored frozen and in revised form July 19, 1976. until used. A single dose of 0.3 to 0.5 ml/100 g of ©1976,by the International Society of Nephrology. body wt was given intravenously, its effectiveness 425 426 vonBaeyer ela! monitored by measuring the 24-hr protein excretion Systolic blood pressure was measured indirectly on prior to experimentation. unanesthetized animals using a tail cuff and pneu- Experimental procedures. Micropuncture studies. matic pulse transducer (Narco), amplified and re- Animals were anesthetized with mactin (80 to 100 corded with a polygraph (Grass, Model 79). When mg/100 g of body wt i.p.) and prepared for micro- checked against direct intraarterial pressures, these puncture in the conventional manner [5]. Urinary measurements agreed within 2 to 3 mm Hg. and peritoneal fluid losses were replaced by the i.v. On completion of the micropuncture studies, the infusion of isotonic saline solution (0.02 to 0.03 kidneys were removed and sectioned coronally. One mI/mm >< 100 g of body wt). The infusion contained piece was fixed in phosphate-buffered formalin and 4 g/l00 ml of synthetic inulin (polyfructosan, Laevo- then imbedded in paraffin for light microscopic san) for the measurement of single and total glomeru- studies. Sections were stained routinely with hema- lar filtration rate (SNGFR and GFR, respectively). toxylin-eosin, and in selected cases also with methe- When SNGFR was measured, urine for concomitant namine silver, Mason trichrome and the PAS GFR was collected by ureteral catherer, outflow re- method. The second piece of the kidney was immedi- sistance being minimized by use of a short segment of ately frozen in chilled isopentane. It was stored in this PE 10 tubing attached to PE 50. Without this precau- until examined by means of the immunofluorescent tion dilatation of the collecting system occurred. When technique [6]. tubular fluid was collected for protein analysis, urine Frozen sections 4z in thickness were stained with for protein and GFR was collected by bladder cath- fluorescein-labelled [6] im munoglobulins to sheep eter (PE 60 tubing). Tail blood samples were taken at IgG, rat IgG and rat C3 (Hyland Laboratories, Los the beginning and end of each urine collection period. Angeles, CA). Sections were examined in a photo- Two types of micropuncture experiments were per- microscope (Zeiss) using the attachments for immu- formed: (a) Determination of SNGFR. Tubular fluid nofluorescence. The intensity of the staining along samples were collected quantitively from proximal glomerular and tubular basement membrane was sites. When protocol required end-proximal samples, graded from 0 to 4. puncture sites were selected near the efferent star, and Analytical methods. Albumin concentration and end-proximal location was verified by following the the presence of other proteins in tubular fluid samples course of injected oil droplets. The appearance of were determined by discontinuous polyacrilamide gel more than one loop before final disappearance of the electrophoresis [7] as modified for capillary columns drop disqualified the sample as end-proximal. Collec- by Oken [8]. Details of methodology have been pub- tion time was three to five minutes under conditions lished [9]; the sensitivity is such as to detect picogram of spontaneous inflow, and sample volume was deter- quantities of albumin in nanoliter samples of tubular mined by its measured length in a constant bore fluid or urine. Tubular fluid samples are transferred capillary. SNGFR was calculated as collected volume directly from the collection pipet to the analytical per minute X the tubular fluid to plasma inulin ratio, capillary tube within one hour of collection. Plasma (TF/P)1. Two or three SNGFR determinations were and urine samples were analyzed by the same elec- made during each of three measurements of GFR trophoresis system after suitable dilution. Standards per experiment. of diluted rat serum in volumes comparable to un- (b) Determination of tubular fluid protein concen- knowns supply reference curves for each experiment. tration and its relationship to fluid reabsorption. The After electrophoresis the gels are extruded into collection technique was similar to that above except acetic acid, fixed, stained with Fast Green, destained that larger samples were needed for analysis of both and scanned directly in an ultramicrodensitometer protein and inulin. (Joyce-Loebl). An integrator unit quantifies the scan Filtration fraction was calculated in some experi- and yields a linear relationship with protein concen- ments from the simultaneous clearances of poly- tration. fructosan and para-aminohippuric acid (PAH, 0.4 Urinary polyfructosan was analyzed by the anth- g/lOO ml of infusion solution). In representative ani- rone method of Fuhr, Kaczmarczyk and Kruttgen mals chosen at random, plasma concentrations of [10], plasma and tubule fluid polyfructosan by a mi- total protein, albumin, creatinine, urea, cholesterol, cromodification of the same method [11]. Color de- sodium, and potassium were measured. Excretion velopment in the micro-samples was read in a spec- rates for total protein and sodium were determined trophotometer (Gilford) adapted for a 3l cuvette. from overnight collections of urine, the animals being PAH in urine and plasma was analyzed according to kept for this purpose in metabolic cages without the method of Smith et al [12].
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