marker, followed by digital limits) 0.04% (0.02%)) but infiltration of macrophages was detected in minimal change nephropathy (0.29% (0.

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1 1181 Jlin Pathol 1997;:118-1 Papers Pathology, Leiester General Hospital, Gwendolen Road, Leiester LE 4PW P N Furness Nephrology K P G Harris Pathology, Leiester Royal nfirmary, PO Box 6, Leiester LE 7LX L Rogers-Wheatley orrespondene to: Dr P N Furness. Aepted for publiation November 1996 Semiautomati quantitation of marophages in human renal biopsy speimens in proteinuri states P N Furness, L Rogers-Wheatley, K P G Harris Abstrat Aims-To develop and validate a rapid and eonomial semiautomated approah to the measurement of immunostainable tissue omponents whih is appliable to routine diagnosti pratie. To apply this approah to the measurement of marophages in renal biopsy speimens in nephroti states, as protein in the renal tubules may indue marophage infiltration, and the morphology of marophages in tissue setions does not lend itself to ell ounting. Methods-Marophages were identified by immunostaining with a panmarophage marker, followed by digital image apture and analysis using a maro proedure written for the freeware image analysis program NH-mage. Results-The method was rapid, robust and aurate to within the limits imposed by sampling error inherent in the use of small needle biopsy speimens. Very few marophages are found in normal kidney (mean volume fration (±9% onfidene limits).4% (.%)) but infiltration of marophages was deteted in minimal hange nephropathy (.9% (.1%)) and in membranous glomerulonephritis (.4% (.11%)). A statistially signifiant orrelation was found between marophage volume fration and weight of proteinuria in minimal hange nephropathy but not in membranous glomerulonephritis. orrelations were found in both diseases between marophage volume fration and serum reatinine at time of biopsy. onlusions-the equipment is inexpensive and measurement takes less than one minute per biopsy speimen. The results indiate that marophage infiltration is part of the pathologial proess in minimal hange nephropathy and membranous glomerulonephritis. The orrelation with reatinine at time of biopsy suggests that renal impairment in minimal hange nephropathy may result from infiltration by immunologially ative ells and not merely from haemodynami hanges in nephrons. However, the orrelation is not lose, indiating that the relation between marophage infiltration and disease severity is not a simple one. ( lin Pathol 1997;:118-1) Keywords: marophage; minimal hange; membranous; glomerulonephritis; nephroti morphometry. The role of the marophage in the indution of fibrosis is beoming learer in a variety of disease proesses. n the kidney, most emphasis has been plaed on marophage infiltration in the glomerulus, with onsequent prodution of ytokines and indution of exess deposition of extraellular matrix. The renal interstitium has been studied omparatively rarely, despite the aknowledged importane of interstitial fibrosis in renal impairment,' and despite the observation of foam ells in the interstitium in biopsy speimens from patients with heavy proteinuria from a variety of auses. Reently, there have been several attempts to predit the ourse of hroni human renal disease from the renal biopsy speimen by measuring the numbers of myofibroblasts in the interstitium. This has been quite suessful in ga nephropathy3 and in membranous glomerulonephritis,' both of whih are normally rather unpreditable in their ourse. However, the method used, manual ounting of ells after immunoytohemial detetion, is laborious and for this reason alone is unlikely to gain wide aeptane in routine diagnosis. n general terms, one may postulate the following as a typial sequene of events in a fibroti reation: an insult leads to marophage infiltration, whih results in ytokine prodution, whih stimulates proliferation and ativity of matrix produing ells (suh as myofibroblasts).' 6 f this is aepted, it seems possible that quantitation of interstitial marophages rather than myofibroblasts might produe an earlier and potentially more sensitive marker of the severity of the insult to the kidney, although resolution may our if the infiltration is short-lived. Therefore, we sought to measure the level of marophage infiltration in renal biopsy speimens from adult patients with idiopathi membranous nephropathy, with minimal hange nephropathy and, as a ontrol group, J lin Pathol: first published as /jp on 1 February Downloaded from on 1 November 18 by guest. Proteted by opyright.

