pathophysiology of experimental glomerulonephritis in … · pathophysiology of experimental...

Pathophysiology of ExperimentalGlomerulonephritis in Rats

MARJORIEE. M. ALLISON, CuRTIs B. WILSON, and CARLW. GOrrSCHALK, with thetechnical assistance of SARAHK. DAVIS

From the Department of Medicine, University of North Carolina School ofMedicine, Chapel Hill, North Carolina 27514 and the Department of ExperimentalPathology, Scripps Clinic and Research Foundation, La Jolla, California 92037

A B S T R A C T Micropuncture, clearance, immunofluo-rescence and light microscopy techniques were used tostudy kidney structure and single nephron function inrats with autologous immune complex nephritis(AICN), a membranous glomerulonephritis develop-ing over 5 to 20 mo, in the more acute and prolifera-tive glomerular basement membrane (GBM) nephritisand in controls. Both models are known to have clinicalcounterparts in human disease. Kidney functional ab-normalities correlated with the degree of architecturalderangement. In both AICN and anti-GBM nephritisfiltration fraction fell in direct proportion to the fall inglomerular filtration rate (GFR), renal plasma flowbeing unchanged. Fractional electrolyte excretion in-creased as the GFR fell. Despite marked heterogeneityof single nephron filtration rate (SNGFR) (AICN, 5-93 nl/min; anti-GBM, 0-50 nl/min) and of proximaltubular hydrostatic pressure (4-48 mm Hg), eachnephron showed almost complete glomerulotubular bal-ance, absolute reabsorption to the late proximal con-volution varying directly with filtration rate. In addi-tion SNGFRcould be related both to proximal intra-tubular hydrostatic pressure and to calculated glomeru-lar capillary pressure (Pg), being lowest in thosenephrons with the highest intratubular pressure. Ne-phrons with very high filtration rates did not appar-ently reach filtration equilibrium. Mean SNGFRwassignificantly lower in the anti-GBM group, while cal-culated Pg was the same in both. This probably re-flects the acute and diffuse involvement of the anti-GBMlesion with different filtration characteristics from

Dr. Allison was a Career Investigator Fellow, AmericanHeart Association. Her present address is the Renal Unit,Glasgow Royal Infirmary, Glasgow, Scotland.

Dr. Gottschalk is a Career Investigator, American HeartAssociation.

Received for publication 22 June 1973 and in revisedform 8 October 1973.

the more chronic AICN disease. Tubular damage wasmore marked in AICN, and extraction of p-aminohip-purate was reduced in this group.

INTRODUCTION

Since the classical description by Richard Bright in1827 of the clinical effects of chronic renal disease amultitude of detailed studies have been made of wholekidney function in such patients (1-5). The pains-taking microdissection studies of Oliver (6) haveshown that such kidneys are "a heterogeneous collec-tion of various and disparate organs, the abnormalnephrons of chronic renal disease" (7). Impressiveoverall functional renal adaptation accompanies thesemarked structural changes (8-11 ). Information onthe function of individual structurally altered nephrons,however, is sparse and apparently contradictory (12-17).

Wehave used micropuncture, clearance, immunofluo-rescence and light microscopy techniques to study kid-ney structure and single nephron function in rats withtwo forms of immunologically induced glomerulone-phritis.

Firstly, autologous immune complex or Heymann'snephritis (AICN) 1 (18, 19) was chosen as a modelresembling human membranous glomerulonephritis.

1Abbreviations used in this paper: ADH, antidiuretic hor-mone; AICN, autologous immune complex nephritis; anti-GBM, antiglomerular basement membrane; BUN, bloodurea nitrogen; C, clearance; CFA, complete Freund's ad-juvant; E, extraction; EFP, effective filtration pressure;FF, filtration fraction; GBM, glomerular basement mem-brane; GFR, glomerular filtration rate; PAH, p-aminohip-purate; PAS, periodic acid-Schiff; PBS, 0.01 M phosphate-buffered 0.15 M NaCl, pH 7.0; Pg, glomerular capillaryhydrostatic pressure; RPF, renal plasma flow; RTA, renaltubular antigen; SNGRF, single nephron glomerular filtra-tion rate; TBM, tubular basement membrane.

The Journal of Clinical Investigation Volume 53 May 1974 -1402-14231402

Secondly, antiglomerular basement membrane (Anti-GBM) nephritis (20) was studied as a more acute,proliferative model of human glomerular injury.

METHODSAICN. 50 100 g male Wistar rats (Research Animals

Inc., Braddock, Pa.) were given rear foot pad injectionstotalling 12.5 mg of partially purified rat renal tubular an-tigen (RTA) in 0.25 ml complete Freund's adjuvant(CFA) containing 2.5 mg of Mycobacterium butyricum(Difco Laboratories, Detroit, Mich.). Intraperitoneal in-jections (18) were not used because we wished to avoidgranulomatous peritonitis with subsequent micropuncturedifficulties. RTA was prepared by a slight modification ofthat described by Edgington, Glassock, and Dixon (21).A suspension of Wistar and Sprague Dawley rat renalcortex (Pel-Freez Biologicals, Inc., Rogers, Ark.) wasprepared by passing cortical fragments through a 150 meshstainless steel screen. Basement membrane and cellularfragments were removed by low speed centrifugation (150g). The supernate was then centrifuged at 78,000 g for 30min and the resultant sediment washed two times in PBS(0.01 M phosphate buffered, 0.15 M NaCl, pH 7.0) andlyophilized.

In 15 rats a second dose of 12.5 mg of RTA in CFAwas given intradermally 51 days (five rats), 93 days (threerats), and 187 days (seven rats) later. In 28 rats, two addi-tional 12.5-mg doses were given 51 and 106 days (19 rats)and 93 and 187 days (nine rats) after the initial injection.24-h urinary protein excretion rates were used to followthe onset of overt glomerulonephritis. Determination ofurine protein concentration was made by using either theBiuret technique or salicylsulphonic acid. Clearance andmicropuncture studies were done 5-20 mo after the firstinjection.

Anti-GBM glomerulonephritis. Rabbit anti-rat GBMantibody was induced by biweekly injections (foot pads andintradermally) of 10-30 mg of rat GBM (22) in CFA.Antibody production was quantitated by the paired-labelisotope technique (23, 24).

Male Wistar rats (100-150 g) were given 1.75-2.25 ml(five rats) and 1.5-2.2 ml (five rats) rabbit anti-rat GBMantibody containing 113 and 130 ,ug of kidney fixing anti-body per ml, respectively, and were studied 10-38 days later.Intensified glomerular lesions were produced by enhancingthe recipients immune response to the heterologous anti-GBMantibody by administering 1.8 mg of rabbit IgG inincomplete Freund's adjuvant 3 days before receipt of anti-GBMantibody (25).

Controls. 17 male Wistar rats, of a similar age to theAICN animals, were used as controls. In eight that were4-wk old, CFA alone was injected intradermally in theproportions given above, and injections were repeated 93days later in four of them. The remaining nine rats re-ceived no injections.

Clearance and micropuncture studies. Clearance and, inmost instances, micropuncture studies were made in 24 ratswith AICN, in all 10 rats with anti-GBM nephritis, andin 12 control rats. All were deprived of food but not ofwater overnight before study, and were anesthetized byintraperitoneal injection of sodium pentobarbital, 50 mg/kgbody wt. The rats were prepared for micropuncture aspreviously described (26), isotonic saline, 0.5%o of body wt,being given during surgery to replace fluid losses. Bothureters were cannulated with PE 10 polyethylene tubing.

Blood pressure was recorded continuously from the carotidartery by using a Statham P23Db transducer (Statham In-struments, Inc., Oxnard, Calif.) and a Beckman Dynographrecorder (Beckman Instruments, Inc., Fullerton, Calif.).

All rats were studied during hydropenia, modified Ring-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) beinggiven i.v. at the rate of 0.4 ml/100 g body wt/h, togetherwith d-aldosterone (Ciba Pharmaceutical Company, Sum-mit, N. J.), vasopressin (Parke, Davis & Co., Detroit,Mich), ['H]methoxy inulin (International Chemical andNuclear Corporation, Burbank, Calif.) and in most in-stances ['C]p-aminohippurate (PAH) (New England Nu-clear Corp., Boston, Mass.). In those rats in which intra-renal pressure measurements were made a priming doseof 3 Ag of d-aldosterone, 60 ,uCi ['H] inulin, and 2 GCi[CIC]PAH was followed by infusion of d-aldosterone, 2.4,ug/h; antidiuretic hormone (ADH), 0.03 ,g/h; ['C] PAH,5-7 ,Ci/h; and ['H]inulin at a rate of 20 ,Ci/100 g bodywt/h. When proximal tubular F/P inulin levels were tobe measured this infusion was increased to 40-80 ,uCi [3H]-inulin/100 g body wt/h.

