Glomerular sclerosis in kidneys with congenital nephrotic syndrome (NPHS1)

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1 & 6 Interntionl Soiety of Nephrology originl rtile Glomerulr slerosis in kineys with ongenitl nephroti synrome (NPHS1) A-M Kuusniemi 1, J Merenmies 1, A-T Lhenkri 1, C Holmerg 1, K Slmel 2, R Krikoski 1, J Rpol 1 n H Jlnko 1 1 Hospitl for Chilren n Aolesents n Biomeium Helsinki, University of Helsinki, Helsinki, Finln n 2 Kiney Trnsplnttion Unit, Helsinki University Hospitl, Helsinki, Finln Congenitl nephroti synrome of the Finnish type (NPHS1) is rre geneti isese use y muttions in the NPHS1 gene enoing mjor pooyte slit-iphrgm protein, nephrin. Ptients with NPHS1 hve severe nephroti synrome from irth n evelop renl firosis in erly hilhoo. In this work, we stuie the evelopment of glomerulr slerosis in kineys remove from 4- to 44-month-ol NPHS1 ptients. The pthologil lesions n expression of glomerulr ell mrkers were stuie in nephretomize NPHS1 n ontrol kineys using light n eletron mirosopy n immunohistohemistry. An nlysis of 128 glomeruli from ptients revele progressive mesngil slerosis n pillry olitertion. Although few inflmmtory ells were etete in the mesngil re, prglomerulr inflmmtion n firosis ws ommon. The pooytes showe severe ultrstruturl hnges n hypertrophy with the upregultion of ylins A n D1. Pooyte prolifertion, however, ws rre. Apoptosis ws hrly etete n the expression of ntipoptoti B-ell lymphom-2 n propoptoti p3 were omprle to ontrols. Moerte mounts of pooytes were serete into the urine of NPHS1 ptients. Shrinkge of the glomerulr tuft ws ommon, wheres olusion of tuulr opening or protrusion of the glomerulr tuft into suepithelil spe or through the Bowmn s psule were not etete. The results inite tht, in NPHS1 kineys, the mge pooytes inue progressive mesngil expnsion n pillry olitertion. Pooyte epletion, glomerulr tuft hesion, n misirete filtrtion, however, seem to ply minor role in the nephron estrution. Kiney Interntionl (6) 7, oi:1.138/sj.ki.1779; pulishe online 3 August 6 KEYWORDS: nephrin; pooyte; proteinuri; glomeruloslerosis; renl filure Corresponene: H Jlnko, Hospitl for Chilren n Aolesents, University of Helsinki, 29 Helsinki, Finln. E-mil: hnnu.jlnko@hus.fi Reeive 17 My 6; revise 22 June 6; epte July 6; pulishe online 3 August 6 Constnt proteinuri is known to result in glomerulr slerosis n tuulointerstitil firosis, often grully leing to terminl renl filure. 1 Reent stuies hve inite tht injury in glomerulr epithelil ells (pooytes) les to proteinuri n lso triggers progressive glomerulr slerosis. Muttions in genes enoing for pooyte proteins suh s pooin, CD2-ssoite protein n -tinin-4 result in proteinuri n the evelopment of lesions lssifie s fol segmentl glomeruloslerosis (FSGS). 2,3 Congenitl nephroti synrome use y muttions in WT1 gene, on the other hn, is ssoite with iffuse mesngil slerosis. 4 In this work, we nlyze the glomerulr hnges in kineys with ongenitl nephroti synrome of the Finnish type (CNF, NPHS1), whih is reessively inherite renl isese use y muttions in the nephrin gene. Nephrin is pooyte-speifi protein lote t the slit iphrgm of kiney glomerulus. Mny of the NPHS1 muttions, suh s Fin-mjor n Fin-minor, le to the sene of nephrin n severe nephroti synrome strting from the irth. 6 The NPHS1 kineys show isrupte slit iphrgms n effement of pooyte foot proesses seen in mny other proteinuri kiney iseses. 6,7 Chilren with NPHS1 re nephretomize s infnts (usully uner the ge of 1 yer) when the renl funtion is still well preserve. These kineys serve s unique humn mteril to stuy the evelopment of glomeruloslerosis. This is emphsize y the ft tht the si efet is restrite to pooytes; it ffets eqully ll glomeruli n the srring in NPHS1 kineys progresses in fst mnner s ompre to mny other isorers. During the pst yers, proteinuri hs een ssoite with numer of morphologi vrints of glomerulr slerosis, oth in humn iseses n niml moels. 8,9 Also, sequene of pthologil events hs een suggeste, whih strts with pooyte epletion n formtion of glomerulr tuft hesions with Bowmn s psule resulting in misirete filtrtion n estrution of the nephrons Bse on these finings, we were espeilly intereste in the lesions etetle in the eno- n extrpillry omprtments of NPHS1 glomeruli in the erly phse of the sleroti proess. Kiney Interntionl (6) 7,

