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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 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 . 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]. food. Total protein concentration in urine was deter- Anti-GBM glomerulonephritis 427 mined by the method of Lowry et al [13] after tn- cells. Glomeruli contain varying numbers of poly- chioracetic acid precipitation. Total plasma protein morphonuclear neutrophiles, from none to four or was determined by a biuret method [14] and albumin five. Eosinophilic deposits, probably fibrin, are seen by electrophoretic analysis (see above). Systemic col- in isolated capillary loops of some glomeruli. In ap- bid osmotic pressure (COP) was calculated using the proximately one-third of cases, mild to moderate in- Pappenheimer-Renkin equations [15] and deter- filtrates of mononuclear cells are scattered through- mined values for albumin and globulins (total protein out the cortex. In about 50% of the kidneys there is minus albumin). COP was calcu- some evidence of tubular damage, dilatation, flat- lated using the following relationship: Efferent arteri- tened epithelium and crowding of nuclei, all varying ole COP =systemicCOP/l-FF, where FF =filtra- from mild to extensive in degree. Casts are present in tion fraction (Cm/CPA11). The use of the whole kidney a number of cases. Blood vessels appear normal. FF for this calculation has been validated by Wein- Immunofluorescence reveals marked linear stain- mann, Kashgarian and Hayslett [16] for non-volume- ing for sheep IgG along capillary walls of all glome- depleted rats and the uniformity of the glomerular ruli (Fig. 1). There is occasional staining of blood lesion in our nephnitic rats supports extension of its vessels in the medullary rays. Positive staining for rat use in this model. Sodium concentrations in plasma lgG first appears four to five days after injection and and urine were read using a flame photometer (Beck- follows the depositional patterns of sheep lgG. Stain- man Klina). Plasma urea and cholesterol were ana- ing for the third component of complement, present lyzed by commercial test kits (Boehringer-Mann- in all cases, followed a similar pattern. Interrupted heim). Plasma creatinine was analyzed after adsorption linear staining along the basement membranes of and elution from Lloyds Reagent [17]. Values in some tubules was also noted. This finding is common text are given as mean SD. even in the normal rat kidney. Staining in blood vessels was negative. A synopsis of these histological findings with con- Results temporary measurements of GFR and protein excre- Histological findings. In most cases there is slight to tion spanning the period of study is presented in moderate proliferation of epithelial cells. No well- Table 1. Judged by the presence of rat lgG in stained defined crescents are seen. In some kidneys there is sections, the autologous phase begins between the also proliferation of endothelial and/or mesangial fifth and seventh days after injection. Cellular pro-

Fig. 1. Section of a kidney studied 17 days after injection of anti-GBM serum, stained for sheep IgG (X200) There is intense linear fluoresence along the glomerular capillary wall. In numerous places there appears to be fragmentation and thickening of the basement membrane. 428 von Baeyer el a!

