Effects of methylprednisolone on glomerular and medullary mRNA levels for extracellular matrices in puromycin aminonucleoside nephrosis

Kidney International, Vol. 40 (1991), pp. 874—881

Effects of methyiprednisolone on glomerular and medullary mRNA levels for extracellular matrices in puromycin aminonucleo side nephrosis TSUKASA NAKAMURA, ISAO EBIHARA, MITsuMINE FuKul, YAsuHIK0 T0MIN0, and HIKARU K0IDE Division of Nephrology, Department of Medicine, Juntendo University School of Medicine, Tokyo, Japan

Effects of methylprednisolone on glomerular and medullary mRNA levels for extracellular matrices in puromycin aminonucleoside nephrosis.

We examined the effects of methylprednisolone (MPSL) on type IV collagen, laminin and heparan sulfate proteoglycan (HSPG) mRNA levels in the renal glomeruli and medulla of puromycin aminonucleoside

(PAN) nephrosis. mRNA levels encoding for type LV collagen and laminin increased markedly, whereas those for HSPG decreased significantly in glomeruli of PAN nephrosis. Administration of MPSL partially ameliorated the abnormal gene expression for basement mem-

brane components. Furthermore, we showed that medullary mRNA levels for all these basement membrane components decreased with age

in PAN nephrosis with or without MPSL treatment, suggesting that neither PAN nor MPSL has any effect on basement membrane component mRNA levels in the renal medulla. In contrast, mRNA levels for the interstitial collagens including al (1) and al (ILL) chains in glomeruli showed little change with or without MPSL treatment, whereas those in medulla increased significantly in PAN nephrosis when compared with the control. MPSL ameliorated the abnormal gene expression of al (I) and al (III) collagen in renal medulla. These results indicate that PAN

affects both glomerular mRNA encoding for basement membrane components and medullary mRNA encoding for interstitial collagens, and that MPSL has marked effects on the amelioration of abnormal gene expression in both glomeruli and medulla of PAN nephrosis

medulla because of difficulties in isolating and solubilizing the basement membrane and mesangium [7]. The identification and characterization of cDNAs coding for basement membrane components including al (IV), laminin and HSPG and for interstitial coLlagens including al (I) and al (III) have provided a highly sensitive and specific means by which the transcription of extracellular matrix (ECM) proteins can be examined in glomeruli and medulla, namely, assaying the steady-state mRNA levels coding for these proteins [81. Puromycin aminonucleoside (PAN) nephrosis in rats is an established model of minimal change disease [9, 10]. Although this disease has been extensively studied for more than three decades, the pathogenesis is still obscure. In a previous study

[11], we showed the abnormal gene expression of al (IV), laminin and HSPG in glomeruli of PAN nephrosis and indicated

that decreased HSPG mRNA levels might contribute to the development of proteinuria. Eddy et a! [12] have demonstrated significant changes in the interstitium of kidneys in rats given a single intraperitoneal injection of PAN. It is well known that the

administration of steroid to PAN rats results in the rapid disappearance of proteinuria and significantly faster recovery of histological changes [131. A recent study has demonstrated that

Basement membranes are extracellular matrices which sepa- dexamethasone increases the HSPG core protein content of rate epithelial and endothelial cells from the underlying stroma glomerular epithelial cells [14]. Ohyama et a! [15] demonstrated [1, 2]. Recent studies have shown that basement membrane is that steady state levels of mRNA encoding for al (I) and al (III) formed by the coassembly of type IV collagen with a triple chains were reduced by dexamethasone in cultured mesangial helical filament of two al chains and one a2 chain [3], laminin cells. However, little is known about the in vivo effect of methylwith a cruciform-shaped glycoprotein [4] composed of A, B 1 prednisolone (MPSL) on mRNA levels for ECM components and B2 chains, and heparan sulfate proteoglycan (HSPG), consisting of one chain with two to three glycosaminoglycans and interstitial collagens in glomeruli and/or medulla of PAN attached to one end [5]. Type I and III collagens are the nephrosis. The purpose of this study was to determine whether MPSL predominant collagens in interstitial tissues and both collagens affects the gene expression of basement membrane components show similar tissue distributions [6]. However, little is known about the synthesis and degradation of these basement mem- and interstitial collagens in the glomeruli and medulla of PAN brane components and interstitial collagens in glomeruli and nephrosis. Methods Received for publication February 7, 1991 and in revised form May 6, 1991 Accepted for publication May 8, 1991