2 o r i g i n l r t i l e A-M Kuusniemi et l.: Glomerulr slerosis in NPHS1 kineys RESULTS The overll histology of the 49 NPHS1 kineys nephretomize t the ge of 4 44 months rnge from nerly norml to severely mge (Figure 1). The finings inlue glomerulr slerosis, interstitil firosis n inflmmtion, tuulr trophy n ilttion, n rteril wll thikening. Vrition in the rte of progression of the pthologil lesions ws evient, so tht firly norml n totlly sleroti glomeruli n tuuli oul e seen next to eh other (Figure 1). Affete glomeruli were sttere roun the whole ortex, without preiletion to juxtmeullry re s often seen in FSGS. Clusters of inflmmtory ells were ommon roun egenerting glomeruli in NPHS1 kineys (Figures 1, 2e, n 3j). Mesngil expnsion n pillry olitertion were erly n onstnt finings Histopthology of 128 glomeruli in NPHS1 kineys from ptients with ifferent ges ws systemtilly nlyze (Figure 2). Mesngium ws ffete in prtilly every glomerulus (99.7%), so tht the lesions rnge from mil wiening of the mesngil stlk to totl slerosis (Figure 3 e). The erly lesions were sttere roun the glomerulr tuft n onsiste of inrese numer of mesngil ells n mtrix (Figure 3). The numer of proliferting ells, s revele y the MIB-1 stining, ws inrese in the NPHS1 ( /glomerulr gross setion) s ompre to ontrols (.27.14/GCS) (Po.). On the other hn, few terminl eoxynuleotiyl trnsferse-meite UTP nik-en leling (TUNEL)-positive poptoti ells were present in the glomerulr tuft oth in NPHS1 kineys n ontrols (.17.1/GCS n.77.7/gcs, respetively). Glomerulr slerosis ( 3) Age t nephretomy (months) Figure 1 Progression of renl mge in NPHS1 kineys. () A typil fining in firly well-preserve NPHS1 kiney (nephretomize t the ge of 7 months): one totlly slerose glomerulus (rrow) surroune y severl glomeruli with only mil mesngil expnsion. () Severe glomerulr slerosis, rteril wll thikening, n tuulointerstitil mge in n NPHS1 kiney remove t the ge of 29 months. () Development of glomerulr slerosis (sore 3) in 49 NPHS1 kineys in reltion to the ptients ge t the time of nephretomy. The rte of progression of glomerulr slerosis vries onsierly mong the ptients (r 2 ¼.). ( n ; PASM stinings, originl mgnifition ). Severe or totl mesngil slerosis Wiene urinry spe More thn one lyer of PECs r 2 =.72 r 2 =.43 r 2 = Age t nephretomy (months) Age t nephretomy (months) Age t nephretomy (months) Thikene Browmn's e Periglomerulr inflmmtion f 1 sement memrne 1 1 Periglomerulr firosis 8 6 r 2 = r 2 = r 2 = Age t nephretomy (months) Age t nephretomy (months) Age t nephretomy (months) Figure 2 Glomerulr histology in reltion to the ge of the ptients t the time of nephretomy. The results re se on semiquntittive nlysis of glomerulr histology (see Mterils n Methos for more etils). One thousn five hunre n twenty-eight glomeruli from PASM-stine smples in kineys of ptients (ge 4 44 months) with NPHS1 were nlyze. () The proportion of glomeruli with extensive mesngil slerosis ws higher in oler kineys. () The prevlene of wie urinry spe ws roun % in ll NPHS1 smples. () A single-ell lyer of PECs ws foun in over 99% of NPHS1 glomeruli. () There ws gret vrition in the rte of Bowmn s psule thikening etween the smples. (e) Development of periglomerulr inflmmtory ell infiltrtion h the strongest orreltion with ge (r 2 ¼.79). (f) Periglomerulr firosis surrouning the glomerulus i not orrelte with ge Kiney Interntionl (6) 7,

3 A-M Kuusniemi et l.: Glomerulr slerosis in NPHS1 kineys o r i g i n l r t i l e e f g h i j Proportion of glomeruli 1% 8% 6% % % % No (n=4) Mil No (n=) Moerte (n=61) Severe (n=239) Totlly slerose (n=179) Periglomerulr firosis n inflmmtion: No Mil Severe Figure 3 Light mirosopi finings in NPHS1 kineys. () Control kiney glomerulus with norml mesngium. ( e) A series of NPHS1 glomeruli with inresing mount of mesngil slerosis. Some egree of mesngil slerosis ws foun in 99.7% of NPHS1 glomeruli. (f) Urinry spe (Bowmn spe) ws wie in 18% of NPHS1 glomeruli nlyze. Hypertrophi single-ell lyer ws oserve in 9% of the glomeruli, n in 87% of these glomeruli, the Bowmn s sement memrne ws thikene. (g) There were very few tip lesions n no perituulr hnges in NPHS1 glomeruli. (h n i) High-mgnifition imges showing ommon fining in NPHS1 n ontrol kineys: glomerulr tuft