Three consecutive timed urine collections, each lastingapproximately 45-0 min, were made from both kidneys.Blood (<30 ,ul) was obtained from the carotid artery and(L) renal vein at approximately 45-min intervals formeasurement of hematocrit, ['H] inulin, and ["C] PAHlevels. Clearances were determined as previously described(27). Glomerular filtration rate (GFR) was calculatedseparately for the left and right kidneys, renal plasma flow(RPF) for the left kidney only.

In those rats not given ["C] PAH the left RPF wascalculated as follows:

RPF = V(U- R)

where V= urine flow rate and U, A, and R= inulin con-centration in urine, arterial plasma, and renal venousplasma, respectively.

In those rats given ["C]PAH the RPF was calculated as

CPAHEPAH

where C = clearance and E = extraction.Two groups of micropuncture experiments were carried

out In the first series (six AICN, five anti-GBM, andthree control rats) the kidney was bathed with mineral oilheated to 36+1'C. Sharpened siliconized glass pipettes, ex-ternal tip diameter 4-6 ,sm, filled with isotonic saline coloredwith lissamine green (K and K Laboratories, Inc., Plain-view, N. Y.) were used to puncture a superficial proximalconvolution, and the free flow hydrostatic pressure wasmeasured by using the "Landis" technique (26). Simul-taneously the last loop of the proximal tubule visible onthe surface of the kidney was identified by following thepassage of the lissamine green colored saline. This lastconvolution was later punctured with a sharpened silicon-ized glass pipette, external tip diameter 11-12 Am, and amineral oil block, 4-5 tubular diameters in length was in-jected. Tubular fluid was then collected, controlled suctionbeing used to keep the oil block in place and the tubulardiameter constant if this was at all possible. The samplewas pulled into the straight part of the collection pipetteby using toluene, the length and diameter measured by usingan eyepiece micrometer, and the volume calculated. The

Pathophysiology of Experimental Glomerulonephritis in Rats 1403

whole sample was then discharged into counting fluid. Thefollowing calculations were made:

SNGFR= F/P inulin X tubular flow rate in nl/minPercent reabsorption to the late proximal convolution

( F/P inulin) XAbsolute reabsorption = fractional reabsorption

X SNGFRnl/minIn the second group (15 AICN, five anti-GBM, and

seven control rats) the kidney was bathed with isotonicsaline heated to 36±10C in order to allow the use of anelectronic servo-nulling device for measurement of hydro-static pressure as previously described (28). Sharpened,siliconized glass pipettes, external tip diameter 3-5 Aum, filledwith 2 M NaCl were used to measure hydrostatic pressurein proximal tubules during free flow, in the large efferentarterioles or stars, in intermediate vessels, which are largediameter straight vessels and which often arise from stars,and in the larger peritubular capillaries.

In an attempt to estimate glomerular capillary hydro-static pressure (Pg), the stop flow hydrostatic pressurewas also measured (28). The Pg was estimated as thesum of the stop flow pressure and the afferent oncoticpressure. In four rats with AICN and in five rats withanti-GBM disease timed collections of proximal tubularfluid from easily accessible proximal convolutions for cal-culation of SNGFRwere made immediately before mea-suring the stop flow pressure in the same nephron. In-advertent contamination of the tubular fluid sample withsuperficial saline is not important in this instance, sincewe are interested only in filtration rate. Hence we haveSNGFR, free flow hydrostatic pressure, and an estimateof glomerular hydrostatic pressure in individual superficialnephrons.

Efferent arteriolar protein concentration in these nephronswas calculated by using the whole kidney filtration fraction(FF) (29).Efferent arteriolar protein concentration

Afferent arteriolar protein concentration1 - whole kidney FF

Arterial plasma and serum protein concentrations weremeasured before, during, and at the end of each experi-ment by an adaptation of the Lowry technique (30) usingrat serum protein standards. These values were used tocalculate oncotic pressure (7ra) by the Landis and Pappen-heimer (31) equation:

7ra = 2.1Pa + 0.16Pa2+ 0.009Pa3In order to validate this equation for rat serum the oncoticpressure was also measured directly in samples from sixAICN and three control rats (G. Navar, University ofMississippi Medical Center, Jackson, Miss.). The calcu-lated mean value of 18.9±1.41 is not significantly differentfrom that obtained directly, 20.2±3.03 mmHg.

The afferent and efferent effective filtration pressures(EFP) were calculated as follows:

Afferent EFP = Pg - (ITP + ira)Efferent EFP = Pg - (ITP + 7re)

where 7ra is the afferent oncotic pressure, 7re the calculatedefferent oncotic pressure, and ITP the intratubular pressure.

These equations assume that the oncotic pressure of thetubular fluid is so small as to be insignificant, a situationwhich is true for the normal undamaged nephron (32). Wehave not measured the concentration of protein in theglomerular filtrate of the diseased nephron in this study,but Lewy and Pesco (33) and Oken, Cotes, and Mende(34) using animals with aminonucleoside damaged kidneysand proteinuria have reported an albumin concentration ofonly 2.7-234 mg/100 ml in the glomerular filtrate, and itseems unlikely that tubular fluid protein levels in our studywere high enough to exert a significant oncotic pressure.

Blood was taken terminally for estimation of blood ureanitrogen (BUN), measured on a Technicon autoanalyzer(Technicon Corporation, Ardsley, N. Y.) by a modificationof the carbamido-diacetyl fraction as applied to the deter-mination of urea nitrogen and cholesterol. Urine and plasmaelectrolytes were measured on an IL direct reading flamephotometer (Instrumentation Laboratories Inc., Watertown,Mass.). Fractional electrolyte excretion was calculatedwithout use of the Donnan Factor.

Microinjection studies. In order to examine the perme-ability of the nephrons to inulin a separate series of micro-injection experiments (35) were performed in nine ratswith anti-GBM nephritis and in two with AICN, preparedas previously described. For microinjection studies theywere infused with 5% mannitol in isotonic saline at 3.1ml/h and PE 50 catheters were placed in both ureters. Ameasured volume (0.8-3.5 nl) of [8H]inulin (InternationalChemical and Nuclear Corporation) colored with nigrosin,was injected slowly over 30-60 s into superficial proximaltubules, 8-min urine collections being made from eachkidney for 24-120 min thereafter into liquid scintillationcounting fluid (PCS, Amersham/Searle, Arlington Heights,Ill.). The total percentage of the measured injectate re-covered from each kidney was calculated. The hydrostaticpressure in each nephron injected was measured by using aservo-nulling pressure apparatus.

Immunopathologic studies.- At the end of each experi-ment, both kidneys from each animal were weighed. Thekidneys were bisected along the anterior-posterior axis soas to assure adequate assessment of the entire thickness ofthe cortex, medulla, and papilla. One half of the rightkidney was snap frozen (liquid nitrogen or a dry ice-alcoholbath) to await immunofluorescent studies. Cryostat sectionsfrom this kidney were overlayed with fluorescein iso-thiocyanate-conjugated IgG fractions of antiserum mono-specific in immunodiffusion for rat IgG and C3, for directimmunofluorescent study (36). The remainder of the kidneytissue was fixed in Bouin's solution and postfixed in 50%oethyl alcohol. Sagittal sections of the entire bisected surfacewere obtained after paraffin embedding. The sections werestained with hematoxylin-eosin and periodic acid-Schiff(PAS) for light microscopy.

All sections were studied as unknowns. The pattern ofimmunoreactant (IgG, C3) deposition was noted and semi-quantitated by using a 0-4 scale (37). The histologic sec-tions were graded 0 to 4+ for the following categories:glomerular hypercellularity, necrosis, sclerosis; GBMthick-ening; overall architectural derangement; interstitial infil-tration; tubular atrophy, luminal cells, casts; and thicken-ing of tubular basement membrane (TBM). The numberof polymorphonuclear leukocytes per glomerulus was re-corded as well as the percentage of glomeruli exhibitingcrescent formation. Vessels were graded similarly (04+)for degree of endothelial proliferation, duplication of theelastica and medial hypertrophy. Perivascular infiltration

1404 M. E. M. Allison, C. B. Wilson, and C. W. Gottschalk

was generally included in the general category of inter-stitial infiltration. Since the micropunctures studies ofnecessity produced data from superficial nephrons, carefulattention was given any possible variation in immunopatho-logic changes throughout the thickness of the cortex.

RESULTS

General characteristicsWeattempted to induce AICN in a total of 50 male

Wistar rats aged approximately 1 mo. 48/50 rats(96%) had histological evidence of glomerulonephritiswhen examined 5-20 mo after 1-3 intradertual injec-tions of RTA; 39 of 46 studied (85%) had protein-uria exceeding 100 mg per day. Only 2/17 of similarlyaged control rats (13%) ultimately developed protein-uria greater than 100 mg per day, when aged 17 moas previously described (37). None of the control ratsgiven CFA alone (8/17) developed proteinuria greaterthan 60 mg per day, although this has been reportedas causing significant proteinuria in previous studies(38, 39). Fig. 1 shows the varying rates of develop-ment of proteinuria in 19 rats with AICN in whichthis was followed serially and in controls. There wasno relationship between the total number of antigen in-jections given and the subsequent degree of protein-uria, the greatest and most rapidly developing protein-uria (885 mg per day) being found in a rat given asingle injection of RTA.

