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448cet, Paris) daily from day 7 to 11 (organogenesis) and from day 14 to18 (beginning of nephrogenesis) of gestation, or saline by i .p. injectionat 4 p.m. Twelve mothers received gentamicin (75 mg/kg body weightper day), 120 neonates; saline was administered to 10 pregnant controlrats (100 neonates). Delivery occurred normally on day 21 of gestation.Neonates were studied on the following day. Four neonates per litterwere used for experiments with HRP and 4 others were used for im-munocytochemistry.Immunocytochemistry. Kidney slices were fixed by immersion in 4paraformaldehyde and 0.5 glutaraldehyde in 0.12 M cacodylatebuffer plus 0.043 M sodium chloride (NaC1), pH 7.40; they were thenrinsed in buffel; dehydrated in alcohol at -20 ~ C and embedded in thehydrophilic resin, LRWhite, at -20 ~ C. Sections were cut with a dia-mond knife and picked up on Parlodion-coated nickel grids. Sectionswere first incubated for 10 rain on a drop of 0.1 gelatin in phos-phate-buffered saline (PBS, 0.9 NaC1 in 10 mM sodium phosphatebuffer, pH 7.40) to reduce non-specific binding of antibodies. Thegrids were then washed in the same buffer and transferred to a drop ofpolyclonal rabbit anti-gentamicin antibody (ICN) diluted 1:50 andincubated for 1 h at room temperature in a moist chamber. After three5-min rinses in PBS, the grids were incubated for 1 h on a drop ofprotein A-gold solution containing 5-nm gold particles prepared bydensity gradient ceutrifugation according to Slot and Gueuze [27].Control grids were stained by omitting the first incubation step withthe antibody. Grids were then washed with PBS, contrasted with uranylacetate and observed with a Philips CM12 electron microscope.Experiments w ith HRP. The protein tracer HRP (type II, Sigma, mo-lecular weight 40,000) was dissolved in saline and perfused via thejugular vein in a small volume to prevent haemodynamic changes.Thus neonatal rats received 0.2 mg HRP/g body weight and were killed15 min after injection of the tracer. Kidneys were removed and cut intoslices 4- 5 mm thick, which were then fixed for 3 h in 4 para-formaldehyde, 0.5 glutaraldehyde in 0.12 M sodium cacodylatebuffer plus 0.043 NaC1, pH 7.40. After washing three times in 0.12 Mcacodylate buffer slices were immersed in the same medium plus di-methyl sulphoxide (10 ) for 1 h at 4 ~ C, then frozen in a solution ofisopentane in liquid nitrogen.Sections (20 gm) were cut with a cryostat microtome (AmericanOptical), washed in 0.1 M TRIS-HC1pH 7.40 and incubated for 10 minin a filtered solution of 0.01 M 3-3' diaminobenzidine in 0.01 MTRIS-HCI buffer, pH 7.40 containing 0.01 hydrogen peroxide. Thesections were then washed in three changes of the same buffer andpost-fixed for 30 miu in 2 ferrocyanide-reduced osmium in caco-dylate buffer; they were dehydrated in graded alcohols, in propyleneoxide and embedded in Epon 812.The sections were cut with a Reicbert-Jung ultramicrotome, stainedwith uranyl acetate and lead and examined with a Philips CM12electron microscope.Measurement of urinary proteins. Urine samples from neonates wereobtained by bladder puncture, and urinary proteins were measured bythe procedure of Lowry et al. [28] using bovine serum albumin as astandard.Statistical analysis. Differences between means were assessed byStudent's t-test.

e s u l t s

Fig. 1. Localization of gentamicin with anti-gentamicin antibody andprotein A-gold (5 nm) in a glomemlus from a neonate exposed togentamicin in utero. Labelling is seen in capillary CAP), n glomerularbasement membrane GBM) and in urinary space US). Arrows, goldparticles, x50,000

len and, next to these areas, membranes appeared closelypacked, resembli ng a small myeloid body.

Label l i ng w i th t he an t i -gen tamic in an t i bodyThe anti-genta micin antibody was localized with proteinA-gold; electron-dense gold particles were visualized byelectron microscopy.

Gentami cin was located in the glomerular capillaries, inthe GBM and urinary spaces of treated animals (Fig. 1). InPTC, some scattered clusters of gold particles were foundin the brush border and in compartments at the periphery ofthe cell membrane and in the perinuclear zone (Fig. 2b).No gol d particles were seen in controls (Fig. 2 a).

Loca l i za t ion o f perox idase i n PTCIn controls, HRP was taken into small vesicles, transferredinto early endosomes and late endosomes and accumulatedexclusively in lysosomes. Differences in the intensity ofstaining of the vesicles indicated differences in HRP con-centratio ns in these structures (Fig. 3 a).

In treated animals, the brush border membrane s showedan increased electron opacity, representing the adsorptionof peroxidase. In addition, many of the apical tubular in-vaginations at the base of the brush border microvillicontained dense accumulations of reaction product, whileearly end osome s did not c ontai n peroxidase (Fig. 3 b).

Proxima l t ubu lar ce l l morpho logyOnly the deep cortical area, con tain ing the fully diffe-rentiated neonatal nephrons, was examined. Subcellularlesions were found in epithelial cells of animals exposed togentamicin in utero: lysosomes frequently contained my-eloid bodies, Golgi apparatus cisternae were locally swol-

UrinalysisAn increase in urinary proteins was observed in gentami-cin-treated rats compared with controls. Mean values were11.10 __ 0.040 and 6.40 + 0,012 gg/g bod y we ight, respec-tively (P < 0.05).