seems to e in ontt with the Bowmn s psule (rrows). (j) Correltion of the mesngil slerosis with periglomerulr firosis n inflmmtion. ( i; PASM stinings, originl mgnifition in () (g) n in (h) n (i)). Inflmmtory ells were rre in the pillry tuft s stuie y immunohistohemistry, with men of n ells/gcs in NPHS1 n ontrol kineys, respetively (Po.). The ells were minly lymphoytes with ells of the monoyte/mrophge linege (expressing CD14, CD13, n CD68 mrkers) s the seon lrgest group (Figure 4). Olitertion of glomerulr pillries oul e seen lrey in glomeruli with mil n moerte mesngil hnges (Figure ). Immunoperoxise stining for the enothelil mrker CD34 lerly emonstrte erese in the totl enothelil surfe re of the pillry tuft (Po.) (Figure e) n nrrowing of the pillry lumens in NPHS1 kineys s ompre to ontrols (Po.) (Figure f). Enothelil ell swelling ws evient in iret mirosopy (Figure 6 n ), ut pillry thromosis or hyline olusion ws not seen mong 23 NPHS1 glomeruli from 11 ptients stuie with light mirosopy of HE stining. Collpse of the pillry tuft ws evient in glomeruli with severe mesngil lesions. Pooytes showe phenotypi hnges n hypertrophy, ut little prolifertion or poptosis Pooytes in NPHS1 kiney show mrke struturl hnges s shown previously. 7 Prominent pooyte ell oies with mirovillous egenertion, elongte primry proesses, n foot proess effement ws most evient in snning eletron mirosopy (Figure 6 n ). Also in light mirosopy, pooytes in NPHS1 kineys looke hypertrophi with prominent nuleus (Figure 6 n ). The numer of WT1-positive ells (pooytes) in wellpreserve NPHS1 glomeruli ws similr to ontrols Kiney Interntionl (6) 7,

4 o r i g i n l r t i l e A-M Kuusniemi et l.: Glomerulr slerosis in NPHS1 kineys Positive ells per glomerulr ross setion CD13 CD14 CD68 CD3 CDy MCT Neutrophils (Leer) Eosinophils (H&E) Figure 4 Light mirosopy n immunoperoxise stining of inflmmtory ells in NPHS1 n ontrol glomeruli. There ws n verge of 3.7 n 1.1 inflmmtory ells per glomerulr rosssetion in NPHS1 n ontrol kineys, respetively. () The numers of ifferent inflmmtory ell types in NPHS1 ( ) n ontrol (&) glomeruli. () CD3-positive T-lymphoytes were the lrgest group, followe y () CD-positive B-lymphoytes. The ells of monoyte/ mrophge linege inluing () CD14-positive ells were the seon lrgest group fter lymphoytes. Smller mounts of (e) neutrophils, mst ells (mst ell Tryptse), n eosinophils were present. Originl mgnifition. ( n /GCS, respetively) (Figure 7 ). The re frtion of the pooyte intermeite filment, vimentin, ws slightly reue in NPHS1 glomeruli with mil lesions s ompre to ontrols (Figure 7 f). In lerly sleroti glomeruli, the expression of oth mrkers ws erese initing pooyte loss (Figure 7 n f). The numer of MIB-1-positive proliferting extrpillry ells ws low (.8/GCS) in NPHS1 kineys (Tle 1, Figure 8). Also, pooyte lyer hyperplsi, s seen in ollpsing glomerulopthy, ws suspete in only 11 of 128 (1%) NPHS1 glomerulr ross-setions, ll of them showing severe mesngil lesions. Even though pooyte prolifertion ws nerly sent, there ws upregultion of ell yle promoters ylin A n espeilly ylin D1 n ownregultion of ell yle kinse inhiitor p7 in NPHS1 ompre to ontrols (Tle 1, Figure 8 h). This inites tht pooytes were in e e Are frtion (%) Control NPHS 1 minor hnges NPHS 1 severe lesions P=.4 CD34 Control NPHS 1 minor hnges NPHS 1 severe lesions P=.2 Cpillry lumen hypertrophi stte, whih ws lso supporte y the finings in light mirosopy (see ove). Apoptosis of pooytes ws not prominent, s no TUNEL-positive extrpillry ells were foun in NPHS1 glomeruli (Tle 1, Figure 8). In orne with this, no extrpillry ells with onense nulei (poptosis or mitosis) were etete in NPHS1 glomeruli (Tle 1). Although the expression of propoptoti p3 ws not inrese in the extrpillry ells (Tle 1, Figure 8i n j), the numer of ells expressing the ntipoptoti B-ell lymphom-2 ws slightly ut not signifintly higher in NPHS1 glomeruli (./GCS) thn in ontrols (.1/GCS) (P ¼ NS) (Tle 1, Figure 8k n l). Possile ethment of pooytes ws evlute y ounting WT1-positive ells in tuulr lumens of formlinfixe tissue smples. There were no WT1-positive ells in 213 NPHS1 n 128 ontrol tuulr ross-setions. Exretion of pooytes into voie urine ws nlyze using immunofluoresene stining of poolyxin n ZO-1- positive ells n grnules in 18 (of whih 1 were use in immunohistology oule stinings) urine smples from NPHS1 ptients (ge rnge 3 1 months) n 12 ontrol f Are frtion (%) Figure Cpillry hnges in NPHS1 glomeruli. ( ) Immunoperoxise stining of enothelil ell mrker CD34 showing nrrowing of the pillry lumen in NPHS1 kineys s ompre to ontrols. (e) The re frtion of CD34 emonstrting the mount of enothelium present in glomerulus ws signifintly lower in NPHS1 thn in ontrol glomeruli. (f) Also, the re frtion of res insie the CD34-stine pillry wlls ws signifintly lower in NPHS1 glomeruli. The re frtions were lulte from totl of 21 glomeruli. Originl mgnifition Kiney Interntionl (6) 7,