2 Renal marophages in nephroti states 119, 4.., 'S,J'. t.' 4{'. a * a,.:'. f*. 4. 'S (* '"- U. ' ( ts, '/. - Th.z b Figure 1 A digital greysale image of an area of relatively heavy marophage infiltration, immunostainedfor marophages using PGM1, but without ounterstain. (a) An image aptured with the ondenser diaphragm losed, to indiate the loation of the (unstained) tubules. Suh an image is not part of the normal analysis proess. (b) The same field aptured with normal illumination, before thresholding. () mage (b) after thresholding. When thresholding has been ompleted, the omputer an alulate the perentage ofpixels whih are blak almost instantaneously; this is taken as representative of the volunme fration of marophages in this area of the biopsy speimen. The software maintains a 'running mean'as multiplefields from a single speinien are examined. biopsy speimens from patients with mirosopi haematuria and no detetable renal abnormality. These diseases were hosen as they are onventionally desribed as not having an inflammatory omponent. Furthermore, it has been suggested that heavy proteinuria might lead to interstitial fibrosis by diretly induing the release of marophage hemoattratants by 'damaged' tubular epithelial ells.7 Published studies in this field have almost invariably used ell ounting to measure the density of ellular infiltrates. This is laborious, and not appliable to routine pratie. Furthermore, immunostaining of marophages in renal biopsy speimens shows ells with omplex ytoplasmi extensions ramifying through the interstitium, making definition of what onstitutes a single ell very diffiult and somewhat subjetive. To failitate this study, and to provide a tool for further work, we sought to develop and validate a rapid omputer-assisted method for measurement of omponents of renal biopsy speimens whih would eliminate the need for laborious ell ounting, and whih ould be applied in routine pratie. Methods Sequential renal biopsy speimens were identified from the files of the Histopathology Department at Leiester General Hospital representing eah of the following three ategories: normal (34 ases); minimal hange nephropathy (31 ases); and membranous nephropathy (43 ases). ases were only aepted if the diagnoses were supported by light mirosopy, immunoytohemistry for immunoglobulins and omplement, and eletron mirosopy. ases in the 'normal' ategory were all biopsies performed for the investigation of mirosopi haematuria without proteinuria in whih all investigations of the biopsy speimen had shown no abnormality. The ases were not speifially mathed for age, but no paediatri ases were inluded. A'., *. S linial data olleted inluded serum reatinine onentrations and the 4 hour urine protein output at the time of biopsy. MMUNOSTANNG * Paraffin wax setions (3 gm) were dewaxed, and treated with 6% H) to blok endogenous peroxidase. Setions were exposed to. 1% trypsin (Difo) in.1% al, ph 7.8, for minutes at 37. A standard indiret immunoperoxidase method was used with PGM1 (Dako), a pan-marophage marker, as the primary antibody applied overnight at 4 at a dilution of 1 in in TRS-buffered saline. A ounterstain was omitted in order to failitate subsequent image analysis. MAGE ANALYSS Setions were viewed on a Zeiss photomirosope with a JV TK 1E video amera attahed. The amera was linked with a Y/ able to the in-built framegrabber board of an Apple Maintosh 71/8 AV miroomputer, and images were imported diretly to the freeware image analysis program NH-mage*, using the 'Plug-in digitizer' software Photoshop-ompatible plug-in. Sequential images were grabbed using the xlo objetive, moving along the entral line of eah biopsy speimen from one end of the available ortex to the other, without overlapping. The presene of glomeruli in the fields was ignored. Renal medulla was not measured. To alulate the volume fration of the marophages, eah image was onverted to greysale, and then a threshold was applied at a level whih distinguished between the stained marophages and the unstained bakground. *The image analysis program NH-mage was written by Wayne Rasband ( wayneqahelix.nih.gov.) and is distributed as freeware (at: or by anonymous ftp from zippy.nimh.nih.gov.). f a Y J lin Pathol: first published as /jp on 1 February Downloaded from on 1 November 18 by guest. Proteted by opyright.