Table 1.Histopathology, immunofluorescence, GFR and protein excretions GFR Protein excretion lgG IgG mi/mmX g of mg/24hr X100g Rat Day Prolif PMNL sheep rat C3 kidney WI of body wi 43 2 1 3 3 0 3 1.00 135 49 2 3 1 4 0 4 0.91 289 57 3 I I 4 0 3 0.32 192 60 3 2 1 4 0 4 0.02 279 44 3 1 2 3 0 4 0.05 73 51 4 3 1 4 0 4 0.23 348 47 5 1 1 4 1 3 0.98 86 6 6 2 2 2 1 4 0.09 247 46 7 1 2 2 1 3 0.93 129 39 7 3 1 4 2 4 0.07 189 8 8 1 2 4 1 3 0.10 228 9 9 2 2 3 2 3 0.10 171 34 12 2 2 3 1 3 0.09 238 35 13 1 3 2 1 4 0.34 269 20 14 2 1 4 0 4 0.64 103 II 15 2 1 4 2 3 0.13 146 Grading scale:proliferation, 0 to 3; IgG, C3 fluorescence, 0 to 4;polymorphonuclear leukocyte accumulation, 0 to3. liferation and polymorphonuclear leukocyte infil- hr X 100 g of body wt) is 30 times control level (5.7 tration, as well as positive immunofluorescent stain- mg/24 hr X 100 g of body wt). ing for sheep lgG and rat C3, are already evident on The time course of sodium retention following ini- the second day. GFR is commonly reduced by day 2, tiation of the nephritic lesion is set out in Table 3. the degree of reduction thereafter being unrelated to The sharp fall in sodium excretion on the days imme- postinjection time. Similarly, no progression in path- diately following injection of antiserum is due to the ological lesions or the magnitude of proteinuria oc- virtual disappearance of sodium from the urine since curs with time. This uniformity has enabled us to urine volume is not diminished. Urinary sodium con- pooi functional studies and tubular fluid protein data centration rises progressively on succeeding days (Tables 2 and 3; Fig. 2) and relate them to these (Table 3) so that by day 8 in this series sodium morphological changes. excretion rates have returned to control values. Ex- Pathophysiology. The renal functional equivalents amination of sodium excretion in our entire nephritic of the morphological changes (Table 1) seen in the diseased animal are summarized in Table 2. Total Table3. Sodiumexcretion in control and nephritic ratsa kidney GFR is depressed to about 40% of control. V UNaV Fractional fluid reabsorption, (U/P)1, is also re- ml/24 hrX 100g mEq/litermEq/24hr X 100g duced so that urine flow rate is diminished to a lesser ofbody of body wt degree. Total protein excretion increased during the Controlday 6.9 77.8 0.48 first 24 hr after induction of the lesion and was main- +2.3 +0.22 tained at the same level for the three weeks of study. 2 3.8 223.0 0.77 The average increase in protein excretion (189 mg/24 3 3.8 214.0 0.83

Nephriticday Table2. Kidneyfunction 2 3.5 1.4 0.004

V (U/P),,, GFR Protein excretion 3 5.5 5.6 0.023 X hr X ti//mm g mi/mmxgmg/24 g 100 12.8 ofkidney WI ofkidney Wt ofbody WI 4 8.3 8.7 0.068 Control 3.30 330 1.07 5.7 8 7.0 59.3 0.50 SD 0.80 76 0.31 2.0 +0.53 N 11 11 11 42 9 7.5 68.8 0.62 +3.8 Nephritic 10 7.2 85.1 0.74 2.37 180 0.43 189 +0.68 SD 1.60 108 0.33 89 11 9.6 116.9 1.03 N 42 42 42 67 P <0.01 <0.01 <0.01 <0.01 Valuesare means SD, N =10. A nti-GBM glomerulonephritis 429

Electrophoreon Alboerin Globulin population reveals that restoration to control levels Bande of sodium excretion usually commences after day 4. p011cm mg/lOGml %

