Pathophysiology of Experimental Glomerulonephritis in Rats MARJORIE E. M. ALLISON, CuRTIs B. WILSON, and CARL W. GOrrSCHALK, with the technical assistance of SARAH K. DAVIS From the Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 and the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California 92037 A B S T R A C T Micropuncture, clearance, immunofluo- the more chronic AICN disease. Tubular damage was rescence and light microscopy techniques were used to more marked in AICN, and extraction of p-aminohip- study kidney structure and single nephron function in purate was reduced in this group. rats with autologous immune complex nephritis (AICN), a membranous glomerulonephritis develop- ing over 5 to 20 mo, in the more acute and prolifera- INTRODUCTION tive glomerular basement membrane (GBM) nephritis Since the classical description by Richard Bright in and in controls. Both models are known to have clinical 1827 of the clinical effects of chronic renal disease a counterparts in human disease. Kidney functional ab- multitude of detailed studies have been made of whole normalities correlated with the degree of architectural kidney function in such patients (1-5). The pains- derangement. In both AICN and anti-GBM nephritis taking microdissection studies of Oliver (6) have filtration fraction fell in direct proportion to the fall in shown that such kidneys are "a heterogeneous collec- glomerular filtration rate (GFR), renal plasma flow tion of various and disparate organs, the abnormal being unchanged. Fractional electrolyte excretion in- nephrons of chronic renal disease" (7). Impressive creased as the GFR fell. Despite marked heterogeneity overall functional renal adaptation accompanies these of single nephron filtration rate (SNGFR) (AICN, 5- marked structural changes (8-11 ). Information on 93 nl/min; anti-GBM, 0-50 nl/min) and of proximal the function of individual structurally altered nephrons, tubular hydrostatic pressure (4-48 mm Hg), each however, is sparse and apparently contradictory (12- nephron showed almost complete glomerulotubular bal- 17). ance, absolute reabsorption to the late proximal con- We have used micropuncture, clearance, immunofluo- volution varying directly with filtration rate. In addi- rescence and light microscopy techniques to study kid- tion SNGFR could be related both to proximal intra- ney structure and single nephron function in rats with tubular hydrostatic pressure and to calculated glomeru- two forms of immunologically induced glomerulone- lar capillary pressure (Pg), being lowest in those phritis. nephrons with the highest intratubular pressure. Ne- Firstly, autologous immune complex or Heymann's phrons with very high filtration rates did not appar- nephritis (AICN) 1 (18, 19) was chosen as a model ently reach filtration equilibrium. Mean SNGFR was resembling human membranous glomerulonephritis. significantly lower in the anti-GBM group, while cal- 1Abbreviations used in this paper: ADH, antidiuretic hor- culated Pg was the same in both. This probably re- mone; AICN, autologous immune complex nephritis; anti- flects the acute and diffuse involvement of the anti- GBM, antiglomerular basement membrane; BUN, blood GBM lesion with different filtration characteristics from urea nitrogen; C, clearance; CFA, complete Freund's ad- juvant; E, extraction; EFP, effective filtration pressure; Dr. Allison was a Career Investigator Fellow, American FF, filtration fraction; GBM, glomerular basement mem- Heart Association. Her present address is the Renal Unit, brane; GFR, glomerular filtration rate; PAH, p-aminohip- Glasgow Royal Infirmary, Glasgow, Scotland. purate; PAS, periodic acid-Schiff; PBS, 0.01 M phosphate- Dr. Gottschalk is a Career Investigator, American Heart buffered 0.15 M NaCl, pH 7.0; Pg, glomerular capillary Association. hydrostatic pressure; RPF, renal plasma flow; RTA, renal Received for publication 22 June 1973 and in revised tubular antigen; SNGRF, single nephron glomerular filtra- form 8 October 1973. tion rate; TBM, tubular basement membrane. 1402 The Journal of Clinical Investigation Volume 53 May 1974-1402-1423 Secondly, antiglomerular basement membrane (Anti- Blood pressure was recorded continuously from the carotid GBM) nephritis (20) was studied as a more acute, artery by using a Statham P23Db transducer (Statham In- struments, Inc., Oxnard, Calif.) and a Beckman Dynograph proliferative model of human glomerular injury. recorder (Beckman Instruments, Inc., Fullerton, Calif.). All rats were studied during hydropenia, modified Ring- METHODS er's solution (NaCl, 0.85 g/100 ml; KCl, 0.042 g/100 ml; CaCl2, 0.025 g/100 ml; NaHCO3, 0.02 g/100 ml) being AICN. 50 100 g male Wistar rats (Research Animals given i.v. at the rate of 0.4 ml/100 g body wt/h, together Inc., Braddock, Pa.) were given rear foot pad injections with d-aldosterone (Ciba Pharmaceutical Company, Sum- totalling 12.5 mg of partially purified