Kidney International, Vol. 45 (1994), pp. 1326—1332

Glomerular macrophage phagocytic activity in experimentalimmune complex nephritis

MARVA M. MoxEy-MIMs and BERNICE NOBLE

Departments of Pediatrics, Microbiology and Pathology, State University of New York at Buffalo, Buffalo, New York, USA

Glomerular macrophage phagocytic activity in experimental immunecomplex glomerulonephritis. In rats with the proliferative immunecomplex glomerulonephritis of chronic serum sickness, kidney functiondeteriorates in three clearly distinguishable and discrete stages: mild,moderate and severe. The macrophage component of glomerular in-flammation in each stage is also quantitatively and qualitatively distinct,with abnormal phenotypic markers appearing in the moderate stage andincreasing in the severe stage. To determine whether there were distinctfunctional differences among macrophages from the three stages, Fcyreceptor-mediated phagocytic capacity was measured. The phagocyticcapacity of glomerular macrophages increased significantly in themoderate stage, then significantly decreased, as rats progressed tosevere chronic serum sickness. This decline in phagocytic function wasnot associated with a decrease in the expression of Fcy receptors on theglomerular macrophage cell surface. Furthermore, the phagocytic func-tion of peritoneal macrophages from rats with severe chronic serumsickness was not impaired. Whether or not this attenuation of glomer-ular macrophage phagocytic capacity is the cause, or result, of renaldisease progression remains unclear. It may indicate a potentiallyprotective role for intraglomerular macrophages, and can serve as anadditional functional marker of disease progression.

Chronic serum sickness in rats is a model of human prolifer-ative glomerulonephritis that allows for the study of the rela-tionship between kidney pathophysiology and immunopathol-ogy [1]. Three discrete stages of nephritis have been defined.Those stages, designated mild, moderate and severe, are eachclearly distinguishable by characteristic immunopathologic andpathophysiologic features [2—6]. A significant glomerular hyper-cellularity develops in the moderate stage that is largely attrib-utable to the accumulation of macrophages, some of whichexpress abnormal phenotypic markers [6]. During the severestage of nephritis, which is characterized by an irreversibledecline in glomerular filtration rate [2], the number of glomer-ular macrophages and the percentage expressing abnormalphenotypic markers, increases even further [2, 4, 6]. There issubstantial evidence that macrophages contribute significantlyto the pathogenesis of both acute and chronic proliferativeimmune complex glomerulonephritis [7—13]. The phagocyticcapacity of glomerular macrophages has been demonstrated bytheir ability to ingest various materials, including preformed

Received for publication July 28, 1993and in revised form November 29, 1993Accepted for publication December 28, 1993

© 1994 by the International Society of Nephrology

immune complexes [14], ferritin [15], immunoglobulin aggre-gates [16] and colloidal carbon [17]. Additionally, in animalmodels of diffuse proliferative glomerulonephntis, it has beenshown that glomerular macrophages play a predominant role inthe phagocytosis of circulating immune complexes [16, 17].This phagocytic function may contribute to renal tissue damageand disease progression [18], but may also have a protectiverole. Persistence of glomerular immune complexes, whethersecondary to a decrease in glomerular macrophage numbers inone animal model [19], or impaired epithelial cell endocytosis inanother [20], has been postulated to contribute to the progres-sion of the lesions of immune complex glomerulonephritis. Wewished to determine if the changes in macrophage phenotypethat accompany the progression of immune complex nephritiswere correlated with functional changes in those cells. For thatreason, Fcy receptor-mediated phagocytosis was measured invitro at each of the three stages of chronic serum sickness; thefunction of glomerular macrophages was compared to that ofthe resident peritoneal macrophages from normal rats as well asthat of peritoneal macrophages from rats in each of the stages ofchronic serum sickness. A specific association between glomer-ular macrophage phagocytic function and disease progressionhas not previously been demonstrated.

Methods

Induction and monitoring of chronic serum sickness

Female LEW rats (Charles River Breeding Laboratories),weighing 125 to 150 g received four subcutaneous injections of3 mg of bovine serum albumin (BSA) in incomplete Freund'sadjuvant at two week intervals as previously described [1, 21],until precipitating antibodies to BSA with titers (log2) � 16wereproduced. Gradually increasing amounts of daily intravenousBSA injections were given until the rats tolerated a single dailyintravenous injection of 2 mg BSA in 0.4 ml isotonic saline [1,21]. Urine samples were collected at least twice per week fromanimals kept overnight (16 hr) in metabolism cages. At the endof each urine collection period, 100 .d of blood were obtainedfrom the tail vein. Protein, albumin, creatinine and sodiumconcentrations in urine and plasma were measured as previ-ously described [2, 4].