Some individuals show evidence of restoration as pro-albumin 3(1074 66 Serum albumin so 534 14 early as day 3. u, fi, y-globulins (42) Table 4 illustrates concomitant changes in plasma Oil. 15l composition. Creatinine and urea (not shown) con- pre-albun±n centrations are slightly elevated but not to a signifi- P;eat cant extent. The increase in phosphate is significant (P < 0.01). Total plasma protein and albumin con- pre—albumin 4 2253 20 centrations are makedly reduced. The decrease in Urine albumin so± 1537 10 albumin concentration is greater than that seen for u, 3-globulins (28) total protein and is reflected in a percentage increase Dii. l5i in the globulin fraction. Cholesterol levels are raised Fig. 2. Electrophoretic analysis of fluid, urine and more than three-fold over normal. Noticeable hy- serum of nephritic rats. perlipemia is a frequent finding. The hematocrit value is diminished. Plasma Na and K concentrations do length (solid circles). The regression line for the neph- not change significantly. ritic values depicts an increase in albumin concentra- Ascitic fluid was present in the majority of rats tion as filtrate is reabsorbed along the proximal tu- although body weight remained relatively constant bule. The correlation coefficient is significant at the (average change = —2%). Mean systolic blood pres- 0.1% level. Extrapolation to the site of zero reabsorp- sure was slightly but significantly (P < 0.01) elevated tion predicts a filtered albumin concentration of 9.6 in animals 8 to 13 days after injection (controls, 106 mg/l00 ml. When the individual TF albumin values + 7; nephritic, 125 13 SD mm Hg). are corrected for fluid reabsorption by their respec- Protein handling. Electrophoretic analyses were tive (TF/P)10 ratios, a similar concentration of 11.9 performed on samples of proximal tubule fluid, urine, 6.9 mg/l0O ml is found. This near identity in the two and serum of the nephritic animals. A summary of estimates of filtrate concentration is possible only if these results is shown in Fig. 2. Proximal tubular reabsorption (or secretion) of albumin is negligible. fluid albumin concentration is greatly increased when We have used the latter relationship to calculate compared to normal [8, 9, 18, 19]. The elec- filtered albumin concentration and to estimate the trophoretic patterns of proximal tubule fluid and of filtered load of albumin. In nine experimental periods urine are quite similar. The serum pattern is dis- GFR and total kidney albumin excretion were meas- tinctively different from normal in that the total ured and two to six tubular fluid samples (N = globulin percentage has increased from 44 (Table 4) 33) were taken during each period. The average fil- to 66% and a y band is evident in the globulin frac- tered albumin concentration of these samples, TFalb tion. Typical electrophoretic patterns of the three corrected for (TF/P)1, was multiplied by GFR to biological fluids are depicted in Fig. 2. give the filtered albumin load. When these values are Simultaneous measurements of tubule fluid albu- plotted against the corresponding excretion rates in min and fluid reabsorption, (TF/P)1, in 38 samples Fig. 4, the points are scattered around the line of from nephritic rats revealed a significant correlation identity. Pair analysis of the data shows no significant (Fig. 3) (open circles). In normal rats TF albumin difference (P > 0.2) and indicates that reabsorption concentration is constant with proximal tubular of albumin is negligible.

Table 4.Plasma composition Inorganic Plasma Plasma Creatinine phosphorus protein albumin Cholesterol Hematocrit mg/lOOml mg/lOOml g/IOOml g/IOOml mg/IOOml % Control 0.37 7.7 6.43 3.60 74 47 so 0.19 1.2 0.45 0.95 13 4 N 13 13 32 35 13 33 Nephritic 0.53 9.8 4.27 1.19 263 41 so 0.26 1.8 1.15 0.66 115 5 N 11 11 48 61 9 61 P NS <0.01 <0.01 <0.01 <0.01 <0.01 430 vonBaeyeret a!

90 0.5). Fractionalvolume reabsorption therefore re- 0 0 mains constant over the entire range of SNGFR (2 to 42 nl/min X g of kidney wt), i.e., a glomerulo-tubular balance is maintained. Variability of values for end- proximal (TF/P)1 is greater at filtration rates below 70 15 nI/mm X g of kidney wt). The technical diffi- E y = 11.9x—2.3 culties of locating end-proximal sites and of tubular r=0.50 fluid collection at very low filtration rates are such P < 0.001 0 that little significance can be attached to this latter observation. The relationship of end-proximal 50 (TF/P)1 to total kidney GFR is shown in Fig, 6, the 0 same technical limitations with respect to very low filtration rates apply here as well. With these values 0 0 0 excluded (open circles), proximal fractional volume 0 0 reabsorption is independent of GFR. 30 0 0 The relationship of lowered albumin concentration to the determinants of filtration rate and tubular reabsorption are examined in Fig. 7, a and b, where filtration fraction is plotted against systemic COP and total kidney GFR, respectively. In Fig. 7b it 10 0 appears that the reduction in GFR is associated with a proportional fall in filtration fraction; that is, there is not a corresponding drop in renal plasma flow 1.0 1.5 2.0 2.5 3.0 3.5 4.0 (RPF). We have used the relationship depicted in (TF/P)11,, Fig. 7a to estimate efferent arteriolar COP and the Fig. 3. Proximal tubule fluid albumin concentration and (TF/P)1,,. derived values have been plotted against average end- Closed circles () represent control rats; open circles (0), nephritic proximal (TF/P)1 for 15 rats in Fig. 8. The corre- rats. lation coefficient of the calculated regression is of borderline significance. In the insert is shown the Proximal tubular function. In Fig. 5 values for end- decrease in COP from a control group to the average proximal (TF/P)1 are plotted against their corre- value for the nephritic animals. The fall in average sponding SNGFR in the nephritic animals. The slope efferent arteriolar COP from 32.05.6 to 11.64.6 of the regression line does not differ from zero (P > mm Hg (insert) is associated with a very modest drop in proximal fractional reabsorption (—5%). Adecrease in SNGFR parallels the reduction in

y =0.87x +0.00006 450 GFR. Data for the nephritic group were calculated r =0.81 from 66 end-proximal collections and 27 random site P <0.01 proximal samplings. A histogram of control and nephritic values (Fig. 9) reveals an asymetric distribu-