© 1991 by the International Society of Nephrology

Animal models

Two hundred male Sprague-Dawley rats, weighting 150 to 200 g, were used for this study. They were housed in individual

874

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Nakamura et a!: Effect of MPSL on ECM gene expression

metabolic cages and had free access to food and water throughout the experiments. One hundred rats were made nephrotic by a single intraperitoneal injection of 15 mgIlOO g body weight of puromycin aminonucleoside (Sigma Chemical Co., St. Louis,

200

Missouri, USA). The other 100 rats were used as controls. Methylprednisolone (Upjohn Co., Kalamazoo, Michigan, USA)

was administered at a dose of 3 mg/100 g body weight in 50 nephrotic rats and 50 control rats every other day for 20 days [161. Ten rats from each group were sacrificed on days 0, 2, 8,

150

14 and 20. The kidneys were immediately removed and glomer-

uli were isolated by sieving through size 60, 150 and 200 meshes. Decapsulated glomeruli were collected from the top of

the 200-mesh sieve for RNA extraction. The preparation of decapsulated glomeruli was greater than 90% pure and contained very few tubular fragments. Remnant medulla was also collected for RNA extraction. Twenty-four-hour urine speci-

1 100

50

mens were obtained from all rats in metabolic cages during the course of the experiments. Urinary protein was measured by the biuret method [17].

Extraction and quantitation of RNA Total RNA was extracted from glomeruli and medulla by the acid guanidinium thiocyanate-phenol chloroform extraction method 1181. Isolated glomeruli from ten rats were mixed and used for RNA extraction. Total RNA concentration was determined spectrophotometrically at 260 nm and 280 nm. RNA samples (15 xg) were denatured and electrophoresed through 0.7% agarose gels containing 2.2 M formaldehyde, and transferred by capillary blotting onto nylon filters (Gene Screen, New England Nuclear, Boston, Massachusetts, USA) [19. 201.

20

Time, days

Fig. 1. Urinary protein excretion in control (A) and PAN-nephrotic

rats with (0) or without (•) methyiprednisolone treatment. Means standard deviation are indicated.

membrane heparan sulfate proteoglycan (clones 7, 54 and 72)

were provided by Dr. D.M. Noonan, National Institute of Dental Research (Bethesda, Maryland, USA) [241. Clones for

Before transferring the samples onto the nylon membrane, the al chain of type I collagen and al chain of type III collagen ethidium-stained gels were visualized under ultraviolet illumi- were provided by Dr. Y. Yamada, National Institute of Dental nation to determine the position of 28s and l8s ribosomal RNA Research (Bethesda, Maryland, USA) [6].

bands to assess the integrity of RNA and verify that equal

The cDNA inserts were excised and labeled by using a

amounts of RNA were loaded. RNA was fixed to the filter by Random Primed cDNA Labeling Kit (Boehringer-Mannheimultraviolet irradiation for five minutes at 254 nm. In addition, we Yamanouchi K.K., Tokyo, Japan) with 32P-deoxynucleotides performed dot blot analysis to ensure the reliability of Northern (New England Nuclear, Boston, Massachusetts, USA) to a blot analysis. Several two-fold dilutions of RNA were applied to specific activity of l0 cpm/g [25]. nylon filters using a 96-well vacuum manifold. RNA was fixed Statistics to the filter by ultraviolet irradiation for five minutes at 254 nm. Statistical calculations were performed by one-way analysis Hybridization was carried out in 1% BSA, 7% SDS, 0.5 M of variance. NaH2PO4, 1 mrvi EDTA and 100 sg/ml sonicated salmon sperm DNA for 20 hours at 65°C [19]. Filters were washed three times Results in 0.5% BSA, 5% SDS, 40 mrvi NaH2PO4 and 1 mM EDTA at Urinary protein excretion 65°C, followed by three washes in 1% SDS, 40 mM NaH2PO4 The urinary excretion of protein is shown in Figure 1. Protein and 1 mM EDTA at 65°C [19]. Filters were then exposed to Kodak X-OMAT-AR film (Eastman Kodak, Rochester, New excretion of control rats remained within 14.8 6.3 mg/day York, USA) at —70°C with the aid of intensifying screens for 6 throughout the experimental period (20 days). PAN-treated rats hours to 7 days. Relative intensities of bands on autoradiograms showed proteinuria within four days after PAN injection. Prowere quantified by the Scanning Shimadzu CS-9000 densitom- teinuria reached a peak on day 8 (210.6 38.4 mg/day) and then declined to the control level by day 20. In PAN-nephrotic rats eter (Shimadzu Manufacturing, Kyoto, Japan). administered 3 mg/l00 g body weight of MPSL, the urinary cDNA probe protein level was ameliorated as shown in Figure 1 (100.4 Clones for the al chain of type IV collagen (pFAC) and 22.8 mg/day on day 8). laminin A (e3), Bi (p24) and B2 (p7) chains were isolated from mRNA analysis a cDNA library constructed from differentiated F9 teratocarciOn day 2, before proteinuria appeared, mRNA levels for the noma cell mRNA as previously reported [7, 20—22]. The $-actin