rat renal tubular an- mit, N. J.), vasopressin (Parke, Davis & Co., Detroit, tigen (RTA) in 0.25 ml complete Freund's adjuvant Mich), ['H]methoxy inulin (International Chemical and (CFA) containing 2.5 mg of Mycobacterium butyricum Nuclear Corporation, Burbank, Calif.) and in most in- (Difco Laboratories, Detroit, Mich.). Intraperitoneal in- stances ['C]p-aminohippurate (PAH) (New England Nu- jections (18) were not used because we wished to avoid clear Corp., Boston, Mass.). In those rats in which intra- granulomatous peritonitis with subsequent micropuncture renal pressure measurements were made a priming dose difficulties. RTA was prepared by a slight modification of of 3 Ag of d-aldosterone, 60 ,uCi ['H] inulin, and 2 GCi that described by Edgington, Glassock, and Dixon (21). [CIC]PAH was followed by infusion of d-aldosterone, 2.4 A suspension of Wistar and Sprague Dawley rat renal ,ug/h; antidiuretic hormone (ADH), 0.03 ,g/h; ['C] PAH, cortex (Pel-Freez Biologicals, Inc., Rogers, Ark.) was 5-7 ,Ci/h; and ['H]inulin at a rate of 20 ,Ci/100 g body prepared by passing cortical fragments through a 150 mesh wt/h. When proximal tubular F/P inulin levels were to stainless steel screen. Basement membrane and cellular be measured this infusion was increased to 40-80 ,uCi [3H]- fragments were removed by low speed centrifugation (150 inulin/100 g body wt/h. g). The supernate was then centrifuged at 78,000 g for 30 Three consecutive timed urine collections, each lasting min and the resultant sediment washed two times in PBS approximately 45-0 min, were made from both kidneys. (0.01 M phosphate buffered, 0.15 M NaCl, pH 7.0) and Blood (<30 ,ul) was obtained from the carotid artery and lyophilized. (L) renal vein at approximately 45-min intervals for In 15 rats a second dose of 12.5 mg of RTA in CFA measurement of hematocrit, ['H] inulin, and ["C] PAH was given intradermally 51 days (five rats), 93 days (three levels. Clearances were determined as previously described rats), and 187 days (seven rats) later. In 28 rats, two addi- (27). Glomerular filtration rate (GFR) was calculated tional 12.5-mg doses were given 51 and 106 days (19 rats) separately for the left and right kidneys, renal plasma flow and 93 and 187 days (nine rats) after the initial injection. (RPF) for the left kidney only. 24-h urinary protein excretion rates were used to follow In those rats not given ["C] PAH the left RPF was the onset of overt glomerulonephritis. Determination of calculated as follows: urine protein concentration was made by using either the Biuret technique or salicylsulphonic acid. Clearance and micropuncture studies were done 5-20 mo after the first RPF = V(U-R) injection. Anti-GBM glomerulonephritis. Rabbit anti-rat GBM where V = urine flow rate and U, A, and R= inulin con- antibody was induced by biweekly injections (foot pads and centration in urine, arterial plasma, and renal venous intradermally) of 10-30 mg of rat GBM (22) in CFA. plasma, respectively. Antibody production was quantitated by the paired-label In those rats given ["C]PAH the RPF was calculated as isotope technique (23, 24). Male Wistar rats (100-150 g) were given 1.75-2.25 ml CPAH (five rats) and 1.5-2.2 ml (five rats) rabbit anti-rat GBM EPAH antibody containing 113 and 130 ,ug of kidney fixing anti- body per ml, respectively, and were studied 10-38 days later. where C = clearance and E = extraction. Intensified glomerular lesions were produced by enhancing Two groups of micropuncture experiments were carried the recipients immune response to the heterologous anti- out In the first series (six AICN, five anti-GBM, and GBM antibody by administering 1.8 mg of rabbit IgG in three control rats) the kidney was bathed with mineral oil incomplete Freund's adjuvant 3 days before receipt of anti- heated to 36+1'C. Sharpened siliconized glass pipettes, ex- GBM antibody (25). ternal tip diameter 4-6 ,sm, filled with isotonic saline colored Controls. 17 male Wistar rats, of a similar age to the with lissamine green (K and K Laboratories, Inc., Plain- AICN animals, were used as controls. In eight that were view, N. Y.) were used to puncture a superficial proximal 4-wk old, CFA alone was injected intradermally in the convolution, and the free flow hydrostatic pressure was proportions given above, and injections were repeated 93 measured by using the "Landis" technique (26). Simul- days later in four of them. The remaining nine rats re- taneously the last loop of the proximal tubule visible on ceived no injections. the surface of the kidney was identified by following the Clearance and micropuncture studies. Clearance and, in passage of the lissamine green colored saline. This last most instances, micropuncture studies were made in 24 rats convolution was later punctured with a sharpened silicon- with AICN, in all 10 rats with anti-GBM nephritis, and ized glass pipette, external tip diameter 11-12 Am, and a in 12 control rats.