All ten rats given anti-GBM antibody and subse-quently studied by micropuncture developed significantproteinuria, so that when studied in the autologousphase 10-38 days later (mean, 20 days) all had pro-teinuria in excess of 100 mgper day.

Table I compares the serum protein, BUN, andcholesterol concentrations according to the final degreeof proteinuria measured in the AICN, anti-GBM, andcontrol rats. There was no evidence of hypoproteinemiain the rats with AICN, even in those with proteinuriagreater than 400 mg per day. In the more acute anti-GBMnephritis group, however, mean serum protein

1971 -4--- 1972

FIGURE 1 Development of proteinuria after one to threeinjections of RTA in 19 rats with AICN and in controlrats of a similar age.

levels were significantly reduced below that of thecontrols.

The general characteristics of that portion of thetotal group in which detailed clearance and micropunc-ture studies were later carried out (24 rats with AICN,5-151 mo after initial injections; 10 rats with anti-GBM nephritis, 10-38 days after injection; and 12control animals) were similar. Daily protein excretionrates varied from 15 to 885 mg (mean 357 mg ±238SD) in AICN and from 137 to 570 mg (mean 356 mg±145 SD) in anti-GBM nephritis. 24 h urine volumewas significantly increased in AICN rats (controls12.7 ml +4.0 SD, AICN 23.7 ml +-11 SD, P < 0.001).Mean plasma protein concentration was significantlyreduced, however, only in the anti-GBM nephritisgroup (controls 5.68 g/100 ml +0.36 SD, Anti-GBM4.88+0.70 SD, P <0.02). None were edematous, al-though mean hematocrits were significantly reduced(controls 48.6%±3.5 SD, AICN 44.4%+5.4 SD, P<0.05; Anti-GBM 41.2%±5.6 SD, P <0.001). Allhad hypercholesterolemia. Mean BUN concentrationwas significantly higher than in control rats only inanti-GBM nephritis (controls 18-44.5 mg/100 ml, mean

TABLE I

Relationship of Proteinuria to Serum BUN, Cholesterol, and Protein Concentrations in Control and Glomerulonephritic Rats

AICN Controls Anti-GBMAge 6-21 mo Age 5-17 mo Age 2-3 mo

Serum Serum SerumProteinuria x BUN Cholesterol protein n BUN Cholesterol protein n BUN Cholesterol protein

mg/day mg/ZOOml mg/100 ml g/100 ml mg/1OO ml mg/1OO ml g/100 ml mg/fOO ml mg/100 ml g/100 ml

0-50 5 22.847.6 69.6435.7 5.89±0.44 12 27.749.1 76.5±-23.4 5.77±4-0.35 - --50-100 2 22.0 120.0 5.78 3 30.7 ± 16.2 84.5 430.4 5.84 ±0.06 - - - -

100-200 7 26.9±9.84 108.3±28.2 5.9740.59 2 11.0 123.5 5.90 1 31 114 5.88200-400 17 19.3±+13.7 168.6457.8 5.67±+0.31 - - - - 4 40.1 ±12.9 216.8±70.7 5.0640.50400+ 15 31.1±412.7 237.4±88.3 5.66+0.56 - - - - 5 50.3413.1 244.0±68.4 4.54±0.86

m = number of rats. All figures are mean4SD.


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FIGuRE 2 Relationship between the filtration rate of the left kidney and the degree of pro-teinuria, BUN concentration, FF, and fractional potassium excretion in 24 rats with AICN,10 rats with anti-GBM nephritis, and in 12 controls.

29.8 mg/100 ml +10.0 SD; anti-GBM 27-69 mg/100ml, mean 44.3 mg/100 ml ±13.4 SD, P<0.001). Bothexperimental groups had significantly increased mean

systemic blood pressure (controls: 106 mmHg ±11 SD,AICN: 118 mmHg ±14 SD, P<0.02; anti-GBM:126 mmHg ±14, P < 0.01).

Clearance studiesClearance data are given for the (L) kidney, there

being no statistically significant difference between the(R) and (L) kidney GFR.

Fig. 2 illustrates the relationship between kidney fil-tration rate and the degree of proteinuria, BUN con-

centration, FF, and potassium excretion. Quantitativeproteinuria increased as the GFR fell (AICN: r=

0.636, n = 24, P < 0.01; anti-GBM: r = 0.869, n = 10,P< 0.01). Filtration. fraction fell in direct proportion

to the fall in GFR, since the RPF, as measured by theclearance (CPAH) and extraction of PAH (EPAHI) or

inulin, did not decrease. Fractional excretion of sodium(not illustrated) and potassium increased, so that whenthe GFR was reduced to approximately 10% of con-

trol over 0.3% of the filtered load of sodium and 50%of the filtered load of potassium was excreted.

Table II compares the clearance data in animals withproteinuria in excess of 200 mg per day with con-

trols. GFR and RPF have been expressed in ml/min,ml/min/g kidney wt and ml/min/100 g body wt sincekidney and body wt varied significantly between theanti-GBM rats and the others (Table III). MeanGFR was significantly reduced in both experimentalmodels irrespective of the method of presentation. RPF,however, was significantly increased in the group withanti-GBM nephritis when calculated /100 g body wt.


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TABLE I IClearance Data (Left Kidney) in Control Rats and in Glomerulonephritic Rats with

Proteinuria Greater than 200 mg per Day (Mean+SD)

AICN Control Anti-GBMn = 17 P n = 12 P n = 9

GFRml/min 1.04±-0.4 <0.001 1.96--0.5 <0.001 0.714-0.36ml/min/g kidney wt 0.40-±0.16 <0.001 0.97--0.24 <0.001 0.43±-0.24ml/min/100 g body wt 0.16-±0.06 <0.001 0.30±-0.08 <0.001 0.16--0.10

RPFml/min 9.18±-4.1 NS 7.97--2.7 NS 7.42-±1.8ml/min/g kidney wt 3.39±-1.29 NS 3.94±-1.31 NS 4.70--1.44ml/min/100 g body wt 1.37-±0.55 NS 1.23-±0.41 <0.001 2.47-±0.74

FF 0.144-0.05 <0.001 0.27-±0.05 <0.001 0.11±-0.05CPAH* 5.17--1.6 NS 6.56±42.17 NS 6.10±-1.5

ml/min/g kidney wt 1.95 -0.68 <0.02 3.22 ±0.89 NS 3.88±-1.29ml/min/100 g body wt 0.78±-0.22 NS 1.03--0.30 <0.01 2.03 -0.62

CIn/CPAH 0.18±-0.03 <0.01 0.29±-i0.07 <0.01 0.14±-0.06EPAH 0.71-±0.14 <0.05 0.86±L-0.06 NS 0.83-±0.08U/P inulin 194±-131 <0.001 624±-133 <0.001 225±-142

Sodium excretionAeq/min 0.21±--0.13 NS 0.18±-0.24 NS 0.19±-0.15%Filtered load Na

excreted 0.14±40.09 <0.05 0.06±-0.07 <0.01 0.19±-0.09

Potassium excretionueq/min 1.084-0.65 NS 1.01±-0.90 NS 0.83±-0.46%Filtered load K

excreted 18.6-- 1 1.8 <0.01 5.88±-2.2 <0.001 29.5± 18.3

P, comparison of control and AICN or anti-GBM data by unpaired Student t test.* CPAHwas measured in eight AICN, six control, and seven anti-GBM rats.

Mean EPAH was within normal limits (40) in anti-GBMnephritis while this was significantly reduced in theAICN rats. Electrolyte excretion rates and serum elec-trolyte concentrations were not significantly differentin the three groups.

Micropuncture studiesSeverely damaged kidneys from rats with either

AICN or anti-GBM nephritis were enlarged, pale, and

granular. Microscopic examination of the cortical sur-face of the living kidney showed marked heterogeneityof single nephron structure, the severity of which cor-related generally with the severity of disease as judgedby the whole kidney filtration rate. Patches of large,dilated, paler proximal tubules alternated with smallerareas of small atrophic-looking nephrons. White speckssometimes visible on the kidney surface were found tobe tubular casts in dilated nephrons. Small whitish

TABLE IIIBody Wt and Left Kidney Wt in Glomerulonephritic Rats and in Control Wistar Rats of Similar Age

AICN AICNProteinuria Proteinuria

Control 200 mg/day 200 mg/day Control* Anti-GBMn = 12 n = 7 P n = 17 P n = 20 n 2 1O P

Body wt 649 634 NS 656 NS 326 290 NSg ±61 471 ±92 ±46 ±51

Left kidney wt 0.320 0.333 NS 0.406 <0.001 0.3803 0.594 <0.001g/100 g body wt ±0.04 ±0.04 ±0.05 ±0.05 ±0.17

* Data obtained from Dr. R. Kramp.


