Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6499-6503, September 1986 Cell Biology Xylosylated-proteoglycan-induced Golgi alterations (glomerular epithelial cells/sulfated glycosaminoglycan/basement membrane) YASHPAL S. KANWAR*, LIONEL J. ROSENZWEIGt, AND MICHAEL L. JAKUBOWSKI* *Department of Pathology, Northwestern University Medical School, Chicago, IL 60611; and tDepartment of Veterinary Biology, University of Minnesota, St. Paul, MN 55108 Communicated by Emanuel Margoliash, May 27, 1986 ABSTRACT The effect of p-nitrophenyl (3-D-xylopyrano- METHODS side on the Golgi apparatus and proteoglycans (PG) ofthe renal glomerulus was investigated in an isolated kidney organ perfu- Radiolabeling of Glomerular Cells and Matrices and Prep- sion system and monitored by utilizing [35S]sulfate as the PG aration of Electron Microscopic Autoradiograms. Glomerular precursor. By electron microscopy, a selective intracytoplas- cells and matrices were radiolabeled under sterile conditions mic vesiculization ofGolgi apparatus ofvisceral epithelium was in an ex vivo organ perfusion system as detailed in our observed in the .-xyloside-treated kidneys. Electron micro- previous publications (11, 12). [35S]sulfate (Amersham) was scopic autoradiography revealed most grains localized to the the precursor for labeling of glomerular PGs or GAGs. intracytoplasmic Golgi-derived vesicles, while very few grains Satisfactory labeling was achieved by constantly recirculat- were associated with the extracellular matrix membranes. ing [35S]sulfate (500 ,uCi/ml; 1 Ci = 37 GBq; specific activity, Biochemically, a 2.3-fold increase in cellular matrix and a >1300 Ci/mmol) contained in a chemically defined perfusion reduction by a factor of 1.7 in extracellular matrix of [15S]sul- medium (11, 12). To perturb the cellular synthesis of fate incorporation was observed. Besides a larger macromo- PGs/GAGs, the perfusate included 2.5 mM ofp-nitrophenyl lecular form (Kayg = 0.25; Mr = 130,000), lower molecular ,B3D-xylopyranoside (Sigma). The kidneys from 1- to 7-hr weight PGs were recovered in the cellular (Ka.g = 0.46, Mr = intervals were processed only for biochemical determination 30,000) and matrical (Kavg = 0.42, Mr = 45,000) compartments of incorporated radioactivity, and each kidney at the final after xyloside treatment. The xyloside treatment increased the time point of 7 hr was additionally processed for electron incorporated radioactivity, mostly includedinfreeglycosamino- microscopy and autoradiography (11, 12). glycans and small PGs, in the media fraction by 3.8-fold. These and Characterization of PGs from Cells, Matrices data indicate that xyloside induces a dramatic imbalance in the Isolation and Media. After 1-7 hr of radiolabeling, the kidneys were de novo-synthesized PGs of cellular and extracellular compart- containing 1% bo- ments and that cellular accumulation of xylosylated (sulfated) perfused with Krebs-Ringer bicarbonate serum albumin and a mixture of protease inhibitors (10 PGs selectively alters the Golgi apparatus of the glomerular vine PGs. mM 6-aminohexanoic acid, 5 mM benzamidine-hydro- epithelial cell, the cell that actively synthesizes chloride, and 1 mM phenylmethylsulfonyl fluoride) at pH 7.0, and glomeruli were isolated by the sieving technique (11-13). The proteoglycans (PGs) are comprised of glycosamino- The cellular PGs were extracted for 12 hr at 40C with 1% glycan (GAG) chains covalently bound by O-glycosidic deoxycholate containing 10 mM sodium EDTA, sodium linkage to the core protein via galactosylgalactosylxylosyl- acetate, and the mixture ofprotease inhibitors at pH 5.8. The serine (for review, see ref. 1). The GAG chains are trans- solubilized cellular PGs were separated from the glomerular ferred onto the core protein in a stepwise manner and are extracellular matrices (GEMs) by sedimenting the latter at initiated by UPD-D-xylose:core-protein xylosyltransferase x 3000 rpm in an IEC (International Equipment) refrigerated followed by additional transferases for completion of these centrifuge. The sedimented GEMs were extracted with 4 M chains. In the final step, these chains are 0 or N-sulfated in guanidine-hydrochloride containing 10 mM sodium EDTA the Golgi saccules (1, 2). The addition ofGAG chains onto the and sodium acetate at pH 5.9 for 48 hr at 4°C in the presence core protein can be competitively inhibited by the presence of protease inhibitors (11-13). The unextracted residue was of D-xylose or ,-xyloside in the culture medium, because the pelleted by centrifugation at x 10,000 rpm in a Beckman xyloside itself acts as an initiator of chain formation, bypass- microfuge. The residue was then reextracted with 0.5 M ing the requirement for the receptor core protein and sodium hydroxide at 600C for 3 