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Fig. 2. Localization of gentamicin withanti-gentamicin antibody and protein A-gold5 nm) in proximal tubular cells PTC) froma control and b gentamicin-treated animals.There is no labelling in controls; in treatedanimals labelling is present in brush borderBB) and in endosomes E). Arrows, goldparticles, x50,000

Fig. 3. Horseradish peroxidase HRP) local-ization in the fully matured PTC fro m a controland b animals exposed to gentamicin in utero.In controls HRP is present in BB, in smallvesicles, in early endosomes EE) and lateendosomes LE). In treated animals HRP isonly present in BB and in some EE. Thesealterations are not focal and are seen in allobserved PTC four neonates examined perlitter). Arrowhead, myeloid bodies, x15,000

i s cus s ion

I t i s we l l known tha t the ra t k idney i s no t com ple te ly m a-tured a t bir th , so that a l l s tages of nephr on d ifferent ia t ioncan be obse rved in the s am e cor tex f rom capsu la r to jux-tam edul la ry reg ions . We s tud ied the fu l ly d i f fe ren t i a tednephrons which , a t th i s age , were loca ted on ly in the deepcortex.

In neona te s born to t rea ted m othe rs , the gen tam ic inf i l t e red th rough the GBM, s ince an t i -gen tam ic in an t ibodywas loca l i zed in g lom eru la r cap i l l a r i e s , in GBM and inur ina ry spaces . In P TC the l abe l l ing pa t t e rn was s im i la r to

tha t p rev ious ly obse rv ed in adu l t s [6 , 7 , 29] : the gen tam ic inwas loca ted in b rush borde r and in endocy t ic vacuo les .

Th is s tudy a l so ind ica ted tha t gen tam ic in l ed to a de fec to f p ro te in reabsorp t ion by the P TC and inh ib i t ed the pas -s age f rom ap ica l invag ina t ions to ea r ly and l a te endo som es ,and consequen t ly to lysosom es . HRP was on ly s een inbrush borde r and in som e ap ica l ves ic le s o f the ve ry f i r s ts t ep o f endocy tos i s . The dec reased tubu la r up take o f HRPin gen tam ic in - t rea ted an im a ls cou ld be in te rp re ted a s adec reased ra te o f endocy tos i s , and the accum ula t io n o fH R P i n th e b r u sh b o r d e r o f P T C m a y b e s e c o n d a r y t o a ninh ib i t ion o f the de l ive ry o f p ro te ins to ea r ly endosom es .

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450A s i n d i c a t e d b y t r a c e r s tu d i e s, P T C f r o m t h e g e n t a m i c i n

g r o u p w e r e u n a b l e t o in c o r p o r a t e H R P i n to e n d o c y t i c v e -s i c le s . A t t h e s a m e a g e , t h e e n d o c y t i c p r o c e s s o c c u r r e dn o r m a l l y i n th e c o n t r o l f u l ly d i f f e re n t i a te d P T C . H R P w a sa b s o r b e d b y t h e e n d o c y t i c v a c u o l e s a n d n e v e r a c c u m u l a t e di n t h e c y t o p l a s m ; t h e r e a c t i o n p r o d u c t w a s s e e n i n b r u s hb o r d e r , i n c o a t e d p i t s a n d v e s i c l e s , i n e a r l y a n d l a t e e n d o -s o m e s a n d i n ly s o s o m e s . T h e r e f o r e , g e n t a m i c i n t re a t m e n ti n u t e ro c o m p l e t e l y o r p a r t i a l l y i n h i b i t e d th e e n d o c y t i cp r o c e s s .

I n n o r m a l r a t f e tu s e s , p r o t e i n r e a b s o r p t i o n i n t h e f u l l yd i f f e r e n t i a t e d P T C b e g i n s a t d a y 1 8 o f g e s t a t i o n w h i c hl a s t s 2 1 d a y s ) [ 3 0 ] . O u r s t u d y s h o w e d t h a t e n d o c y t o s i s i n1 - d a y - o l d n e o n a t e s e x p o s e d t o g e n t a m i c i n i n u t e r o w a ss i m i l a r to t h a t i n n o r m a l f e t u s e s 3 d a y s b e f o r e b i r t h .T h e r e f o r e g e n t a m i c i n i s l i k e l y t o s lo w d o w n t h e m a t u r a t i o no f e n d o c y t i c p r o t e i n r e a b s o r p t i o n .

W e h a v e a l r e a d y r e p o r t e d t h a t in n e o n a t e s e x p o s e d t og e n t a m i c i n i n u t e r o t h e i n c r e a s e d p r o t e i n u r i a is la r g e l y d u et o a d e l a y i n t h e m a t u r i t y o f t h e g l o m e r u l a r f i l t r a t i o n b a r r i e r[ 2 2 , 2 3 ] . W e d e m o n s t r a t e i n t h i s s t u d y t h a t t h i s p r o t e i n u r i am a y a l s o b e r e l a t e d t o a n i m m a t u r i t y o f t h e t u b u l a r re -a b s o r p t i o n s y s t e m .

I n c o n c lu s i o n , g e n t a m i c i n g i v e n t o t h e m o t h e r d u r i n gp r e g n a n c y l e a d s , i n n e o n a t e s , t o a d e c r e a s e o f t h e P T Ce n d o c y t i c p r o c e s s , w h i c h e v o k e s a m a t u r a t io n d e l a y a n d i sp a r t i a l l y r e s p o n s i b l e f o r t h e p r o t e i n u r i a .Acknowledgements H. Smaoui is grateful to CNRS France for a PhDfellowship.

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