5 A-M Kuusniemi et l.: Glomerulr slerosis in NPHS1 kineys o r i g i n l r t i l e smples. Urine from NPHS1 ptients ontine more poolyxin-positive ells (mein 1.9 vs.7 ells/ml) n grnules (totl sores: 24.9 vs 1.) thn ontrol urine smples (Figure 9,, n e). Doule immunostining for poolyxin n ZO-1 revele mein of 13.9 n.3 opositive ells in NPHS1 n ontrol urine smples, respetively (Figure 9,, n f). Figure 6 Pooytes in NPHS1 glomeruli. (, ) Snning eletron mirosopy of NPHS1 glomeruli showing prominent ell oies, unnt mirovillr strutures, long primry proesses (white rrow), n foot proess effement. () Light mirosopy of NPHS1 kineys showing pooytes with lrge, hypertrophie ell oies n lrger nulei tht were lote fr from the sement memrne (rrowhes). Enothelil ells were lso hypertrophi in NPHS1 glomeruli (rrows). () Norml pooytes (rrowhes) n enothelil ells (rrows) in ontrol kiney. ( n ; PASM stinings, originl mgnifition ). Prietl epithelil ells were resistnt to protein overlo Prietl epithelil ell (PEC) lyer looke norml in 91% (197/1) of the glomeruli tht were inlue in the nlysis. Hypertrophi single-ell lyer ws oserve in 9% of the glomeruli, mostly (87%) in ssoition with thikene sement memrne (Figure 3f). Only one glomerulus with more thn one PEC lyer ws oserve. Eighteen n five PECs with onense nuleus (poptosis/mitosis) per 1 glomeruli were oserve in NPHS1 n ontrols, respetively (Po.). PECs positive for the prolifertion mrker MIB-1 were somewht more frequent in NPHS1 kineys thn ontrols (18 vs 3 per 1 glomeruli, P ¼ NS). On the other hn, only one TUNEL-positive poptoti PEC ws foun in 836 NPHS1 glomeruli. The results inite tht PECs were quite resistnt to the unusully high protein onentrtion of the primry filtrte. No protrusion of the pillry tuft into perituulr or periglomerulr spes ws oserve Urinry spe (Bowmn spe) ws wie in 18% (221/1227) of the NPHS1 glomeruli (Figure 2). Two-thirs of these showe t lest moerte slerosis, suggesting tht the wiening ws use y shrinkge of the glomerulr tuft. This ws supporte y the ft tht the verge re of glomeruli with norml (n ¼ 4) n wie urinry spe (n ¼ 13) ws quite similr ( vs pixels; P ¼ NS), respetively. Contt of the pillry tuft with the Bowmn s psule ws quite ommon oth in NPHS1 n ontrol kineys (Figure 3h n i). Protrusion of the pillry tuft through Bowmn s psule, however, ws not seen in ny of the 141 glomeruli speifilly nlyze. Ahesion of the pillry tuft n Bowmn s psule t the tuulr opening (tip lesion) ws suspete in only four of the 7 (2%) glomeruli in whih tuulr opening ws visile (Figure 3g). Similrly, seprtion Positive ells/gcs P=NS WT1 Controls NPHS1 minor hnges NPHS1 severe lesions e f Are frtion % P=.7 Controls NPHS1 minor hnges NPHS1 severe lesions Figure 7 The expression of WT1 n vimentin in NPHS1 n ontrol kineys. ( ) The numer of WT1-positive ells per glomerulr ross-setion in NPHS1 n ontrol kineys. ( f) Are frtion of vimentin stining in NPHS1 kineys. Originl mgnifition. Vimentin Kiney Interntionl (6) 7,

6 o r i g i n l r t i l e A-M Kuusniemi et l.: Glomerulr slerosis in NPHS1 kineys Tle 1 Expression of mrkers for ell yle n poptosis in NPHS1 n ontrol glomeruli NPHS1 Control P-vlue MIB (2/34).37.2 (/18) NS TUNEL (/68) B (1/2) NS Conense nuleus (/63) B (2/414) NS p (282/16) (2438/1).3 Cylin A.37.3 (/82) (/31).4 Cylin D (17/337) (18/224).3 p (19/31).17.1 (1/164) NS Bl (/378).17.1 (3/281) NS Arevitions: Bl, B-ell lymphom; MIB, min om homolog; NPHS1, nephroti synrome of the Finnish type; NS, nonsignifint; TUNEL, terminl eoxynuleotiyl trnsferse-meite UTP nik-en leling. Results re expresse s the men7s.. of positive ells per glomerulr ross-setion. In prenthesis: positive