hr to solubilize the remaining xylosyltransferase. Not incorporated onto the core protein, GAGs. GEM-associated PGs were designated to be in the these xyloside-initiated chains are discharged readily into the guanidine-hydrochloride and the cellular PGs were designat- medium. Thus, in the presence of xyloside, an incomplete ed to be in the deoxycholate extract. All the extracts were macromolecular form of PG occurs along with a net "stim- extensively dialyzed against distilled water containing 1 mM ulation" of "free" GAG chain synthesis. Such intriguing phenylmethylsulfonyl fluoride and diisopropyl fluorophos- biochemical effects of xyloside on PG synthesis have been phate at 4°C. The PGs/GAGs from the media fractions investigated in a wide variety of systems (3-10). However, (xyloside and control) were isolated by applying them to a the effect of this increased synthesis and intracellular accu- Sephadex G-50 column and purified by DEAE-Sephacel mulation of sulfated GAG chains on the cellular organelles chromatography (13, 14). Aliquots were assayed for protein remains undocumented. We report discrete cellular changes contents and total radioactivities. Techniques for character- in the renal epithelium that may be induced by intracellular ization ofPGs/GAGs (11-13) and determination of molecular accumulation of sulfated GAG chains under the influence of weight (15-17) have been previously reported. xyloside. Abbreviations: GEM, glomerular extracellular matrix; GBM, The publication costs of this article were defrayed in part by page charge glomerular basement membrane; HS-PG, heparan sulfate-pro- payment. This article must therefore be hereby marked "advertisement" teoglycan; CS-PG, chondroitin sulfate proteoglycan; PG, pro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. teoglycan; GAG, glycosaminoglycan. 6499 Downloaded by guest on September 24, 2021 6500 Cell Biology: Kanwar et al. Proc. Natl. Acad. Sci. USA 83 (1986) RESULTS endothelial and mesangial cells were unremarkable, and the morphology of the extracellular matrices-i.e., GEM-was General Morphology and Autoradiography of the Renal not significantly altered; the electron density ofthe GBM was Glomerulus. The renal glomerulus is a network of capillaries as usual with epithelial and endothelial components firmly (Fig. 1A) made up of epithelial, endothelial, and mesangial adherent, and the mesangial matrix maintained its normal cells-and their cytosecretory products [basement mem- appearance and relationship to the mesangial cells. brane (GBM) and mesangial matrix; the latter two together By tissue autoradiography, a drastic increase in the are referred to as extracellular matrices (GEM)]. Heparan [35S]sulfate-associated silver grains was observed over the sulfate-proteoglycan (HS-PG) is regarded as the major PG of glomerular epithelium of the xyloside-treated kidneys (Fig. the GEMs, although minute amounts of chondroitin sulfate- 1B). These grains were distributed throughout the cytoplasm proteoglycan (CS-PG) have also been reported (18, 19). but, for the most part, were excluded from the nucleus. They The general architecture ofthe glomerulus remained intact were associated with intracytoplasmic vesicles and Golgi with no observable deterioration in the morphology resulting saccules-the region where sulfotransferases seem to be from perfusion with or without xyloside over a 7-hr period. localized and sulfation probably occurs. The autoradio- Changes occurred mainly in the visceral epithelium in the graphic grains were found in the Golgi saccules of the form of cytoplasmic vesiculation (Fig. LA). Devoid of endothelial and mesangial cells; however, the experimental clathrin-coat, the vesicles measured 10-100 nm and were and control groups did not differ appreciably. distributed throughout the cytoplasm, appearing to originate The dramatic increase in the autoradiographic grains over from the terminal saccules of the Golgi apparatus. Besides the epithelium after xyloside treatment meant that a differ- this cytoplasmic vesiculation, no alterations in the mitochon- ential accumulation of xylosylated-sulfated products had dria, rough endoplasmic reticulum, or nucleus were recog- occurred. Conversely, a significant decrease in the autora- nized. No vesicles associated with the plasmalemma were diographic grains over the GBM was seen (Fig. 1B), thereby seen. Foot processes, emanating from the visceral epitheli- indicating a dramatic reduction in the incorporation of de um, had a regular interdigitating arrangement with intact novo-synthesized PGs/GAGs into the extracellular matrical intervening slit diaphragms, and these processes remained compartments. firmly attached to the peripheral aspect of the GBM. No Characterization of Glomerular and Media PGs/GAGs.