14.0 a- I • 0.04 U. 3.0 •: • E2° y=0.Olx+2.25 0.02 a. 1.O r=O.19NS

10 20 30 40 0.02 0.04 0.06 0.08 Singlenephron GFR, ni/rn/n X gof kidney wt Albumin load, mg/rn/n Fig.5. Proximal fractional fluid reabsorption (end-proximal /TF/PJ,,,) and single nephron filtration rate in nephritic rats. The Fig.4. Excreted albumin and filtered albumin in nephritic rats. The equationdescribes the regressionline drawn through the closed thick line is the regression line; the thin line is the line of identity. circles ,nephriticrats). The dashed line represents control data. Anti-GBM glomerulonephriiis 431

4.0 0.5 00 y0.02x—0.02 • Q =0.71 r S 3.0 0.4 0 C- 0 U- 0 2.0 0.3 S C . E 0 . 0 y0.43x+2.33 0.2 a.10 r=0.27NS ••. wC 0.1 .I 0.2 0.4 0.6 0.8 1.01.21.4 Totalkidney GFR, mi/rn/n X g of kidney wt 0 5 10 15 20 a Fig.6. Proximal fractional fluid reabsorption (end-proximal Systemic colloid osmotic pressure, mm Hg /TF/P/1,,)and totalkidneyfiltration rate in nephritic rats. The 0.5 regression line is drawn through the closed circles (I). y= 0.27x+ 0.07 c r=0.87 tion, more marked for the nephritic group. There- 0.4 fore, the median values (control, 25; nephritic, 7.5 0.3 nl/min X g of kidney wt) better characterize the . S change in SNGFR than the average (control, 27.6 0.2 10.3; nephritic, 14.1 9.4nl/min X gofkidneywt). U- In Fig. 10 all determined values for SNGFR are 0.1 . plotted against the corresponding measurements of GFR. Considerable scatter is apparent, particularly in the vicinity of mean values. The smallest values are 0 0.2 0.4 0.60.8 1.01.21.4 b more closely aggregated. There appears to be less Total kidney GFR, mi/rn/n X g of kidney wt heterogeneity of function in the most severely af- Fig.7. a, Filtration fraction and systemic colloid osmotic pressure. fected . Open circles (0) represent control rats. The regression line is drawn through the closed circles •,nephriticrats). b, Filtration fraction and total kidney filtration rate in nephrizic rats. Discussion A marked and sustained proteinuria characterizes similarly prepared. Significant leakage of inulin from the onset and course of this anti-GBM nephritis dur- damaged tubules was excluded in the experiments of ing both the heterologous and autologous phases. Rocha, Marcondes and Malnic [22] by the identity of Protein excretion reaches maximal rates by the sec- SNGFR's determined from proximal and distal sites. ond or third day following the injection of heterol- Allison et al [2] were able to recover 98% of prox- ogous anti-serum and is maintained throughout the imally microinjected inulin from ureteral urine in all period of study (2 to 17 days). The histological and but the most severely damaged nephrons. In these, immunofluorescent findings tabulated (Table 1) are tubular fluid was nearly stationary, the injectate re- representative of 36 kidneys read as unknowns and include all for which both GFR and protein excretion rates were measured. The linear distribution of IgG and complement in the glomerular basement mem- brane typifies the lesion as it is seen experimentally 54 [20] and in certain cases of human glomeruloneph- cop ntis [21]. Absence of crescents and the moderate U- I- degree of cellular proliferation distinguish this mor- phology from that seen in a later phase [2] and may in E part reflect a greater specificity of the antigens used in y 0.04x+ 1.94 21 r =0.50 the present report. From Table 1 it appears that no systematic progression of the lesion or of functional wOC derangement occurs after the third postinjection day. 5 10 15 20 25 The functional expressions of these morphological Efferentarteriole colloid osmotic pressure, mm Hg changes are summarized in Table 2. The reduction to Fig.8. Proximal fractional fluid reabsorption (end-proximal /TF/PJ1,,) and efferent arteriole colloid osmotic pressure in nephritic 40% of normal total GFR compares closely to the rats. The insert presents the average nephritic value ()andan findings of others [2, 221 in studies of animal models average control value (0). 432 vonBaeyer at a!