ells/glomerulr ross-setions exmine. Positive extrpillry ells ounte only (exept in the se of p7). All p7-positive glomerulr ells ounte, s p7 is expresse only in pooytes. e f g h i j k l Figure 8 Cell yle mrkers of extrpillry ells in NPHS1 n ontrol glomeruli. () There were very few (.8/GCS) proliferting extrpillry ells (rrow) in NPHS1 kineys se on the expression of MIB-1. () Apoptosis ws even more unommon mong extrpillry ells in NPHS1. Few (.1/GCS) TUNEL-positive ells (rrow) were however foun within the tuft. () There were signifintly less p7-positive ells in NPHS1 thn in () ontrol glomeruli. (e h) On the other hn, there were more ylin A- n ylin D1-positive extrpillry ells in NPHS1 glomeruli thn in ontrols (Po.). (i l) On verge, one p3- n one B-ell lymphom-2-positive extrpillry ell ws foun mong GCS. Although they were more frequent in NPHS1, the ifferene to ontrols ws not sttistilly signifint. ( n l; immunohistohemil stinings). Originl mgnifition. of tuulr sement memrne from its epithelium n formtion of perituulr spe ws not seen mong the tuulr openings oserve. Segmentl resent ws seen in only.% (/116) of the glomeruli nlyze. DISCUSSION We evlute the evelopment glomerulr lesions in NPHS1 kineys lking the mjor pooyte slit-iphrgm protein nephrin. The nlysis inite tht onstnt hevy proteinuri in these kineys inevitly resulte in totl glomerulr slerosis n estrution of the nephrons. The progression rte vrie from nephron to nephron, suggesting tht protein lekge, ffeting eqully ll glomeruli, ws not the only ontriuting ftor for slerosis. Impt of the mge pooytes on the mesngil n enothelil ells ws most proly ruil for the pthologil proess. The most onstnt glomerulr fining ws mesngil slerosis s reporte previously. 13,14 Aumultion of extrellulr mtrix proteins n ells in the mesngium ws evient in prtilly every glomerulus. Immunohistohemistry 1428 Kiney Interntionl (6) 7,

7 A-M Kuusniemi et l.: Glomerulr slerosis in NPHS1 kineys o r i g i n l r t i l e e Cells/ml Poolyxin Cells/ml Poolyxin n ZO-1 NPHS1 Control NPHS1 Control Figure 9 Immunofluoresene stining of pooytes in the urine of NPHS1 ptients n ontrols. () Immunofluoresene for poolyxin in NPHS1 kineys. Poolyxin is foun in pooytes n enothelil ells. () ZO-1 is speifi for pooytes in the glomerulus. () Poolyxin- n () ZO-1-positive ells in NPHS1 urine (imges from immunofluoresene oule stining). (e) Urine from NPHS1 ptients ontine more poolyxin-positive ells (mein 1.9 vs.7 ells/ml) thn ontrol urine smples. (f) Doule immunostining for poolyxin n ZO-1 revele mein of 13.9 n.3 o-positive ells in NPHS1 n ontrol urine smples, respetively. The line inites the mein (in e n f). Originl mgnifition. revele tht the glomerulr tuft ontine low numers of inflmmtory ells, initing tht the inrese ellulrity ws use y retive mesngil ells. Espeilly the low numer of monoytes/mrophges ws interesting, s these ells hve een suggeste to ply entrl role in resent formtion n glomeruloslerosis. 1,16 The erly lesions in the glomerulr tuft i not show preiletion to perihilr or urinry pole, s is the se in some types of FSGS. 17,18 Also, tip lesions, tht woul olue the opening of the tuulr lumen, were prtilly non-existing in NPHS1 kineys. Mesngil lesions were ssoite with olitertion of the glomerulr pillries. This ws oserve in the iret mirosopy n immunohistohemil stining n ws evient lrey in glomeruli with mil lesions. Also, the thikening of rteriolr wlls in NPHS1 kineys is onstnt fining 19 n further highlights the importnt role of efetive loo irultion in the srring proess.,21 Bse on morphologil nlyses of FSGS kineys, Kriz n others hve evelope wiely epte theory tht, in proteinuri kineys, poptosis or ethment of pooytes le to enue res of glomerulr sement memrne, f whih here to the Bowmn s psule. This then results in misirete filtrtion of urine into the prglomerulr n prtuulr spe, finlly leing to nephron loss This pooyte epletion theory is supporte y finings tht pooytes re foun in the urine from ptients n experimentl nimls with proteinuri In NPHS1 kineys, pooytes unoutely ply mjor role oth in the protein lekge n in triggering the sleroti proess. Our previous eletron mirosopi nlysis 7 n the results in this work, however, suggest tht it my not e question of simple pooyte epletion, ut rther more omplex interply of pooytes with mesngil n enothelil ells. Similr phenomen hve een oserve in rts with nti-thy 1.1 glomerulonephritis when trete with puromyin minonuleosie injetions s well s in trnsgeni mie where pooyte mge hs een use y pooyte-speifi immunotoxin. 