3 1 Furness, Rogers-Wheatley, Harris 1. r- 1. f. 1 Figure Results of measuring marophage volume fration using 1 serial setions, all stained on separate oasions. Mean (entral line), range (vertial line) and quartiles (shaded bar) are shown. Results are shown for measurements made on two separate oasions. The proportion of blak to white pixels in the image (exluding the edges) was then alulated as a perentage. As sequential fields were assessed, a running mean of the marophage area fration was maintained automatially. Proessing of a typial image is illustrated in fig 1. A set of maros was written in the NH- mage programming language to automate this proess, suh that sequential fields an be measured with just two keystrokes per field. These maros are available as a simple text file, free by on request to pnfl@le.a.uk. METHOD VALDATON To test the reproduibility of the image analysis method, 1 sequential setions were ut from a single renal biopsy speimen. These were immunostained with PGM1 on 1 separate oasions, eah oasion separated by at least three days. The volume fration of marophages in the resulting set of 1 slides was then measured, and the measurements were repeated the following day. STATSTAL ANALYSS The regression oeffiients were alulated as part of the graphing proess by the program A-riket graph ; p values for these linear orrelations were alulated using Systat. omparisons between the mean marophage volume frations in normal biopsy speimens and those in the disease groups were made using Student's t test (unpaired), as alulated by Mirosoft Exel; 9% onfidene limits were also alulated using Exel. Results METHOD VALDATON The results of measurements of 1 repliate setions are shown in fig. The means, quartiles and maximum/minimum figures show little variation between the two oasions on whih they were measured. The overall variation is onsiderably less than one might 3. r.h.k 1.K 1. K. n apsule -o--- --D % Volume Aumulated mean 4-..-O ortio-medullary juntion Figure 3 Measurements of volume fration in adjaent non-overlappingfields of a single biopsy speimen (membranous glomerulonephritis), runningfrom renal apsule (left) to ortio-medullary juntion (right). Dotted line, atual measurements; solid line, aumulated mean. t is evident that in this ase there is a gradient of marophage infiltration aross the ortex, whih is of onsiderably greater magnitude than the measurement error as shown in fig g. -.4 E.3 E >. E.1 Normal K-i 1 1 Minimal hange Membranous Figure 4 Marophage volume frations in normal biopsy speimens, in minimal hange nephropathy and in membranous glomerulonephritis. Error bars represent 9% onfidene limits. expet with a quantitative immunoytohemial method, and we felt use of the method in the main part of the study was justified. n most biopsy speimens, the exat depth of the ortial tissue ould not be judged. n a few speimens, however, the renal apsule and ortio-medullary juntion were both visible. Suh ases were too few for statistial analysis, but there seemed to be a gradient of marophage numbers whih dereased from outer to inner ortex (fig 3). This finding probably justifies further study; if onfirmed, it suggests that sampling error, rather than measurement error, is likely to be the biggest soure of random variation with this method. MAROPHAGE VOLUME FRATONS The mean marophage volume frations are shown in fig 4. Marophage infiltration in minimal hange nephropathy and in membranous nephropathy was signifiantly greater J lin Pathol: first published as /jp on 1 February Downloaded from on 1 November 18 by guest. Proteted by opyright.