40 greater [23]. The pathogenesis of the syndrome is generally believed to be urinary loss of protein, the 30 Nephritic consequence of increased permeability of the gb- N=93 merular capillary. The 30-fold increase in daily pro- 20 tein excretion documented in Table 2 is pre- dominantly albumin (Fig. 2) and exceeds the entire 10 plasma albumin pool in the normal rat (product of plasma albumin concentration and plasma volume C 0 [24]). Although this daily loss of albumin is also in > excess of the normal rate of synthesis, 79 to 96 mg/24 a, 0 hr X 100 g of body wt, reported by Freeman and 0 0 Gordon [25] and by Kirsch et al [26], respectively, it Control is not itself sufficient cause for hypoalbuminemia. As N=25 shown by Drabkin and Marsh [27], plasma albumin production in the nephrotic rat may be three- to four- fold greater than normal and accelerated rates of synthesis by liver slices from nephrotic rats have been demonstrated in vitro[28].In an immune-complex model of experimental nephritis, albumin loss greater than that reported here was sustained without evi- 10 20 30 40 50 60 70 dence of hypoproteinemia [2]. Singlenephron GFR, ni/rn/n X g of kidney wt The concentration of albumin in the gbomerular filtrate of the normal rat has been defined by micro- Fig. 9. Frequency distribution of single nephron filtration rate in control (lower panel) and nephritic (upper panel) rats. puncture studies in this and other laboratories [1, 8, 9]. In 94 proximal tubular fluid samples collected maining visible for one to two hours. No comparable during a previous study [9], the average albumin disturbance was noted in any of our preparations and concentration was 1.35 1.06 SD mg/l00 ml. As we are confident that the clearance of inulin is a valid shown in Fig. 3 (solid points), the albumin concentra- measure of filtration rate in these animals. tion in control rats does not increase with distance Features of the nephrotic syndrome develop rap- along the tubule, an observation that delineates the idly in this animal model (Tables 2 and 4) and are extent of albumin reabsorption in that segment of the well established within the first week after injection. nephron. The nephritic values (open circles) correlate Hypoproteinemia is accompanied by hyperlipemia significantly with fluid reabsorption, and extrapola- and generalized edema, including ascites. The slight tion of their calculated regression line predicts a fil- rise is serum creatinine concentration is less than tered albumin concentration of 9.6 mg/100 ml. The expected for a 60% reduction in GFR, whereas the near identity of filtered and excreted albumin for a elevation in inorganic phosphate is proportionately nephritic group (Fig. 4) reinforces the inference of Fig. 3, that negligible albumin is reabsorbed prox- imally at this filtered concentration and supplies the a, y 19.6x + 5.2 r = 0.64 additional connotation that there is no measurable 50 P<0.001 distal reabsorption. Nearly 90% of normally filtered 0 albumin is reabsorbed and we believe this normal x40 load approaches the reabsorptive capacity of the kid- ney [9]. 30 C The sieving coefficient [29, 30] for albumin is given

U- by the ratio of the concentrations of albumin in gb- 20 C merular filtrate and plasma, (alb)F/(alb)P. It is 0 markedly increased in our nephritic rats. For the 10 a, normal rat, our average of 1.35 mg of albumin/100 C a, ml of proximal tubular fluid and a plasma albumin a, C 0.5 1.0 1.5 concentration of 3600 mg/100 ml (Table 4) yield a Totalkidney GFR, rn//rn/n X g of kidney wt value for this ratio of 3.6 X 10-i. In deriving the Fig.10. Single nephron fill ration rate and total kidney flit ration rate coefficient for the nephritic series, we used the aver- in nephritic rats. age (U/P)1 ratio and urine albumin concentration Anti-GBM glomerulonephritis 433