A ICN

60r 15 RATS

__CONTF

4 8 12 16

ROL

20 24 28 32 36 40 44 48

ANTI-GBM NEPHRITIS

7 RATS

-~~~ ~~~~~~~~~~~~~~~~~~~r.T--4 8 12 16 20 24 28 32 36 40 44 48PROXIMAL INTRATUBULARPRESSURE

mmHg

10 RATS

SNGFRni/min

FIGURE 3 Distribution of proximal intratubular hydrostatic pressure and SNGFRmeasure-ments in 15 rats with AICN, eight rats with anti-GBM nephritis, and in controls.

pieces of material could at times be seen passing downtubules or adhering to the walls. The postglomerularcirculation appeared less plentiful, with fewer "stars"and smaller peritubular capillaries. Less severely dam-aged kidneys, in animals with proteinuria less than 200mg per day, showed only occasional patches of dilatednephrons and a normal looking post-glomerular vas-

culature.Impressive functional heterogeneity was revealed by

single nephron studies of filtration rate and proximaltubular hydrostatic pressure in both AICN and anti-GBMrats with proteinuria over 200 mg per day (Fig.3). Thus in rats with AICN, SNGFRvaried overallfrom 0 to almost 100 nl/min. More importantly thispattern of functional heterogeneity was also found inindividual kidneys. Although the mean SNGFRwas

significantly lower in the smaller rats with anti-GBMdisease individual filtration rates again varied greatly,

1408 M. E. M. Allison, C. B. Wilson, and C. W.

ranging from 0 to 50 nl/min. These findings are to becompared with the much smaller scatter found in con-

trol rats, in which over 50% of all nephrons had filtra-tion rates between 32 and 40 nl/min.

Similarly proximal tubular hydrostatic pressurevaried from 4 to 44 mmHg in AICN and from 4 to48 mmHg in anti-GBM disease. In general thosenephrons with high intratubular pressures appearedlarge and dilated on microscopic examination of theliving kidney. In contrast, in control rats, all pressuresrecorded were between 8 and 16 mmHg. We havepreviously shown (27) that proximal intratubular pres-

sure is the same in normal rats weighing 230-275 gand 545-740 g, similar to the weights of the anti-GBMand AICN rats, respectively.

Despite this marked heterogeneity of single nephronfiltration rate absolute reabsorption to the late proxi-mal convolution was in direct proportion to the filtra-

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r---n

tion rate, so that fractional reabsorption was uniform(Fig. 4). Thus in both forms of glomerulonephritisindividual nephrons demonstrated almost completeproximal glomerulotubular balance at all levels ofSNGFR.

Simultaneous measurements of SNGFRand wholekidney filtration rate were made in 52 nephrons in 11animals with AICN, in 50 nephrons of 10 rats withanti-GBM nephritis, and in 15 nephrons of four controlrats (Table IV). It is interesting to note that, althoughthe mean whole kidney glomerular filtration rate wassignificantly reduced below control levels in the animals

TABLE IVComparison of Left Kidney Filtration Rate and mean SNGFRI

Kidney in Control and Glomerulonephritic Rats

Wholekidney

Group GFR SNGFR

mi/mn nm/minAICN 0.88 39.6

0.86 36.01.78 63.11.10 29.51.27 54.51.24 52.11.61 64.20.63 40.70.91 42.40.84 28.41.04 46.2

Mean 1.11 43.3SD 4-0.35 i 12.6P <0.05 NS

Control 1.42 43.81.42 34.12.07 47.11.50 37.2

Mean 1.60 40.6SD 4-0.31 i6.0

Anti-GBM 0.80 20.20.35 12.61.13 32.40.23 6.40.57 17.01.02 21.70.70 17.61.10 31.20.70 24.10.32 11.1

Mean 0.69 19.4SD 4-0.33 :4-8.4P <0.001 <0.001

P, comparison of GFRand SNGFRdata by unpaired Studentt test.

AICN8 RATS

y-2.803O0.42Xr *0.855,Pc0.01

0

30 -

0

201

lop

ABSOLUTEREABSORPTION

TO LATEPROXIMAL

CONVOLUTIONnllnin 50

40

ANTI -GBM NEPHRITIS8 RATS

y-0.01 0.463Xr * 0.9080.01

301

20_

101_a U

20 40 60SNGFRnl/min

80 100

FIGuRE 4 Correlation between absolute reabsorptive rateto the late proximal convolution and SNGFR in super-ficial nephrons of kidneys of rats with AICN and anti-GBMdisease.

with AICN, mean SNGFRwas not different from con-trols. This could be due either to a sampling error inthe selection of remaining nephrons for puncture in thesick rats, with a tendency to choose the more normallooking ones with the higher filtration rates, or to afall in the whole kidney filtration rate in AICN re-sulting from the loss of substantial numbers of ne-phrons. Wedo not think that there was a major sam-pling error, since there was a good correlation betweenthe mean SNGFRper rat and the whole kidney filtra-tion rate (Fig. 5A). For any given level of whole kid-ney filtration rate, however, a wide range of SNGFRwas found (Fig. 5B). Thus while the overall numberof surviving nephrons was reduced those remainingwere heterogeneous in function. In anti-GBM nephritiswhole kidney and single nephron filtration rates werereduced to a similar degree, probably due to the acute-ness and uniformity of the injury, suggesting that lossof more damaged nephrons had not yet occurred.

There was no significant difference between the meanvalues found for SNGFR, F/P inulin, and absolute re-

Pathophysiology of Experimental Glomerulonephritis in Rats

*:;

1409

8o F * AICN* ANTI-GBMA CONTROL

60 1

SNGFRni /min 40

20 _

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LA* :

UU t

aa

0a

1.0

80 F

60 _

SNGFRnl/min

40 F

20

0 AA *A^

0

8

Om.* *

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FIGURE 5 (A) Relationship of mean SNGFRwhole kidney filtration rate in 11 rats with AI(anti-GBM nephritis and in controls. (B) Rel;individual SNGFRmeasurements to whole kidnrate in the same rats.

absorptive rate to the late proximal convohAICN and control rats (control SNGFRmin -7.2 SD, F/P inulin = 2.71+±0.8 SSNGFR= 47.8 nl/min ±18.5 SD, F/P iI

±0.4 SD). Animals with anti-GBM disease had sig-nificantly lower SNGFRand absolute reabsorptive ratesthan either AICN or controls (SNGFR= 17.7 ml/min +8.6 SD, F/P inulin = 1.81±0.2 SD), a finding

£ not entirely explained by their smaller size since pre-vious studies have shown the normal hydropenic Wis-tar rat kidney to have superficial SNGFRvalues of25-35 nl/min. Mean F/P inulin values were not sig-

A nificantly different, probably due to the small numberof observations involved in the control group.

Table V gives the mean values, calculated per rat, forthe hydrostatic pressure in the carotid artery, efferentarteriole, large peritubular capillary, and proximal tu-bule in rats with AICN and in controls, together withthe carotid pressure and free flow proximal tubularpressure in rats with anti-GBM nephritis. The latterhad significantly higher systemic and proximal tubularpressures than the other two groups. No statisticallysignificant difference in efferent arteriolar or peri-tubular capillary pressures were seen in the controland AICN rats, although it should be noted that all

A mean postglomerular hydrostatic pressures were lowerin the animals with AICN. This tendency was evenmore marked in individual severely damaged kidneys,where the lowest peritubular capillary pressure recorded

B was 4.5 mmHg (lowest control 7.5 mmHg).Mean values for estimated glomerular oncotic and

hydrostatic pressures, calculated per rat, in 11 animals2.0 with AICN, five with anti-GBM nephritis and in five

control rats are given in Table VI. Animals with anti-Lper rat to GBMdisease had significantly lower afferent and ef-CN, 10 withationship of ferent oncotic pressures, but significantly higher cal-ley filtration culated Pg than the controls. Filtration equilibrium

apparently was not reached in any of the three groups,this being especially marked in the rats with anti-GBM

ition in the nephritis, mean estimated efferent EFP being 25.1 mm=41.7 nl/ Hg.

;'D; AICN In an attempt to determine the factors regulatingnulin = 2.04 single nephron filtration rate we examined the relation-

TABLE VHydrostatic Pressure (mm Hg) in Carotid Artery, Proximal Tubules, Efferent Arterioles, and Peritubular Capillaries in Control

and Glomerulonephritic Rats (Mean±SD)

AICN P Control P Anti-GBM

Carotid artery 119±11.3 (15)* <0.02 106±11.5 (9) <0.0025 125±6.8 (7)

Efferent arteriole 11.241.8 (11) <0.05 12.7±1.0 (9)

Large peritubular capillary 7.6±2.8 (2) NS 10.3±2.0 (5)Proximal tubule 13.1±5.4 (15) NS 12.1±1.8 (9) <0.0025 16.2±2.9 (7)

P, comparison of means by unpaired Student t test.* Figures in brackets refer to number of rats.