29,3 Pooytes in NPHS1 kineys unergo phenotypi hnges, suh s foot proess effement, whih is typil for nephroti iseses. They lso show hypertrophy, whih ws evient oth in eletron n light mirosopy n further supporte y the finings tht upregultion of ell yle promoters ylin A n D1, n ownregultion of ell yle kinse inhiitor p7 (together sign of hypertrophi n hyperplsti phenotype) were foun in prllel with low rte of prolifertion in pooytes. 31 Importntly, the pooytes in NPHS1 kineys i not proliferte, s is the se in the ollpsing vrint of FSGS. 32 Sheing of pooytes into urine is elieve to reflet the pooyte injury, n hs een reporte in severl types of humn kiney iseses Anlysis of urine from NPHS1 ptients revele tht the numer of poolyxin-positive ells in NPHS1 ptients (mein 1.9 ells/ml) ws quite lose to tht previously foun in ptients with FSGS (4.2 n 1.3 ells/ml) 2,26 n lupus nephritis (6.2 ells/ml). 27 So, it seems ler tht pooytes eth from the glomerulr sement memrne in NPHS1 kineys. Whether this reflets some primry event or is onsequene of the glomerulr tuft mge remins to e solve. We were espeilly intereste in possile synehie of the glomerulr (enue) pillries with the Bowmn s psule leing to misirete filtrtion of pillry loo or urine. Atthment of the glomerulr tuft with the Bowmn s psule ws frequently seen in NPHS1 n ontrol kineys in iret mirosopy n this fining ws iffiult to interpret. However, if misirete filtrtion n filtrte spreing were n importnt erly event leing to glomerulr slerosis n nephron egenertion, segmentl lesions shoul hve een seen in sustntil proportion of the hunres of wellpreserve (funtionl) NPHS1 glomeruli nlyze. This ws lerly not the se. We foun no lol protrusions of the glomerulr tuft into the prtuulr spes or prglomerulr spe, s reporte in FSGS In onlusion, the results suggest tht in NPHS1 kineys the role of mesngil slerosis n stenosis of glomerulr pillries re pivotl for the estrution of the glomerulus, n possily the ownfll of the entire nephron. Whih Kiney Interntionl (6) 7,

8 o r i g i n l r t i l e A-M Kuusniemi et l.: Glomerulr slerosis in NPHS1 kineys meitors re importnt in the interply of mge pooytes with mesngil n enothelil ells remins to e solve. MATERIALS AND METHODS Tissue n urine smples A totl of 49 hilren with NPHS1 (ge 4 44 months) were nephretomize t the Hospitl for Chilren n Aolesents, University of Helsinki, Finln, etween the yers 1986 n 3. Twenty-nine of the ptients were homozygotes for Fin-mjor muttion, nine h Fin-mjor/Fin-minor genotype n four were Fin-minor homozygotes.,6 In ition, one ptient h Fin-mjor/ C46Y, one h Fin-mjor/IVS26-1 g-, n one Fin-mjor/C46Y genotype. No knowlege of the muttions in four smples ws ville. All ptients h severe nephroti synrome from the irth n were trete with ily lumin infusions to supplement the ontinuous hevy protein losses. Routine formlin-fixe prffin-emee smples were prepre from the kineys n the rest of the renl ortex ws snp-frozen in liqui nitrogen n store t 71C. The glomeruli for snning eletron mirosopy were isolte uner issetion mirosope n proesse s esrie previously. 7 As ontrols, we use eight norml ult kineys (ge 47 8 yers) remove for trnsplnttion. These kineys prove unsuitle for trnsplnttion minly euse of vsulr normlities. Formlinfixe prffin-emee setions n snp-frozen smples were ollete. The ver kineys h to e use s ontrols, s fresh tissue smples from norml infnt kineys were not possile to otin. Fresh urine smples were otine from four NPHS1 hilren ge 3 1 months, five peitri ptients with norml kiney funtion, n five helthy ults. Fifteen smples were ollete from NPHS1 hilren n 12 smples from the ontrol group. The urine speimens were proesse within 2 h fter voiing. 