(Table 2 and Fig. 2) to calculate a filtrate concentra- human glomerular disease [34—36]. Although the his- tion, (alb)F, of 12.5 mg/tOO ml. The mean plasma togram (Fig. 9) and the plot of SNGFR against GFR albumin concentration, (alb)p, for nephritic animals (Fig. 10) reveal considerable variation in the filtra- is taken from Table 3. Use of the urinary concentra- tion rate of single nephrons, it is apparent that the tion index (U/P)1 to arrive at the albumin concentra- lower nephritic values are less widely dispersed. This tion in filtrate is appropriate because of the equality finding differs from reports of others [22, 37] who between filtered and excreted albumin (Fig. 4). In- found greater heterogeneity of single nephron func- troduction of these mean values into the ratio yields a tion in the diseased kidney. All agree, however, that sieving coefficient of 105 X l0- and describes a 30- normal gbomerulo-tubular balance is preserved and fold increase in permeability to albumin in this di- this is reconfirmed in the present study (Fig. 5). seased model. The gbomerular lesion is characterized functionally The structural basis for acute alterations in gb- by a dissociation between permeability of the mem- merular membrane permeability is the subject of in- brane to macromolecules, which is increased, and to tense contemporary interest. Most evidence favors smaller solutes and water which is diminished. This the basement membrane as the primary barrier to the paradoxical diversity of permeability alteration has passage of macromolecules [31], and the 30-fold in- been described in minimal change nephrotic syn- crease in protein excretion (Table 2) within hours drome in adults [38] and children [36]. The determi- after the injection of anti-GBM serum is consistent nants of glomerular filtration have been quan- with an immunologically mediated change in the bar- titatively defined by Brenner et al and Robertson et al rier function of that structure. Three hours following in the normal rat [39—41] and in a model of nephritis induction of an anti-GBM nephritis, Gang et al [32] similar, though of lesser degree, to that reported here could demonstrate increased deposition of lantha- [42]. In the latter study the authors found a marked num hydroxide aggregates (diameter =150A)in the fall in single nephron filtration rate per unit of effec- basement membrane of the rat. In the present study tive pressure difference across the membrane. This animals with severe disease showed some fragmenta- relationship defines the glomerular capillary filtration tion and moderate thickening of the basement mem- coefficient (Kr) and its reduced value describes a se- brane but this did not appear to be associated with lective impedance to the filtration process. The im- greater than average proteinuria. pedance was compensated by an increase in trans- Ryan and Karnovsky have recently examined the capillary hydrostatic pressure so that normal distribution of albumin in the successive layers of the filtration rate was maintained. In the present in- glomerular membrane as affected by hemodynamic vestigation a progressive fall in K most likely under- factors [33]. Interruption of normal blood flow by lies reduction in GFR. The anatomical correlate of clamping resulted in a marked increase in the depression in K is presently undefined. That it is the penetration of albumin throughout the GBM and not exclusively the expression of a fixed permeability into the urinary space. Release of the clamp rapidly barrier is evidenced by the ability of the nephritic restored the albumin to its intraluminal confines. nephron to increase its filtration rate in response to This "functionally dependent restriction" may be contralateral nephrectomy [43]. modified by the abrupt reduction in GFR that char- It is of interest that the marked fall (64%) in effe- acterizes the onset of anti-GBM nephritis (Table 1). rent arteriolar COP (corresponding to an average Its rapid reversibility in the acute model of Ryan and decrease in protein concentration from 10.9 to 4.8 Karnovsky suggests a nonstructural change in per- g/lOO ml) shown in Fig. 7 is associated with no more meability as might be induced by a flow-dependent than an insignificant depression in proximal frac- variation in the electrical charge on the glomerular tional reabsorptive rate. Absolute reabsorption is di- membrane. minished in proportion to the reduction in GFR. The The parallel reduction in GFR and SNGFR (Fig. observed maintenance of fractional reabsorptive rate 