*

0 011

-0

a

ship between each nephron's measured filtration rateand intratubular hydrostatic pressure, calculated Pg andafferent and efferent EFP for that nephron. Fig. 6shows the relationship between SNGFRand free flowhydrostatic pressure. In animals with AICN nephronswith higher intratubular pressures had low filtrationrates. However in most instances no relationship wasobserved between SNGFR and pressure, a fivefolddifference in SNGFRbeing found in nephrons of simi-lar intratubular pressure. In the anti-GBM group athreefold difference in intratubular pressure was notassociated with any difference in filtration rate.

A closer relationship was seen between single ne-phron filtration rate and calculated Pg (Fig. 7), therelationship being statistically significant, however, onlyfor the rats with anti-GBM disease. It is interestingto note that although the mean calculated capillarypressure was almost the same in the two groups (55.3mmHg in AICN and 58.0 mmHg in anti-GBM)mean SNGFRwas significantly greater in the AICNgroup (37.0 nl/min in AICN, 21.6 nl/min in anti-GBM) suggesting, perhaps, a difference in glomerularmembrane filtration characteristics in the two diseasestates, as in permeability and/or area.

SNGFRwas also closely related to calculated affer-ent and efferent EFP (Fig. 7). Filtration equilibriumwas apparently approached only in those nephrons withvery low filtration rates and the higher free flow pres-sures. Nephrons with higher filtration rates did notappear to reach equilibrium, estimated efferent EFPbeing as high as 45-65 mmHg.

Microinjection studiesStructural and functional changes in the kidneys of

the animals used for microinjection studies were simi-lar to those previously described. Proteinuria rangedfrom 133 to 550 mg per day (mean 305 mg+123 SD)and BUN from 14 to 116 mg/100 ml (mean 44.8 mg/100 ml ±36.4 SD). Two animals developed grossascites and edema with serum protein concentrationsof 1.9 and 1.8 mg/100 ml.

Al CN10 RATS

2001

80

SNGFRnl/min

60

40

20

A

* -

* 9

* 0-u

0 % 0

ANTI-GBM NEPHRITIS8 RATS

100

80

SNGFRn l/min

60

40

20

B

*u a*

EU. U.* U..

5 10 '5 20 25 30

PROXIMAL INTRATUBULARPRESSURE rnmHg35 40

FIGURE 6 Relationship of SNGFR to proximal intra-tubular free flow hydrostatic pressure in (A) rats withAICN and in (B) rats with anti-GBM disease.

No evidence of significant leakage of inulin wasfound in those nephrons which appeared normal invivo and in which the intratubular hydrostatic pressurewas less than 19 mmHg. In 26 microinjected proximaltubules (20 anti-GBM, 6 AICN) hydrostatic pressureaveraged 13.0 mmHg +2.7 SD and inulin recoveryfrom the injected kidneys averaged 98.2%+9.1 SD. Thelatter is not different from the average value of 98.4+2.6 SD found in seven control tubules. There was no

TABLE VICalculated Glomerular Capillary Hydrostatic and Oncotic Pressures (mm Hg) in

Control and Glomerulonephritic Rats (Mean±LSD)

AICN Control Anti-GBMn = 11 P n =5 P n =5

Afferent oncotic pressure 16.1A1.04 NS 15.4±0.96 <0.02 13.8±0.67FF 0.1440.02 <0.001 0.22 40.02 <0.001 0.124±0.01Efferent oncotic pressure 20.1±1.84 <0.02 22.9±2.2 <0.001 16.6±0.93Pg 55.9±7.4 <0.02 47.4±-3.3 <0.02 58.5±9.4Afferent EFP 24.7±8.5 NS 19.9±3.1 NS 27.6±10.9Efferent EFP 20.3±8.7 <0.1 12.4±3.0 NS 25.1425.0

Pathophysiology of Experimental Glomerulonephritis in Rats

* 9

*0

.m

a0

0 a 0

1411

A ICtN

60

40

20

100 - C

80 _

00

0

0

0

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00

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so F

40

20

30 40 50 60 70 80 90 l0 20 30 40 50

ANTI-GBM NEPHRITIS

B 100F D

80 _

60 -

* a

a aa .

a

30 40 50 60 70 90

Pg mmHg(SFP+.o)

20

10 20 30 40 50 60

AFFERENTEFPmmHg(SFP-ITP)

60

40

20

0 /s- 0

aa

70 10 20 30 40 50 60 70

EFFERENT&FP mmHg(Pq-[e + ITP])

FIGuRE 7 (A and B) Correlation between calculated Pg and SNGFRin AICN (A, r =

0.362, P> 0.05) and in anti-GBM nephritis (B, r = 0.556, P < 0.01). (C and D) Correlationbetween calculated afferent EFP and SNGFRin AICN (C, r = 0.754, P <0.01) and anti-GBMnephritis (D, r = 0.453, P <0.01). (E and F) Correlation between calculated efferentEFP and SNGFRin AICN (E, r = 0.759, P <0.01) and in anti-GBM nephritis (F, r =0.414, P < 0.05). ITP, intratubular pressure; vra, afferent oncotic pressure; 7re, efferent oncoticpressure; SFP, stop flow pressure.

statistically significant excretion of inulin by the rightkidney. Urine flow rate averaged 13 A4/min, and theinjectate appeared in early distal tubules 0.8-4 minafter proximal injection, except in one nephron wherethe flow rate was very slow, 25 min elapsing beforethe injectate reached the early distal segment.

The eight microinjected anti-GBM nephrons withhigher intratubular pressures (mean 24.4 mmHg ±6.2SD) appeared dilated and all had very slow flow rates.In most the injectate remained visible in the super-

ficial proximal tubule for 1-2 h. In three no significantradioactivity was recovered from either kidney. In theother five, however, 19.8%±4.3 and 19.6±7.2 of themicroinjected inulin was recovered in the urine fromthe left and right kidneys, respectively. Although we

are unable to exclude the possibility of a significanttranstubular leakage of inulin in this circumstance, theleakage may well have occurred, in view of the highintratubular pressure and very slow tubular flow rate,at least in part, at the site of puncture and thus haveno physiological significance.

Immunopathologic studiesHistologically, the renal lesions induced either by

autologous immune complex deposits or by anti-GBM

antibodies were quite characteristic as were the im-munofluorescent findings. Varying severities of involve-ment were present in both models so that attempts tocorrelate physiologic and immunopathologic variationswere possible.

AICN. The glomerular lesions in the AICN ratsdeveloped slowly and were typified by uniform thick-ening of the glomerular basement membrane (GBM)with only minimal glomerular hypercellularity and in-crease in polymorphonuclear leukocytes characteristicof membranous glomerulonephritis. Proliferation ofBowman's capsule (crescent formation) and glomerularsclerosis were infrequent. The severity of glomerularinvolvement was quite uniform throughout the cortexof each individual kidney (Fig. 8). Although the GBMwas obviously thickened in all rats studied in this group,

the degree of thickening was extremely difficult toestimate even semi-quantitatively from PAS-stainedsections and correlated poorly with the severity ofeither proteinuria (r = 0.105, n = 23) or fall in GFR(r = 0.245, n = 23) (Table VII).

These glomerular lesions were accompanied bychanges in the renal tubules and interstitium whichled to different degrees of derangement in overall renalarchitecture. This latter parameter, which, in addition


A

80

60

SNGFRni/min

40

20

100r

60SNGFRni/min

40

000

00

0 0

0 0

00 0

moor F

FIGUE 8 The uniformity of membranous thickening of the GBMis shown at different levelsin the cortex from a rat with AICN. Virtually no proliferation accompanied the thickeningof the GBMas seen in glomeruli near the arcuate vessels (A) in the mid-cortex (B) or inthe superficial cortex (C). PAS stain. A-C, X 220.

to the glomerular pathology, was comprised of varyingdegrees of tubular atrophy and dilatation, interstitialmononuclear cell infiltration, tubular casts, and thepresence of intraluminal cells confining PAS-positivematerial either singly or in multinucleated form (Fig.9), correlated directly with the fall in GFR (r = 0.747,n = 23, P < 0.01) as well as with proteinuria (r =

0.711, n= 23, P <0.01) (Table VII). Similar corre-lations could be made when each component was ana-lyzed separately. Surprisingly, for the degree of overallrenal damage, only minimal medial thickening of smallarterioles was observed in the most hypertensive rats.