The criteria for distinguishing the stages of chronic serumsickness were as previously described (Table 1) [2—6]. Briefly,normal protein excretion ( 25 mg124 hr), normal creatinineclearance (� 0.50 ml/min x 100 g body wt) and normal sodium

1326

Moxey-Mims and Noble: glomerular macrophage phagocytosis 1327

Table 1. Some key features of chronic serum sicknessglomerulonephritis

Mild Moderate Severe

Serum titer of � 1:16 None Noneprecipitating BSAantibodies

Number of macrophagesper glomerular crosssection (mean)Total 8 25 50ED1+a 8 23 47ED2+a 0 0 5ED3+a 0 9 41

Number of T cells per 0 2—4 0glomerular cross section

Kidney function Normal Proteinuria(25—500 mg/day)

Proteinuria(>500 mg/day)

GFRNa retention

a The monoclonal antibody EDI recognizes monocytes and mostmacrophages; ED2 recognizes many mature tissue macrophages; ED3is a macrophage antigen normally expressed only in lymphoid tissue[25].

excretion (� 0.25 mEqI24 hr x 100 g body wt) were character-istic of the mild stage. The moderate stage begins with thesudden onset of proteinuria, while normal creatinine clearancewas maintained. The severe stage begins with an abrupt de-crease in both creatinine clearance (0.25 mllmin) and sodiumexcretion (� 0.20 mEq/24 hr), accompanied by further in-creases in urinary protein excretion. In addition to thesepathophysiologic parameters, there are also quantitative andphenotypic changes in the glomerular macrophage populationthat distinguish each stage of nephritis (Table 1) [61.

Buffers and mediaIsotonic veronal buffered saline (VBS), 0.01 M EDTA buffer

and dextrose veronal buffered saline (DVBS) were prepared aspreviously described [22, 23]. Hanks balanced salt solution withand without calcium and magnesium (HBSS and HBSS,respectively) and RPMI 1640 were obtained from GIBCO(Grand Island, New York, USA). Hypotonic saline buffercontaining 0.66 M ammonium chloride, 3.7 x i0 M potassiumcarbonate and 1.1 >( lO M EDTA (lysing buffer) was preparedin our laboratory.

Preparation of opsonized sheep erythrocytesSheep erythrocytes (E) were washed with 0.01 M EDTA

buffer and VBS buffer before suspension in VBS at 1 x iocells/mi. A portion of E was suspended in DVBS and a 1:100dilution of rabbit anti-sheep IgG (Diamedix, Miami, Florida,USA) or rat anti-sheep IgG (provided by Dr. William Cham-bers, Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania,USA) was added followed by a 15 minute incubation at 37°C toform opsonized sheep erythrocytes (EIgG). These cells werethen washed three times in DVBS. Both E and EIgG wereresuspended in DVBS at 1.5 x 108 cells/ml for use in phagocy-tosis and rosetting assays.

Isolation of glomerular macrophagesGlomerular macrophages were isolated from the kidneys of