10) conforms to the histological evidence of a uni- is at variance with most experimental models in form membranous lesion affecting all glomeruli. The which the influence of peritubular oncotic forces on fall in GFR is not accompanied by a corresponding proximal fluid reabsorption were investigated [44—49, drop in effective renal plasma flow but relates directly 51]. Brenner et al [46], using a rapid intraaortic injec- to filtration fraction (Fig 7b) as noted by Allison et al tion of isotonic Ringer's solution to produce a 21.6% [2] whose measurements included renal plasma flow decrease in peritubular capillary plasma protein con- (CPAH/E?AH). These investigators found no difference centration, found that proximal fractional reabsorp- in EPAH between their anti-GBM group and controls. tion was reduced by 36.2%. A smaller response to Depression in filtration fraction is characteristic of changes in peritubular COP was obtained by Spitzer 434 von Baeyer et a! and Windhager [47]. They perfused peritubular capil- excretion rate. The ability of the chronically diseased laries with a colloid-free solution and found a 19% kidney to maintain sodium balance by natriuresis of reduction in fractional reabsorption using re-collec- surviving nephrons is well-known [55] but cannot be tion techniques. A later study by Brenner, Troy and the adaptive mechanism operating in the present Daugharty [50] demonstrated a 50% decrease in study since SNGFR is reduced in proportion to proximal fractional reabsorption when there was a GRF. It is possible that volume expansion resulting 26% reduction in efferent arteriole protein concentra- from the acute sodium-retaining period in our neph- tion induced by acute volume expansion with isotonic ritic rats is itself the signal for a compensatory Ringer's solution. These results, derived from acute distal rejection of sodium. Such a response has, in changes in the peritubular oncotic force, ascribe a fact, been ascribed specifically to the collecting ducts much greater role of COP in regulating fractional in the volume-expanded rat [56—58]. reabsorptive rate than our calculations or the con- clusions of Allison et al [2] and Oken, Cotes and Acknowledgments Mende [18] derived from nephritic rats allow. This study was presented in part at the 1973 Meet- It would appear that some adaptive alteration in ing of the American Society of Nephrology, Wash- reabsorptive rate takes place in protracted hypo-al- ington, D.C., and the 1974 Meeting of the American buminemia and this is nicely demonstrated in the Physiological Society, Albany, New York. Hans von experiments of Kuschinsky et a! [52], wherein the Baeyer was a National Kidney Foundation Fellow, a plasma protein concentration of rats was either Buswell Fellow at the State University of New York chronically or acutely reduced by saline infusion. at Buffalo, and Stipendiat der Deutschen For- When plasma protein was diluted by 38% over a schungsgemeinschaft. seven-day period, these authors found a decrease of only 5% in fractional reabsorptive rate, a result com- Reprint requests to Dr. I. B. Van Liew Department of Medicine, parable to the present study. Their findings in the Veterans Administration Hospital—IOB, 3495 Bailey Avenue, Buf- acute volume-expanded rat were similar to the obser- falo, New York 14215. U.S.A. vations of others [44—5 1]. Willis et a! [53] confirm a normal proximal fractional reabsorption in the References chronically salt-loaded dog. Neither of these studies 1. BALDAMUSCA,GALASKER,EISENBACI-IGM,KRAUSEHP, of chronic animal models in which proximal function STOLTEH:Glomerular protein filtration in normal and neph- was directly assessed by micropuncture [52, 53] sup- riticrats. ContrNephrol 1:37—49, 1975 ports evidence obtained indirectly for a diminished 2. ALLISONME,WILSON CB, GOTTSCHALKCW:Pathophy- siology of experimental glomerulonephritis in rats. J Clin In- proximal fractional reabsorption in hypoalbumi- vest 53:1402—1423, 1974 nemic man [36]. The latter conclusion was based on 3. MASUGIM;CJber die experimentelle Glomerulonephritis clearance experiments during diuretic-induced distal durch das spezifische Antinierenserum: Em Beitrag Zur Path- blockage of sodium transport. ogenese der diffusen Glomerulonephritis, Beit Pat ho! Although filtered sodium load remains at less than XCII:429—466, 1933 4. KRAK0WER CA, GREENSPONSA:Localization of the nephro- one-half its normal value and proximal fractional toxic antigen within the isolated renal . 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