Granular deposits of IgG and C3 were present dif-fusely along the GBM(Fig. 10) in the 23 AICN ratsavailable from the 24 undergoing clearance studies.This deposition correlated poorly with amounts of pro-teinuria and only in a general way with GBMthicken-ing assessed by light microscopy. The deposits weremost clear in rats with less structural glomerular dam-age. As glomerular sclerosis developed, the depositionlessened in both intensity and extent. IgG eluted (36)

from these kidneys reacted with antigens present in thebrush border of normal proximal renal tubules whentested by indirect immunofluorescence on normal kidneysections.

Of additional interest, evidence from the immuno-fluorescent studies suggested an immune nature forsome of the renal tubular lesions observed in these ani-mals. IgG and C3 were found deposited along thebrush border of 8/23 of the more heavily proteinuricAICN rats studied (Fig. 11). This observation wouldsuggest that antibodies specific for rat RTA (residentin the brush border) of the proximal convoluted tubulewere being filtered and bound in vivo. This findingcorrelated well with the presence of chunks of IgGand C3 within the tubular lumens, unrelated to tubularcasts, and the presence of cells containing PAS-positivematerial adjacent to the brush borders and free withinthe tubular lumens (Fig. 9). In addition to brush bor-der and occasional intraluminal deposits of IgG andC3 (unrelated to albumin-containing casts) granulardeposits of these immunoreactants were observed in


the tubular cytoplasm near the TBM. This suggeststhat immune complexes either filtered into or formedin the tubular urine were being taken up by the tubu-lar cells or that antibody had gained access to the cellsto combine with intracellular antigens. Similar ob-servations have been made in Sprague Dawley ratswith AICN augmented by pertussis (41). No evidenceof anti-TBM antibodies was found by immunofluores-cence in this study.

Anti-GBM glomerulonephritis. In rats with experi-mental anti-GBM glomerulonephritis, the degree ofGBM irregularity, crescent formation, and architec-tural derangement assessed by light microscopy corre-lated well with the fall in GFR (Table VII). Theglomerular pathology typified by increased cellularity,polymorphonuclear leukocyte infiltration, proliferationof Bowman's capsule (crescent formation), and irregu-larly thickened GBMwas accompanied by correspond-ing degrees of general architectural derangement in-cluding tubular atrophy and interstitial cellular infil-tration. Glomerular alterations were quite uniformthroughout the cortex (Fig. 12), possibly with aslightly greater hypercellularity in the more superficialglomeruli. Virtually no vascular changes were encoun-tered, although the animals had significantly increasedmean systemic blood pressure.

Immunofluorescent studies revealed similar intensi-ties of linear IgG and C3 deposits in the nine kidneys

available for study. In kidneys with more GBMdam-age, the linear IgG and C3 deposits were increasinglyirregular (Fig. 10) corresponding to the loss of thenormal smooth, ribbon-like structure of the GBM. Thepattern of deposition, however, was always easily dis-tinguishable from the granular immune complex de-posits typifying the AICN rats. Virtually no IgG de-posits were noted outside the glomeruli.

Controls. Seven of the control rats had minimalglomerular hypercellularity, irregularity of the GBM,and architectural derangement. These rats also hadmild proteinuria and slight decreases in renal function.The histological and functional abnormalities corre-lated with advancing age but not with the administra-tion of CFA. Spontaneous glomerulonephritis withadvancing age has been described in Wistar rats (42).The remaining control rats were free of histologic orfunctional abnormalities.

Small amounts of IgG and/or C3 in a segmentalgranular pattern were found in glomeruli of the ratswith histologic glomerular abnormalities suggestingthe presence of a low-grade spontaneous immune com-plex glomerulonephritis as has been reported in manyanimal species. No attempts were made to identify theantigenic portion of the presumed immune complex inthese animals. Similar IgG deposits were found in twoadditional rats that remained histologically and func-tionally normal.

TABLE VI IRelationship of Whole Kidney Function to Changes Seen on Light Microscopy of Right Kidney

in Control and Glomerulonephritic Rats*

Glomerular GBM Architectural U,/PGroup hypercellularity thickening Crescents derangementl GFR Inulin Proteinuria

%glomeruli ml/min g mg/24 hkidney Wt

AICNn = 11 0.4 2.0 0-10 1.7 0.30 122 535n = 12 0.5 1.8 Rare 0.9 0.74 350 205

Controln = 7 0.6 0.4 0 0.3 o.78§ 550§ 66n= 10 0.1 0 0 0 1.1411 63111 10

Anti-GBMn = 5 1.9 1.7¶ 10-70 1.8 0.19 100 426n = 5 1.4 1.3 0-5 0.7 0.71 359 287

All figures are means.* Animals in each group have been subdivided on the basis of GFR. Mean glomerular hypercellularity, GBMthickening, and:wiritectural derangement were calculated from individual animals graded on a 0-4+ scale (see Methods) where 1 = 0.25,trace = 0.5, 1 = 1, etc.X Includes gross glomerular changes, tubular atrophy and dilatation, interstitial mononuclear cell infiltration, casts, and intra-luminal cells.§ Data from four rats.

Data from nine rats.¶ Irregularity and fraying.


r 11pirw-r4k!T;,-- AM m e-0s

FIGURE 9 Accumulations of cells were noted in the lumens of tubules in the AICN rats. ThePAS-positive brush border area of the tubular cells appeared to be stripped from segmentsof the lining epithelium and phagocytized by mononuclear cells which tended to becomemultinucleated. Mononuclear cell infiltrates were also noted adjacent to some renal tubules (D).Hematoxylin-eosin stain (A) and PAS stain (B-D) A and B, X 220; C and D, X 350.

DISCUSSIONThis study was designed to measure single nephronfunction in histologically different experimental glo-merulonephritides morphologically resembling humanglomerulonephritis. Examples of the two major im-munopathogenic mechanisms of glomerulonephritiswere chosen. Firstly, AICN was used as a model ofimmune complex (nonglomerular antigen-antibody) -in-duced glomerular injury, a mechanism which appearsto be responsible for approximately 80% of humanglomerulonephritis (43). The RTA-antibody systemwhich typifies AICN has recently been identified inthree patients with membranous glomerulonephritisfrom Japan (44). Secondly, anti-GBM antibodies wereused to induce glomerular injury typified by an acuteonset (within hours) of a diffuse, proliferative glomer-ular lesion similar to the infrequently found (approxi-mately 5%) anti-GBM antibody-induced glomerulo-nephritis in man (36). It should be noted that, althoughAICN has the morphology of a membranous glomerulo-

nephritis, immune complex glomerulonephritis can as-sume virtually any type of glomerulonephritic histo-logic alteration, apparently influenced more by theintensity and tempo of the immune reaction and thesubsequent host response than by the inciting event.Both antibody-induced forms of glomerular injury uti-lize common mediation pathways, namely complementand polymorphonuclear leukocytes, so that observationsmade in this study dealing with diffuse proliferativeglomerulonephritis induced by anti-GBM antibodiesmight also apply to similar morphologic lesions inducedby immune complexes.

Measurement of whole kidney function in our glo-merulonephritic rat models revealed changes very simi-lar to those previously reported in extensive clinicalstudies of renal function in human glomerulonephritis.Thus whole kidney GFRand FF fell, RPF, determinedby the clearance and extraction of PAH, remainingunchanged in the more chronic AICN disease or in-creasing slightly in the acute, proliferative anti-GBM


FIGURE 10 Granular deposits of immunoglobulin were observed in the glomeruli of the AICNrats. The fine granularity was present diffusely along the GBMwhere little thickening (A)or after extreme thickening (B) had occurred. Linear deposits of immunoglobulin typifiedthe glomerulonephritis introduced by heterologous anti-GBM antibodies. (C) The patternwas very smooth and continuous in kidneys with well preserved glomerular architecture. (D)With greater degrees of glomerular damage, the immunoglobulin pattern became increasinglyirregular with apparent fraying of the GBM. Fluorescein isothiocyanate-conjugated anti-ratIgG. X 250.

nephritis. Such observations have been frequently re-ported in acute, subacute, and early chronic glomerulo-nephritis in humans (1, 2, 45). Absolute electrolyteexcretion rates were not significantly different fromcontrols, but this was achieved by an increase in thefractional electrolyte excretion as the GFR fell, a neces-sary adaptive response previously documented in pa-tient studies (3, 10, 46). Only two of our rats developededema or ascites, although this has been reported asoften accompanying experimental immune complex andanti-GBM disease in rats (18, 47).