normal, age-matched control rats and from each stage of

chronic serum sickness glomerulonephritis, as previously de-scribed [24]. Briefly, rat kidneys were perfused in situ withsterile PBS, then removed. Glomerular isolation was accom-plished by compressing slices of cortex through 60-, 100- and200-mesh steel sieves. Cell dissociation was accomplished by athree-stage procedure. First the glomeruli were incubated for 20minutes at 37°C with shaking, in a cocktail consisting of 5 mgtrypsin type II, 10 mg collagenase type I and 1 mg deoxyribo-nuclease type IV (all from Sigma) in 10 ml HBSS. Secondly, thecells were incubated in 2 mM EDTA in HBSS for 30 minutesat 37°C. The supernatant was removed and placed on ice, andthe precipitate was incubated for 20 minutes at 37°C in 10 mlHBSS containing 10 mg collagenase. The suspension waswashed with HBSS then passaged through a 23 gauge needlethree times. The cells were allowed to settle on ice for fiveminutes, then the supernatants were washed twice with 5%heat-inactivated FCS (GIBCO) in HBSS. These cells were thensuspended at 1.5 x 106 cells/mI in HBSS with 10% heat-inactivated FCS then plated in slide-mounted, eight-chambertissue culture plates (Nunc, Inc., Naperville, Illinois, USA).After an overnight incubation at 37°C in 5% CO2 chamber, theslides were washed vigorously three times with RPM! toremove any non-adherent cells. Staining with the monoclonalantibody ED! (Bioproducts for Science) confirmed that > 95%of the adherent cells were macrophages [25]. There were nosignificant differences in the number of glomeruli isolated fromeach stage of disease (approximately 4 x l0 per rat in eachcase, with kidneys from rats with severe disease yielding anaverage of 5 x 1O glomeruli per rat). However, as expectedfrom histologic studies [6], larger numbers of macrophageswere isolated from the moderate and severe stages of disease.Means SEM were as follows: 9 x 106 0.5 x 106 macro-phages per rat with severe chronic serum sickness; 5 x 1060.4 x 106 per rat with moderate chronic serum sickness; 1.5 x106 0.4 x 106 per rat with mild disease; 1 X 106 0.3 x 106per normal, age-matched rat. The pattern of isolated glomerularmacrophage staining with the monoclonal antibodies ED2 andED3 were the same as previously reported in situ (Table 1) [61.Specifically, approximately 40% of glomerular macrophagesfrom the moderate stage and 80% of glomerular macrophagesfrom the severe stage stained positively with ED3. Approxi-mately 10% of glomerular macrophages from the severe stagealso stained positively with ED2.

Isolation of peritoneal macrophages

Resident peritoneal macrophages were obtained from normalrats and rats at each stage of chronic serum sickness byrepeated gentle lavages of the peritoneal surface with RPM!1640, after exsanguination of the animals by cardiac punctureunder anesthesia. The peritoneal cells were washed three timesin RPM! 1640 and suspended at 5 x l0 cells/ml. 250 pi of thecell suspension were added to each chamber of slide-mounted,eight-chamber tissue culture plates (Nunc) for phagocytosis androsetting experiments. Staining with the monoclonal antibodyED! confirmed > 95% of these cells to be macrophages.Peritoneal macrophages from normal rats stained positivelywith ED! and ED2, but not ED3. However, >90% peritonealmacrophages from rats at each stage of chronic serum sicknessstained positively for ED2 and ED3.

1328 Moxey-Mims and Noble: glomerular macrophage phagocytosis

Activated peritoneal macrophages were obtained from nor-mal rats following intraperitoneal injection of 2.5 ml completeFreund's adjuvant (CFA). Two weeks after the adjuvant injec-tions, cells were harvested as described above for residentmacrophages, for use in phagocytosis and rosetting assays.More than 90% of those peritoneal macrophages stained posi-tively with ED1, ED2 and ED3.

Phagocytosis assaysTwo hundred and fifty microliters of RPMI 1640 were added

to each chamber of the slide-mounted tissue culture plates onwhich the macrophages were adhered. The cells were thenplaced in a 37°C, 5% CO2 incubator for 30 minutes to allow forequilibration. To assess Fcy receptor (FcyR)-mediated phago-cytosis, 100 d of either E (to, determine nonspecific ingestion)or EIgG were then added to each chamber and the plates werecentrifuged at 50 x g for five minutes at 4°C. After centrifuga-tion, the cells were again placed in a 37°C incubator with 5%CO2 and phagocytosis was allowed to proceed for one hour.Phagocytosis was stopped by rinsing with 0°C lysing buffer toremove non-internalized E. After two exposures to lysingbuffer, the cells were fixed with 1% glutaraldehyde and stainedwith Giemsa. The percentage of macrophages in each chamberthat had ingested one or more E was determined by lightmicroscopy (l000x; oil immersion). The total number of Eingested by these macrophages was also counted. The results ofthe latter were expressed as the phagocytic index (P1), the totalnumber of ingested E per 100 macrophages.

Peritoneal macrophages, exposed to the three-step enzymaticprocess used to isolate glomerular macrophages for phagocyto-sis assays, did not differ from untreated cells in phagocyticcapacity or phenotypic marker expression. Therefore, subse-quent experiments with peritoneal macrophages did not incor-porate those procedures.