33 The stuy protool ws pprove y the ethil ommittee of the Hospitl for Chilren n Aolesents of the University of Helsinki n onforme to the priniples outline in the Delrtion of Helsinki. Antioies The following ntioies were purhse from Dko (Glostrup, Denmrk): B-ell lymphom-2 (M887), CDy (M7), CD34 (M716), CD68 (M876), MIB-1 (M72), p3 (M71), n vimentin (M7). Antioies ginst ylin A (NCL-Cylin A), ylin D1 (NCL-ylin D1-GM), n WT1 (NCL-WT-1-6FH2) were ought from Novostr (Newstle upon Tyne, UK). From Am (Cmrige, Cmrigeshire, UK) were ntioies ginst CD3 (828) n CD14 (8679). Antioies ginst CD13 (MS-179) n p7 (MS-162-P) were ought from Neomrkers (Fremont, CA, USA). In ition, we use mst ell tryptse (Mo 347) (Dignosti BioSystems, Plesnton, CA, USA), poolyxin (AF168) (R&D Systems, Minnepolis, MN, USA), n ZO-1 (61-73) (Zyme Lortories, Sn Frniso, CA, USA) ntioies. All these primry ntioies were unonjugte, n ilutions from 1:1 to 1:2 were use. We use the following mrkers for ifferent inflmmtory ell types: CD13 for monoytes, CD68 for mrophges, CD14 for monoytes n mrophges n some of their supopultions, mst ell tryptse for tivte mst ells, CD3 for T-lymphoytes, n CDy for B-lymphoytes. Light mirosopy The histologil lesions in the NPHS1 glomeruli were evlute y light mirosopy from prffin-emee tissue setions stine with hemtoxylin n eosin or perioi i silver methenmin (PASM). Glomerulr slerosis ws orrelte to the ge t nephretomy in 49 NPHS1 kineys s esrie previously (Figure 1). 19 The progression of glomerulr mge ws nlyze y gring 1 fetures of glomerulr histology in 128 glomeruli of NPHS1 kineys (ge rnge 4 44 months, mein ge 8 months). The prmeters were: the inrese of mesngil mtrix n ell ontent, the size of the urinry spe, the thikness of the Bowmn s sement memrne, the size of the mesngil hilus, hypertrophy n the numer of PEC, n the mount of periglomerulr firosis n periglomerulr inflmmtion. In ition, the presene of resents, tipping of the glomerulr tuft into the tuulr pole, perituulr hnges, n possile hyperplsi of the pooyte lyer were note on eh glomerulus. Immunohistohemistry For the immunofluoresene stinings, the ryosetions ( mm) of the kiney smples were fixe with 3.% prformlehye or etone, epening on the ntioy use. The stinings were performe in tritionl wy. Setions use s negtive ontrols were inute in phosphte-uffere sline inste of primry ntioy. Poolyxin n ZO-1-positive ells n grnulr mteril in the urine seiments of NPHS1 ptients n ontrols were stuie y immunofluoresene s esrie previously. 28 Immunoperoxise stinings were performe on the setions of formlin-fixe, prffin-emee renl smples in onventionl wy. To improve ntioy penetrtion, mirowve tretment in 1 mm itri i or Dko Trget Retrievl Solution (Dko) were use. Amplifition of the primry ntioy retion ws hieve y inuting the setions with iotinylte seonry ntioy (Vetor Lortories In., Burlingme, CA, USA). Immunoperoxise stining of ryosetions, fixe with 3.% prformlehye or etone, ws performe similrly. The imge nlysis progrm NIH ImgeJ 1.32j (Ntionl Institutes of Helth, Bethes, MD, USA) ws use to lulte the re frtion of prtiulr immunostine omponent. The re frtion of pillry lumens ws lulte using the sme progrm with mnul seletion of the lulte re. The proportion of lk-to-white pixels in the imge ws lulte s perentge. 34 All immunohistohemil t were nlyze from t lest five ontrol n six NPHS1 kineys. TUNEL The ourrene of poptosis in glomerulr ells ws nlyze y monitoring the presene of DNA frgmenttion. 3 Slies were nlyze y light mirosopy fter ounterstining with hemtoxylin. Cells exhiiting rk rown stining from the olorimetri retion were onsiere positive for DNA frgmenttion. Nine NPHS1 kineys with histology rnging from nerly norml to severely mge were nlyze together with five ontrol kineys. As positive ontrols, we use testis smples with unnt poptosis. Sttistis Dt re presente s men7s.. Two-tile P-vlues o. (Stuent s t-test) were onsiere signifint. 143 Kiney Interntionl (6) 7,

9 A-M Kuusniemi et l.: Glomerulr slerosis in NPHS1 kineys o r i g i n l r t i l e ACKNOWLEDGMENTS This work ws supporte y grnts from the Aemy of Finln, the Sigri Juselius Fountion, the Peitri Reserh Fountion, the Päivikki n Skri Sohlerg Fountion, Helsinki University Centrl Hospitl Reserh Fun, the Finnish Meil Fountion, Frmos Reserh n Siene Fountion, n the Kiney Fountion. We thnk Tuij Heinonen, Tuoms Jlnko, n espeilly Tuike Helmiö for exellent tehnil ssistne. REFERENCES 1. Ey AA. Progression in hroni kiney isese. Av Chroni Kiney Dis ; 12: Boute N, Griouvl O, Roselli S et l. NPHS2, enoing the glomerulr protein pooin, is mutte in utosoml reessive steroi-resistnt nephroti synrome. Nt Genet ; 24: Kpln JM, Kim