The most striking physiological feature in bothmodels of experimental glomerulonephritis was thefinding that each nephron studied showed almost com-plete glomerulotubular balance, despite marked hetero-geneity of SNGFR, proximal free flow intratubularhydrostatic pressure, and calculated glomerular capillarypressure. It might be argued that the heterogeneity of

SNGFRcould, in part, be due to a variable loss ofinulin from structurally damaged nephrons. From ourown and other observations, however, we do not be-lieve that inulin leakage contributed substantially toour findings. No evidence of inulin leakage was foundin nephrons with intratubular hydrostatic pressure lessthan 19 mm Hg, despite marked heterogeneity ofSNGFRin such nephrons (Fig. 6). A significant lossof inulin was seen in only five nephrons with highintratubular pressure and very slow flow rates, threeother nephrons with high pressure and slow flow show-ing no evidence of leakage after proximal microinjec-tion. It is technically very difficult to exclude the possi-bility of a small but significant leakage of tubular fluidonto the surface of the kidney around the site of in-jection in nephrons with high intratubular pressurewhich might have caused, at least in part, the calcu-lated inulin leakage.


FIGURE 11 Heavy deposits of IgG (A) and C3 (B) were observed in the area of the brushborder (arrows) of foci or "islands" of renal tubular cells in some of the AICN rats withmore extensive architectural abnormalities. Fluorescein isothiocyanate-conjugated anti-rat IgG(A) and C3 (B). X 250.

Moreover, even if the calculated inulin leakage re-sulted from the transtubular movement of microinjectedinulin, it does not necessarily follow that a similarfraction of inulin filtered in that nephron would leak.Fractional inulin leakage is a function of contact timeand SNGFRwould have to be vanishingly small, lessthan 0.3 nl/min, for tubular transit time to be as long,circa 1 h, as the observed contact time in these dilatedand obstructed high pressure nephrons studied by mi-croinjection. Accepting the average leakage observedin the five obstructed tubules and assuming a tubulardiameter twice normal, one can calculate that lessthan 3% of filtered inulin would leak at an SNGFRof5 nl/min and less than 1.5% at an SNGFRof 10nl/min.

In addition, if transtubular inulin leakage at highintratubular pressure were a significant factor onemight expect to find a correlation between SNGFR,late proximal F/P inulin level, and intratubular hydro-static pressure, high pressure nephrons having lowerSNGFRand F/P inulin values. No such relationshipwas observed in anti-GBM nephritis, although in AICNrats mean SNGFRin high pressure nephrons was sig-nificantly lower (26.0 nl/min ±22.4 SD) than thatfound in more normal pressure nephrons (48.6 nl/min+-19.9 SD, P <0.005). Late proximal F/P inulinratios, however, were not significantly different. In arecent study of experimental glomerulonephritis pro-duced by rabbit anti-rat kidney serum, a model pre-sumably quite similar to our anti-GBM rats, Rocha,Marcondes, and Malnic (17) found that SNGFRwas

not different when calculated from proximal or distalsamples and concluded that there was no evidence ofinulin loss from the nephritic tubules. Lastly, micro-injection studies in animal models with severe tubulardamage have revealed no evidence of inulin leakage3-4 wk after the simultaneous injection of potassiumdichromate and mercuric chloride (16) or 144 h afteraminonucleoside administration (48).

In any event only a very small number of the micro-puncture collections here reported were from tubuleswith high pressure and very slow flow rates and hencepossibly suspect in regards to transtubular inulin leak-age. Exclusion of these few data in no way controvertsour conclusions about nephron heterogeneity.

Structural and functional heterogeneity among ne-phrons in the chronically diseased kidney was firstdescribed by Oliver, Bloom, and MacDowell in 1941(12) using specific histological staining techniques inspontaneous chronic canine glomerulonephritis. Morerecently micropuncture studies in rats with chronicpyelonephritis (13), heavy metal poisoning (16), andanti-GBM nephritis (17) have confirmed this markedfunctional heterogeneity. From the detailed single neph-ron observations reported in the present study, togetherwith those previously described, we believe that therecan now be no doubt that single nephrons in the ex-perimental chronically diseased kidney show markedheterogeneity both in function and structure.

Each superficial nephron studied, however, demon-strated almost the same glomerulotubular balance, ab-solute reabsorption to the late proximal convolution


FIGURE 12 The uniformity of glomerular proliferative changes are seen at various levelsthrough the cortex in a rat with experimental anti-GBM glomerulonephritis. A glomerulusadjacent to an arcuate artery and vein (A), three glomeruli in the mid-cortex (B), and twoglomeruli in the superficial cortex (C) are seen. Hematoxylin-eosin stain. A-C, X 220.

varying in direct proportion to the nephron filtrationrate at all levels from 5 to 93 nl/min. Similar mainte-nance of single nephron glomerulotubular balance de-spite marked heterogeneity of filtration rates has alsobeen found by Rocha et al. (17) in rats with anti-GBMnephritis, except in those nephrons with lowfiltration rates (25 nl/min) when F/P inulin levelswere unusually high. Important implications concern-ing the mechanism (s) regulating glomerulotubular bal-ance in the diseased nephron follow from our observa-tions, since such a mechanism(s) must obviously workon a single nephron basis.

There has recently been great interest in the roleof the so called peritubular physical factors (i.e., hy-drostatic and oncotic pressure in the tubular environ-ment) in controlling tubular reabsorption. Experimentalvariation of peritubular capillary hydrostatic or oncoticforces can apparently alter proximal reabsorptive rates(49, 50). For these factors to be important in thesetting of glomerulotubular balance in single nephrons

in our heterogeneous diseased kidneys it must first beshown that each proximal tubule is surrounded only byperitubular capillaries from its own efferent arteriole,since the oncotic pressure in each efferent arteriole willinitially be dependent on that nephron's FF. There arecomparatively few detailed anatomical studies of vas-cular-tubular relationships. Some evidence of a closerelationship between each efferent arteriole and its ownproximal tubule has been reported recently by Stein-hausen, Eisenbach, and Galaske (51) in the rat kidneyand by Beeuwkes (52) in the superficial c'ortex of thedog kidney. Both agree however that there are manyfree communications between the peritubular capillaries.On direct microscopic examination of the living kidneythese appear to form a freely anastomotic network. Also,it might be supposed that nephrons with high intra-tubular pressures and low filtration rates should haveadjacent peritubular capillaries with similar high hy-drostatic pressures, assuming that this force couldinfluence transport across the proximal tubule. Inter-


stitial fluid pressure remains unknown, and since it ispresumably the gradient between this compartment andthe peritubular capillary on the one hand and the proxi-mal tubule on the other which would determine re-absorptive rates, absolute peritubular capillary pres-sures may not be of importance. Although a signifi-cantly increased variability in efferent arteriole andperitubular capillary hydrostatic pressures was foundin the diseased kidney compared with controls, thehighest values found did not exceed normal limits. In-deed, we were impressed by the finding of postglomer-ular vessels with very low hydrostatic pressures. Thisobservation was predicted by Bradley (53) because ofthe marked obstructive disease of the glomerular ves-sels found in glomerulonephritis. We therefore find itdifficult to accept that the reabsorptive rate per neph-ron is influenced solely by that nephron's peritubular"physical" environment in the absence of evidence thatit is specific for individual nephrons.

Secondly, it has been suggested that SNGFRmightbe regulated within individual nephrons by a tubular-vascular feedback system involving the renin-angio-tensin system, such that an increase in sodium deliveryto the macula densa might result in increased reninproduction and hence angiotensin release which in turnwould so alter afferent or efferent vascular resistanceto result in a fall in GFR (54-56). Recent studies haveshown that GFR calculated from distal tubular fluidcollections are consistently lower than those calculatedfrom proximal tubular fluid collections in that nephron,when distal tubular sodium concentration are presuma-bly very low due to the proximal oil block (57, 58).Others have failed to substantiate these findings (59).All our tubular fluid collections were made in proxi-mal tubules after placing an oil block of at least 4-5tubule diameters in length distal to the collection pip-ette. Despite complete blocking of the tubular fluidflow beyond this point, however, marked heterogeneityof single nephron filtration rate with maintenance ofglomerulotubular balance was found.

One of the earliest and simplest proposals made toexplain the phenomenon of glomerulotubular balancewas that some factor(s) related to proximal intra-luminal load determined tubular absorption, completelyindependent of peritubular environment (60, 61). Invitro and in vivo single nephron microperfusion stud-ies, however, suggested that glomerulotubular balancewas not an intrinsic property of the proximal tubule,since experimental alteration of proximal tubule per-fusion rates with artificial perfusates did not resultin any significant change in absolute reabsorptive rates(62, 63). It is also now accepted that intratubularvolume (64) is not the primary determinant of proxi-

mal reabsorptive rate (65, 66). Recently, however, Bar-toli, Conger, and Earley (59), using a partial proximaltubule collection technique, have found that a fall in thedelivery rate of normal filtrate along the proximaltubule is associated with a decrease in the absolutereabsorptive rate. They suggest that this might be dueeither to variation in the mixing of unstirred diffusionlayers or to a change in the availability of some ultra-filterable plasma constituent with change in intra-luminal flow rates. Similarly in our studies it is pos-sible that some factor or factors related to proximalintraluminal flow or load was responsible for appro-priate alignment of absolute reabsorptive rate in in-dividual nephrons with widely differing filtration rates.