4 Renal marophages in nephroti states 11 D o u a) 4 _ 3 _ F 1! F 4 U 3. u 1 n 7 H a) U U U1 [ F D A { 4 B r =.99 p=ns r =.398 p< ov *, r =.393 v p<. * %* o o, Figure Satter graphs showing orrelations between marophage volume fration and reatinine at time of biopsy in (A) normal speimens, in (B) minimal hange nephropathy and in () membranous glomerulonephritis. than in normal ontrols (p <.1). Although the mean level of marophage infiltration in membranous glomerulonephritis was greater than in minimal hange disease, the differene was not statistially signifiant (p =.13). There was a signifiant orrelation between marophage volume fration and serum reatinine at the time of biopsy in membranous nephropathy and minimal hange disease (fig ). A signifiant orrelation was also found with the 4 hour urine protein output in minimal hange disease (fig 6). However, it is obvious from the satter plots that the regression value is low and the orrelation is poor. These orrelations have only just reahed signifiane, even though our automated measuring system permitted the analysis of relatively large numbers of speimens. s (N,.'1 L u, -z, - N._- a1) - \J, - 4 ) r ' r 1K 1 A r =.17 p=ns B + / r~~~~~~r=.433 bo/6 p=~~~p= r=.34 4 p=ns n, u.v Figure 6 Satter graphs showing orrelations between marophage volume fration and 4 hour urine protein output immediately before biopsy in (A) normal speimens, in (B) minimal hange nephropathy and in () membranous glomerulonephritis. Disussion These results onfirm that a signifiant interstitial infiltrate of marophages is a part of the pathologial proess in membranous nephropathy and in minimal hange nephropathy. The existene of a orrelation between marophage volume fration and serum reatinine in both diseases implies that immunologial mehanisms may be involved in the redued glomerular filtration rate whih is ommonly seen at presentation of both diseases. This is not surprising in the ase of membranous glomerulonephritis; but in minimal hange nephropathy, the presene of interstitial marophages is not widely reognised as part of the disease proess. ndeed, one argument whih has been used against the puromyin aminonuleoside (PAN) model of minimal hange nephropathy is that PAN indues an interstitial o J lin Pathol: first published as /jp on 1 February Downloaded from on 1 November 18 by guest. Proteted by opyright.

5 1 Furness, Rogers-Wheatley, Harris infiltrate of marophages, on whih the renal impairment seen in the model depends.8 Our results suggest that this ritiism is invalid, and also argue against the widely held view that any renal impairment in minimal hange nephropathy has a haemodynami ause,9 refleting irulation volume hanges in nephrosis, rather than an immunologial one. Furthermore, it shows that the presene of a moderately heavy interstitial infiltrate of marophages does not neessarily indiate the presene of an irreversible fibroti proess, as minimal hange nephropathy rarely, if ever, progresses to renal fibro- SiS Ȧ lose orrelation has previously been reported between marophage infiltration and proteinuria in mesangial proliferative glomerulonephritis with mesangial gm.' Tubular expression of monoyte hemoattratant protein-i (MP-1) is known to be inreased in membranous glomerulonephritis and to orrelate with the interstitial marophage infiltrate." t has been suggested that the mere presene of large quantities of filtered protein in the tubules represents an insult to the tubular epithelium whih an result in hemokineindued marophage infiltration. This ertainly seems to be the ase in the protein overload model of proteinuria in the rat." We have shown that MP-1 is released by ultured tubular epithelial ells after the addition of protein to the medium bathing the apial surfae of the ells.7 However, marophage infiltration is indued in many animal models of fibroti disease, in the kidney and other organs.'4 This inludes the fibrosis aused by ureteri obstrution, where proteinuria is not an issue.' 