Rosetting assaysMacrophages were plated in slide-mounted, eight-chamber

tissue culture plates (Nunc), as described for the phagocytosisassays (see above). After the addition of E (to determinenonspecific binding) or EIgG, however, the cells were incu-bated at 37°C with 5% CO2 for only 30 minutes to allow forattachment but preclude ingestion. The slides were then rinsedgently with RPMI x 3 to remove non-adherent E. The slideswere fixed with 1% glutaraldehyde, stained with Giemsa andexamined with a light microscope at 400 x. Macrophages with

3 erythrocytes attached were counted as positive, as previ-ously described [26]. Exposure of peritoneal cells to the three-step enzymatic process used to isolate glomerular macrophagesdid not alter their rosetting capacity in pilot control studies.Subsequent experiments did not include those enzymatic steps.

Statistical analysisIn all experiments each sample was run in triplicate. Six to

eight rats were used from each stage of chronic serum sicknessfor glomerular macrophage experiments; three rats from eachstage for peritoneal macrophage experiments. Data are ex-pressed as the mean SEM. Analysis of variance was used forcomparison of different progressive stages of chronic serumsickness, followed by Scheffe (post-hoc) adjustments for thedata. P values � 0.05 were considered significant.

Results

Phagocytic capacityAs expected, peritoneal macrophages from normal rats acti-

vated by complete Freund's adjuvant had a significantly in-creased phagocytic capacity as compared to nonactivated nor-mal peritoneal macrophages (53.6% vs. 25.3%; P = 0.02),validating the experimental protocol. The phagocytic capacityof glomerular macrophages from rats with mild chronic serumsickness glomerulonephritis was indistinguishable from that ofnormal pentoneal macrophages or normal glomerular macro-phages (Fig. 1). It was, however, significantly less than that ofperitoneal macrophages from the same disease stage (31.91.8% vs. 61.7 2.0%, respectively; P < 0.01). Glomerularmacrophages from rats with moderate chronic serum sicknessglomerulonephritis had a significantly increased phagocyticcapacity compared with those from rats with mild disease (49.1

7.8% vs. 31.9 1.8%, respectively; P < 0.05). There was nostatistically significant difference in the phagocytic capacity ofperitoneal and glomerular macrophages from rats with moder-ate disease. Glomerular macrophages isolated from rats whichhad progressed to severe chronic serum sickness showed asignificant reduction (P < 0.0001) in phagocytic capacity com-pared with cells from rats with moderate disease (22.2 2.9%vs. 49.1 7.8%, respectively; Fig. 1). There was no differencebetween the phagocytic capacity of glomerular macrophagesisolated from rats with severe chronic serum sickness glomer-ulonephritis and resident peritoneal macrophages isolated fromnormal rats. However, there was a significant decrease inphagocytosis compared with peritoneal macrophages from an-imals with severe disease. The phagocytic capacity of perito-neal macrophages from rats at each stage of chronic serumsickness was elevated compared with normal peritoneal mac-rophages and equivalent to that of CFA-activated peritonealmacrophages from normal rats (Fig. 1). Ingestion of nonop-sonized E was < 3% in all experiments.

The phagocytic index was also increased for glomerularmacrophages from rats with moderate chronic serum sicknessand peritoneal macrophages from all three stages of disease(Fig. 2). In addition, during moderate chronic serum sicknessnephritis, individual glomerular macrophages ingested a greaternumber of opsonized erythrocytes than during the other stagesof disease (Table 2).

Macrophage Fc7R expressionEIgG rosette formation was measured as an index of macro-

phage FcyR expression. Normal resident peritoneal macro-phages made significantly fewer rosettes than peritoneal andglomerular macrophages obtained from animals at each stage ofchronic serum sickness (Fig. 3). There was no significantdifference between the number of rosettes formed with normalglomerular and normal peritoneal macrophages. There was nosignificant difference between the percent of glomerular orperitoneal macrophages forming rosettes at each stage ofchronic serum sickness glomerulonephritis. Rosette formationwith nonopsonized E was <2% for all experiments.