SH, North KN et l. Muttions in ACTN4, enoing lph-tinin-4, use fmilil fol segmentl glomeruloslerosis. Nt Genet ; 24: Hi R, Loirt C, Guler MC et l. The nephropthy ssoite with mle pseuohermphroitism n Wilms tumor (Drsh synrome): istintive glomerulr lesion, report of 1 ses. Clin Nephrol 198; 24: Kestil M, Lenkkeri U, Mnnikko M et l. Positionlly lone gene for novel glomerulr protein nephrin is mutte in ongenitl nephroti synrome. Mol Cell 1998; 1: Ptrkk J, Kestil M, Wrtiovr J et l. Congenitl nephroti synrome (NPHS1): fetures resulting from ifferent muttions in Finnish ptients. Kiney Int ; 8: Lhenkri AT, Lountm K, Ptrkk J et l. Pooytes re firmly tthe to glomerulr sement memrne in kineys with hevy proteinuri. J Am So Nephrol 4; 1: Fogo AB. Animl moels of FSGS: lessons for pthogenesis n tretment. Semin Nephrol 3; 23: D Agti V. Pthologi lssifition of fol segmentl glomeruloslerosis. Semin Nephrol 3; 23: Gssler N, Elger M, Krnzlin B et l. Pooyte injury unerlies the progression of fol segmentl glomeruloslerosis in the f/f Zuker rt. Kiney Int 1; 6: Kriz W, Gretz N, Lemley KV. Progression of glomerulr iseses: is the pooyte the ulprit? Kiney Int 1998; 4: Kriz W, LeHir M. Pthwys to nephron loss strting from glomerulr iseses insights from niml moels. Kiney Int ; 67: Huttunen NP, Rpol J, Vilsk J, Hllmn N. Renl pthology in ongenitl nephroti synrome of Finnish type: quntittive light mirosopi stuy on ptients. Int J Peitr Nephrol 198; 1: Vts AN, Costello B, Muer M. Glomerulr struturl ftors in progression of ongenitl nephroti synrome. Peitr Nephrol 3; 18: Nikoli-Pterson DJ, Ln HY, Hill PA, Atkins RC. Mrophges in renl injury. Kiney Int Suppl 1994; : S79 S Hooke DH, Gee DC, Atkins RC. Leukoyte nlysis using monolonl ntioies in humn glomerulonephritis. Kiney Int 1987; 31: Le Hir M, Besse-Eshmnn V. A novel mehnism of nephron loss in murine moel of resenti glomerulonephritis. Kiney Int 3; 63: Thoms DB, Frneshini N, Hogn SL et l. Clinil n pthologi hrteristis of fol segmentl glomeruloslerosis pthologi vrints. Kiney Int 6; 69: Kuusniemi AM, Lptto R, Holmerg C et l. Kineys with hevy proteinuri show firosis, inflmmtion, n oxitive stress, ut no tuulr phenotypi hnge. Kiney Int ; 68: Fine LG, Orphnies C, Normn JT. Progressive renl isese: the hroni hypoxi hypothesis. Kiney Int Suppl 1998; 6: S Kng DH, Knellis J, Hugo C et l. Role of the mirovsulr enothelium in progressive renl isese. J Am So Nephrol 2; 13: Petermnn AT, Pippin J, Krofft R et l. Vile pooytes eth in experimentl ieti nephropthy: potentil mehnism unerlying glomeruloslerosis. Nephron Exp Nephrol 4; 98: e114 e Petermnn AT, Krofft R, Blonski M et l. Pooytes tht eth in experimentl memrnous nephropthy re vile. Kiney Int 3; 64: Vogelmnn SU, Nelson WJ, Myers BD, Lemley KV. Urinry exretion of vile pooytes in helth n renl isese. Am J Physiol Renl Physiol 3; 28: F F Nkmur T, Ushiym C, Suzuki S et l. The urinry pooyte s mrker for the ifferentil ignosis of iiopthi fol glomeruloslerosis n miniml-hnge nephroti synrome. Am J Nephrol ; : Hr M, Yngihr T, Kihr I. Urinry pooytes in primry fol segmentl glomeruloslerosis. Nephron 1; 89: Nkmur T, Ushiym C, Suzuki S et l. Urinry pooytes for the ssessment of isese tivity in lupus nephritis. Am J Me Si ; 3: Hr M, Ymmoto T, Yngihr T et l. Urinry exretion of poolyxin inites glomerulr epithelil ell injuries in glomerulonephritis. Nephron 199; 69: Moriok Y, Koike H, Ikezumi Y et l. Pooyte injuries exerte mesngil prolifertive glomerulonephritis. Kiney Int 1; 6: Mtsusk T, Xin J, Niw S et l. Geneti engineering of glomerulr slerosis in the mouse vi ontrol of onset n severity of pooyte-speifi injury. J Am So Nephrol ; 16: Mrshll CB, Shnkln SJ. Cell yle n glomerulr isese: minireview. Nephron Exp Nephrol 6; 12: e39 e Detwiler RK, Flk RJ, Hogn SL, Jennette JC. Collpsing glomerulopthy: linilly n pthologilly istint vrint of fol segmentl glomeruloslerosis. Kiney Int 1994; : Segsothy M, Lu TM, Birh DF et l. Immunoytologi issetion of the urine seiment using monolonl ntioies. Am J Clin Pthol 1998; 9: Furness PN. The use of igitl imges in pthology. J Pthol 1997; 183: Tilly JL. Use of the terminl trnsferse DNA lelling retion for the iohemil n in situ nlysis of poptosis. In: Celis JE (e). Cell Biology: A Lortory Hnook. vol. 1 Aemi Press: Sn Diego, 1994 pp Kiney Interntionl (6) 7,