Marked heterogeneity of SNGFRwas found in bothmodels of glomerulonephritis, but more especially inthe AICN group. Also mean SNGFRwas significantlylower in the acute proliferative anti-GBM antibody-induced glomerulonephritis than in the AICN rats withchronic membranous glomerulonephritis. No "super-nephrons" with very high filtration rate was found inanti-GBM nephritis, a finding similar to that of Rochaet al. (17). This probably reflects the acute diffuseinvolvement of glomeruli in this model. In contrast, themuch greater heterogeneity of SNGFRin AICN ratsprobably was related to the chronicity of the experi-mental model allowing adequate time for adaptationof nephron function.

The primary factors influencing individual GFR, i.e.afferent and efferent EFP, RPF, permeability and sur-face area of glomerular capillary bed available for fil-tration, are complex and are presumably altered in thediseased glomerulus. Recently, advances in micropunc-ture technology and finding of accessible superficialglomeruli in a mutant strain of Wistar rats has en-abled direct study of these factors (32, 67). It hasbeen shown that in the normal superficial rat glomerulusPg are lower than previously supposed and that, fil-tration equilibrium is generally reached under a varietyof experimental conditions (68). Wethave attempted tostudy these factors indirectly in the diseased nephron.

Firstly, no consistent relationship was found betweenSNGFR and proximal intratubular hydrostatic pres-sure in either AICN or anti-GBM nephritis, although atendency toward lower SNGFR in nephrons withhigher intratubular pressures was seen in AICN rats.These latter nephrons appeared dilated and were pre-sumably blocked downstream by fibrosis or proteinace-ous debris and casts.

Secondly, we found a positive correlation betweencalculated Pg and SNGFR, but this was statisticallysignificant only for animals with anti-GBM disease.Pg was calculated as the sum of the directly determined


stop flow pressure and the afferent oncotic pressure(69, 28). This calculation involves three important as-sumptions, none of which is as yet directly proven.First, glomerular filtration must be completely stoppedso that the protein concentration in the systemic cir-culation can be taken as the efferent anteriolar proteinconcentration. Secondly, any possible effect of reduc-tion of flow at the macula densa after blockage of thenephron with oil is negligible. Thirdly, appropriatealterations in afferent/efferent arteriolar resistance musthave occurred in order to accommodate the increasedcapillary flow due to cessation of filtration withoutchange in glomerular hydrostatic pressure. That thelatter can occur under certain circumstances has beenshown by Brenner, Troy, Daugharty, Deen, and Rob-ertson (68) when a 100% increase in glomerular capil-lary plasma flow due to plasma volume expansion re-sulted in a change in directly measured Pg of only 1.8mmHg. Values reported for stop flow pressures inhydropenic adult rats from different laboratories havevaried considerably, from 26 to 29 mmHg (17, 70)to 55 mmHg (69), most values lying between 32 and40 mmHg. In our studies the mean value calculatedfor Pg from stop flow pressure measurements in con-trol rats was 47.4 mmHg, which is similar to thaltreported by Blantz, Israelit, Rector, and Seldin (67)obtained by using direct capillary punctures in hydro-penic "Munich" Wistar rats with superficial glomeruli(47.4 mmHg), but slightly higher than that reportedby Brenner, Troy, and Daugharty (32) (44.4 mmHg)in similar animals. Calculated mean Pg was signifi-cantly higher in both AICN and anti-GBM nephritickidneys than in controls, a finding different than thatof Rocha et al. (17) who reported a lower calculatedmean glomerular capillary pressure in rats with anti-GBMnephritis than in control animals. Measurementsmade by Rocha et al. were at random points in theproximal tubule and stop flow pressures are lower by,on average, 7 mmHg, when measured in late proximalrather than early proximal convolutions (28). Obvi-ously direct measurements of hydrostatic pressure insuperficial diseased glomeruli are required. It is pos-sible that a significant fall in hydrostatic pressure occursdue to increased resistance in the damaged capillarybed in experimental glomerulonephritis. Furthermore,the hemodynamic changes occurring in damaged glo-merular capillary beds during stop flow are also un-known and may differ from that seen under normalconditions.

Thirdly, it seems safe to assume that the filtrationcharacteristics of the GBMmay be altered to differentextents in the two histologically different models ofglomerular injury under study. Thus we found that

1420 M. E. M. Allison, C. B. Wilson, and C. W.

although the mean calculated Pg was similar in bothAICN and anti-GBM nephritis, mean SNGFRwas sig-nificantly lower in those animals with anti-GBM dis-ease. This suggests a decrease either in glomerularpermeability or in blood flow due to luminal encroach-ment in anti-GBM rats. Histologically the AICN ratshave uniformly thickened GBMand large amounts ofimmune complex (IgG, C3) deposits in the absence ofan obvious acute phlogogenic response. By electronmicroscopy, these immune complex deposits appear aselectron-dense masses under the fused epithelial cellfoot processes, giving the GBMa rather uniform thick-ened appearance (71, 72). The administration of anti-GBMantibodies, on the other hand, produced a moreacute proliferative response with polymorphonuclearleukocyte infiltration and very early (within hours)changes in glomerular permeability as manifested byproteinuria. Indeed, gaps have been identified in theGBMby electron microscopy (73) and fragments of theGBMhave been detected in the urine (74). Little isknown about the functional characteristics of the remain-ing somewhat irregular GBM; however, our observationswould suggest that its usefulness as an ultrafilter isimpaired.

Fourthly, in AICN and anti-GBM disease there wasa direct relationship between SNGFRand both afferentEFP, obtained by direct measurement of stop flowpressure and intratubular free flow pressure, and cal-culated efferent EFP. Nephrons with high filtratiornrates had high afferent and efferent EFP and did notapparently reach filtration equilibrium. Only those withvery low filtration rates came near to equilibrium. Incontrol rats, mean efferent filtration pressure was sig-nificantly lower than in the diseased kidney, but filtra-tion equilibrium did not appear to be reached. Beforespeculating on the significance of these observationsit should be noted that the calculation of efferent EFPis dependent on estimation of the efferent oncotic pres-sure which we obtained using the whole kidney FFand the afferent protein concentration. Whole kidneyFF in our control rat kidneys was 0.22, lower thanthat generally reported in hydropenic adult rats. As-suming that the estimated Pg is correct, we calculatethat for these animals to reach filtration equilibrium anFF of approximately 0.38 would be required. In thediseased rats whole kidney FF were much lower. FFof individual glomeruli were not measured. If oneassumes a fairly constant glomerular capillary plasmaflow per nephron, then nephrons with high filtrationrates would have significantly higher FF than thosewith low SNGFR, perhaps resulting in a smaller dis-equilibrium at the end of the glomerular capillary bed.More likely, however, both plasma flows and FF in

Gottschalk

individual glomeruli are variable in the diseased kidney.Resolution of these problems obviously awaits mea-surements of single nephron FF in diseased glomeruli.

Finally, the possible contribution of immunologictubular damage to derangement of single nephronfunction should be considered. Previous morphologicstudies have described tubular abnormalities in AICN(40), and tubular abnormalities often considered sec-ondary to glomerular damage are universal in advanc-ing experimental and human glomerulonephritis. Noclearcut immunologic role has been established for theselesions; however, certain observations would suggestthat immune tubular as well as glomerular injury doesoccur in these diseases. Tubular deposits of immuno-globulin and complement have been observed in experi-mental and human renal disease (75-77). Human anti-GBMnephritis frequently has linear deposits of IgGin the TBMsuggesting the presence of anti-TBM anti-bodies as well. We have in addition observed thepresence of anti-TBM antibodies in renal allograftswith specificity restricted to the TBMon elution studies(Wilson, unpublished observations). Irregular granu-lar deposits of IgG and C3 are commonly seen in renaltubules (often associated with the TBM) of patientswith immune complex glomerulonephritis, particularlywhen nearing endstage (Wilson, unpublished observa-tions). The significance of these deposits is as yet un-

known. It is not surprising therefore that immuno-pathologic tubular abnormalities would be present inour experimental models. The AICN rats had evidenceof IgG and C3 deposits on the brush border as well as

intracellularly accompanied by apparent stripping ofthe PAS-positive brush border of the proximal tubulesby cells within the tubular lumen, suggesting immuno-pathologically induced tubular injury. No direct im-munofluorescent evidence of immune deposits was foundin the anti-GBM rats. These observations correlateddirectly with the difference in PAH extraction ob-served in the two models in which only the. AICN rats

had a significant decrease in this monitor of tubularfunction.

ACKNOWLEDGMENTSThis study was supported by a grant-in-aid from the Amer-ican Heart Association, by grant HE-02334 from the Na-tional Institutes of Health, by Public Health Service Con-tract PH 43-68-621 and by U. S. Public Health Servicegrant A 1-07007. Scripps Publication Number 720.

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