16 Of ourse, we do not know for how long our patients had proteinuria before biopsy was performed; variation in this time may aount for some of the variation in marophage numbers, but it seems likely that other aspets of the disease proess are involved in the variation we have seen. f assessment of marophages in renal biopsy speimens is to be relevant to the severity and progression of disease, it is evident that we should be measuring marophage funtion rather than simply their presene. Assessment of ytokines suh as tumour growth fator,b may be more promising." There has been onsiderable interest reently in quantitative or semiquantitative approahes to prediting the rate of disease progression from renal biopsy speimens, both native34 and transplant.' - Most of these studies have used either semiquantitative soring on a sale of to 3 by a 'blinded' observer, or laborious manual morphometry, or ell ounting. Having previously undertaken a study requiring point-ounting,' we have been very favourably impressed with the method desribed here, whih an be applied to any tissue omponent that an be loalised by immunostaining. The high speed and low expense of the method mean that if the linial signifiane of suh methods an be demonstrated, it will be easy to onvert this information into a routine quantitative assessment-a feature urrently laking in most diagnosti histopathology reports. Part of this work was supported by a grant from the Medial Researh ounil to PNF. 1 Risdon RA, Sloper J, DeWardener H. Relationship between renal funtion and histologial hanges found in renal biopsy speimens from patients with persistent glomerular nephritis. Lanet 1968;ii: Shainuk L, Striker G, Luther R, Benditt E. Struturalfuntional orrelations in renal disease.. The orrelations. Hum Pathol 197;1: Goumenos DS, Brown B, Shortland J, el Nahas AM. Myofibroblasts, preditors of progression of mesangial ga nephropathy? Nephrol Dial Transplant 1994;9: Roberts S, Burrows, Shanks JH, Venning M, MWilliam U. nterstitial myofibroblasts; preditors of progression in membranous nephropathy [abstrat]. J Pathol 1996;178S: 44A. Eddy AA. nterstitial marophages as mediators of renal fibrosis. Experimental Nephrology 199;3: Furness PN. Extraellular matrix and the kidney. J lin Pathol 1996;49: Burton, Walls J, Harris K. Tubular MP-1 prodution; a mehanism for proteinuria-indued interstitial inflammation [abstrat]. Pro Eur Dial Trans Asso 1996;33:71A. 8 Harris K, Lefkowith J, Klahr S, Shreiner G. Essential fatty aid defiieny ameliorates aute renal dysfuntion in the rat after the administration of the aminonuleoside of puromyin. J lin nvest 199;86: Lieberthal W, Levinsky NG. Aute linial renal failure. n: Seldin DW, Giebish G, eds. The kidney: physiology and pathophysiology. New York: Raven Press, 199: Garia del Moral R, Gomez-Morales M, ortes V, Aguayo ML, Gigosos RL, Lardelli P, et al. Mononulear ell subsets ingm mesangial proliferative glomerulonephritis. A omparative study with minimal hange nephroti syndrome and immunonegative mesangial proliferative glomerulonephritis. Nephron 1993;6: Prodjosudjadi W, Gerritsma JS, van Es LA, Daha MR, Bruijn JA. Monoyte hemoattratant protein-i in normal and diseased human kidneys: an immunohistohemial analysis. lin Nephrol 199;44: Burton, Harris K. The role of proteinuria in the progression of hroni renal failure. AmJKidney Dis 1996;7: Eddy AA, Giahelli M. Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria. Kidneynt 199;47: El Nahas AM. Pathways to renal fibrosis. Exp Nephrol 199; 3: Shreiner G, Harris K, Purkerson M, Klahr S. The immunologial aspets of aute ureteral obstrution: haraterisation, loalisation and kinetis of the immune ell infiltrate in the kidney. Kidneynt 1988;34: Diamond JR. Marophages and progressive renal disease in experimental hydronephrosis. Am J Kidney Dis 199;6: Yamamoto T, Noble NA, Miller DE, Border WA. Sustained expression of TGF-beta 1 underlies development of progressive kidney fibrosis. Kidneynt 1994;4: soniemi H, Taskinen E, Hayry P. Histologial hroni allograft damage index aurately predits hroni renal allograft rejetion. Transplantation 1994;8: Dimeny E, Wahlberg J, Larsson E, Fellstrom B. an histopathologial findings in early renal allograft biopsies identify patients at risk for hroni vasular rejetion? lin Transplant 199;9: Rush DN, Jeffery JR, Gough J. Sequential protool biopsies in renal transplant patients: repeated inflammation is assoiated with impaired graft funtion at 1 year. Transplant Pro 199;7: Mulloh TA, HarperSJ, Donnelly PK, Moorhouse J, Bell PR, Walls J, et al. nfluene of nifedipine on interstitial fibrosis in renal transplant allografts treated with ylosporin A. J lin Pathol 1994;47: Furness PN. Morphometry in pathology [letter]. J Pathol 199;177:441. J lin Pathol: first published as /jp on 1 February Downloaded from on 1 November 18 by guest. Proteted by opyright.