Discussion

Intraglomerular macrophages have been well documented toplay a major role in the phagocytosis of immune complexes

Moxey-Mims and Noble: glornerular macrophage phagocytosis 1329

0,0)

80

70

60

50

40

30

20

10

0

Stage of chronic serum sickness

Fig. 1. Macrophage phagocytic capacity.Glomerular macrophage phagocytic capacityfrom mild chronic serum sickness nephritiswas not different from that of normalperitoneal niacrophages. * P < 0.0001 vs. all 3stages of chronic serum sickness; ** p0.0001 vs. moderate and severe chronic serumsickness; *** p < 0.0001 vs. moderatechronic serum sickness. There were nosignificant differences in the phagocyticcapacity of peritoneal macrophages from eachstage of chronic serum sickness. Symbols are:(D) peritoneal; () glomerular.

Fig. 2. Macrophage phagocytic index. Thephagocytic index of normal glomerularmacrophages was no different than that ofmacrophages from mild chronic serumsickness. The phagocytic index of normalperitoneal macrophages was significantly lessthan at each stage of chronic serum sickness,* P < 0.001; ** P < 0.01 vs. moderatechronic serum sickness; *** p < 0.01 vs.normal, mild and moderate chronic serumsickness. Symbols are: (0) peritoneal; ()glomerular.

present in the kidney [16, 17]. This function has generally beenthought to contribute to renal damage through the productionand/or release of proteinases, oxidants, prostaglandins, leuko-trienes, cytokines, complement components and coagulationfactors [181. There has been much support for this position, asit has been shown that depletion of macrophages prior toinduction of glomerulonephritis abrogates cellular infiltrationand the associated proteinuria [12, 13, 27]. Additionally, there isdocumentation that intraglomerular macrophages are activatedin immune complex glomerulonephritis [6, 28]. It was thereforenot surprising to find increased phagocytic capacity of intra-glomerular macrophages to levels equal to that of peritoneal

Table 2. Number of erythrocytes ingested per glomerularmacrophage

Stage ofnephritis

Number of EIgG permacrophage1 2—4 5—7 8—10 >10

Normal 18 64 16 2 0Mild 24 76 0 0 0Moderate 18 45 19 12 6Severe 94 5 1 0 0

Data are tabulated as mean percentage of phagocytic cells ingesting aspecified number of erythrocytes. The percentage of macrophages fromeach stage of nephritis that actually ingested � 1 erythrocyte is shownin Figure 1.

Normal Mild Moderate Severe

C

I0

400

300

200

100

0Normal Mild Moderate Severe

Stage of chronic serum sickness

(0a)

0)(00Cci)C.)

a)0

macrophages exposed to complete Freund's adjuvant. Whatwas unexpected, however, was the decreased glomerular mac-rophage phagocytic capacity found as chronic serum sicknessprogressed from the moderate to severe stage, in light ofphenotypic markers suggesting a possible increase in activationstate. It is unclear whether the decrease in phagocytic capacityis the cause, or effect, of disease progression; an inability toclear immune complexes deposited in the kidney has beenpostulated to contribute to the progression of immune complexnephritis [19, 20]. At the very least, the decrease in phagocy-tosis may be considered an immunopathologic marker of gb-merular macrophage dysfunction. This new data correlate afunctional anomaly with the phenotypic abnormalities previ-ously reported (Table 1) [61.

Our data also show that this attenuation of glomerular mac-rophage phagocytic capacity in the severe stage of chronicserum sickness results from decreased FcyR function, notdecreased FcR expression. Gbomerular macrophage FcyR ex-pression was elevated at each stage of nephritis compared withboth normal and activated peritoneal macrophages, yet only30% of the glomerular macrophages which expressed Fc7Rdemonstrated phagocytic activity in severe nephritis. Thiscompares with 53% for normal peritoneal macrophages, 55%for normal glomerular macrophages, 67% for moderate glomer-ular macrophages and 68% for severe peritoneal macrophages.The combination of a larger percentage of phagocytic glomer-ular macrophages and the greater number of erythrocytesingested by individual macrophages during moderate chronicserum sickness nephritis may be a subtle indicator of "super-activation." On the other hand, the low level of glomerularmacrophage phagocytic capacity during severe nephritis, de-spite increased Fc7R expression, may either reflect the actionof a direct or indirect inhibitor of FcyR function, or a general-ized exhaustion of cell energy sources due to continual immunecomplex ingestion.

The production and possible role of the cytokines IL-i and

TNF in some animal models of glomerulonephritis, includingchronic serum sickness, has been previously reported [6, 29—33]. Both of these soluble mediators may act to increasemacrophage phagocytic capacity [34]. However, other immu-nologically active substances may also be secreted during thesevere stage of disease by the highly abnormal ED3 + glomer-ular macrophages, or other glomerular cells, which counteractthe known stimulatory effects of IL-i and TNF. One potentialcandidate for abrogation of an IL-i effect is IL-i receptorantagonist (IL-iRa). Adherent IgG can induce production ofthis cytokine by human mononuclear cells [35]. IL-IRa specif-ically binds to the IL-l receptor, inhibiting the actions of IL-iand could possibly result in a decrease of the high macrophagephagocytic activity noted in the moderate stage of disease.Studies are currently underway to test this hypothesis. Anotherpotential inhibitory candidate is IL-2, secreted by T cells, whichare known to be present during the moderate stage of disease(Table 1) [6]. IL-2 has been shown to inhibit TNF stimulation ofphagocytosis in polymorphonuclear leukocytes [36], and asimilar phenomenon may be occurring with the glomerularmacrophages in this study.

Fc receptor function of peripheral human mononuclear cellsin patients with glomerulonephritis secondary to various auto-immune diseases has been shown to be significantly reducedboth in vivo and in vitro [37—39]. Continual ingestion of immunecomplexes with consequent exhaustion of cell energy sourcehas been postulated as the explanation for the impaired phago-cytic capacity. Most of the glomerular macrophages in thesevere stage of chronic serum sickness have abnormal pheno-types [6], which is suggestive of an activated metabolic state.This lends support to the hypothesis of cell exhaustion. Addi-tionally, circulating immune complexes are highest in the mildstage and lowest in the severe stage [40]. However, it is unclearwhether gbomerular macrophage phagocytic capacity is reflec-tive of peripheral mononuclear cell function as chronic serumsickness progresses, since a large number of the gbomerular

1330 Moxey-Mims and Noble: glomerular macrophage phagocytosis

100

80

60

40

20

0Normal Mild Moderate Severe

Stage of chronic serum sickness

Fig. 3. Macrophage rosette formation.Glomerular and peritoneal macrophages fromeach stage of chronic serum sickness nephritisformed significantly more rosettes than normalperitoneal macrophages, * P < 0.05. Symbolsare: (D) peritoneal; () glomerular.

Moxey-Mims and Noble: glomerular macrophage phagocytosis 1331

macrophages arise by local proliferation [41]. In addition, thephagocytic capacity of peritoneal macrophages from animalswith severe disease is not impaired, as compared with otherdisease stages or normal animals. It would therefore appear thatthere is local, rather than general inhibition of macrophagephagocytic function. This type of differential response at sitesof local inflammation has been described in other situations.Differences in local expression of complement genes in amunne model of lupus nephritis has been reported [42], andrecently, differences in the response of synovial fluid macro-phages and peripheral blood monocytes to IL-4 have beenreported [43]. A recent report from our laboratory indicates thatthe expression of phenotypic markers of macrophage activationcan be prevented in chronic serum sickness by treatment withcyclosporin A [44]. The effect of this treatment on mononuclearcell phagocytic function could be used to evaluate the activa-tion/exhaustion hypothesis.

In summary, macrophages present in the glomerulus duringthe severe stage of chronic serum sickness have decreasedphagocytic capacity when compared to those present during themoderate stage. On the other hand, peritoneal macrophagesfrom rats with severe chronic serum sickness showed no suchattenuation. This may indicate that depression of glomerularmacrophage function can contribute to disease progression inthis model. The presence of glomerular mononuclear cells mayhave multiple effects (some harmful and some beneficial), and inall likelihood, there are multifactorial causes for the attenuatedphagocytic capacity described here. Studies are ongoing to testthe hypotheses discussed.

Acknowledgments

These data were presented in part at the XIIth International CongressOf Nephrology, Jerusalem 1993. The work was supported by a supple-ment to NIH Grant DK41218 and the American Heart Association,New York State Affiliate Grant 92-003GB. The authors thank Dr. LindaDuffy for her expertise and help in the statistical analyses.

Reprint requests to Marva M. Moxey-Mims, M.D., Children's Kid-ney Center, Children's Hospital of Buffalo, 219 Bryant Street, Buffalo,New York 14222, USA.

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