cDNA probe was provided by Dr. D.W. Cleveland, Johns ctl (IV) chain and laminin Bl and B2 chains were already Hopkins University School of Medicine (Baltimore, Maryland, increased, and mRNA levels for HSPG were decreased in USA) [23]. The cDNA clones for the core protein of basement

PAN -nephrotic glomeruli. On day 8, in the nephrotic stage, the

876

Nakamura et a!: Effect of MPSL on ECM gene expression

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Fig. 2. Northern blot analysis of mRNA for /3-actin (A), aI chain of collagen IV(B), A chain of laminin (C), Bi chain of laminin (D), B2 chain of laminin (E) and HSPG (clone 7) (F) in glomeruli. Lanes 1-5: control glomeruli on days 0, 2, 8,1 4, and 20, respectively. Lanes 6-10: PAN nephrotic glomeruli with methyiprednisolone treatment on days 0, 2, 8, 14 and 20, respectively. Lanes 11-15: PAN nephrotic glomeruli on days 0, 2, 8, 14 and 20, respectively. Total cellular RNA was extracted and transferred onto a nylon membrane, hybridized with specific cDNA probe and quantitated as in the experimental methods. Data are expressed as a percentage of the control glomeruli on day 0. The means standard error are based on observations for 10 individual rats. Symbols are: (0) control; (A) PAN + MPSL; (•) PAN; < 0.001. < 0.05; ** < 0.01;

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glomerular mRNA levels for the al (IV) chain and laminin BI control level. mRNA levels for the al (I) chain and al (III) and B2 chains were increased significantly. On day 14, mRNA chain showed little change in glomeruli of PAN nephrosis levels for the al (IV) chain and laminin BI and B2 chains throughout the experimental period. declined, while the HSPG mRNA level increased. On day 20, in These abnormal mRNA levels for the al (IV) chain, laminin the remission stage of PAN nephrosis, mRNA levels for the al BI and B2 chains, and HSPG in glomeruli of PAN nephrosis (IV) chain and laminin BI and B2 chains in the glomeruli were were ameliorated by treatment with MPSL (Fig. 2). In contrast, decreased markedly, but were still higher than those of con- mRNA levels for the al (I) chain and al (III) chain were not trols. In contrast, the HSPG mRNA level was increased to the affected by MPSL treatment (Fig. 3).

877

Nakamura et a!: Effect of MPSL on ECM gene expression

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on days 0, 2, 8, 14 and 20, respectively. Lanes 6-10: PAN nephrotic glomeruli with methylprednisolone treatment on days 0, 2, 8, 14 and 20, respectively. Lanes 11-15: PAN nephrotic glomeruli on days 0, 2, 8, 14 and 20, respectively. Data are expressed as a percentage of the control glomeruli on day 0. Symbols are: (0) control; (ti) PAN ÷ MPSL; (•) PAN.

The mRNA levels for these genes in glomeruli of control rats

decreased with time, whereas the 13-actin mRNA level in

nephrotic stage, and the elevated mRNA expression of these genes was ameliorated by treatment with MPSL (Fig. 5). The

glomeruli exhibited little change in control and PAN-nephrotic /3-actin mRNA level in medulla showed little change in control

rats with or without MPSL treatment (Fig. 2). Laminin A and PAN nephrotic rats with or without MPSL treatment. mRNA was not detected in glomeruli of control or PAN- Medullary mRNA levels for these basement membrane compo-

nephrotic rats with or without MPSL treatment, except on the nents, interstitial collagens and /3-actin of control rats treated eighth day of PAN nephrosis (Fig. 2). Glomerular mRNA levels with MPSL were similar to those of control rats without MPSL for these basement membrane components of control rats treatment. The results of dot blot analysis coincided with those of treated with MPSL were similar to those of control rats without Northern blot analysis (data not shown). MPSL treatment. In addition, we examined the medullary mRNA levels for the a! (IV) and laminin A, Bl and B2 chains and HSPG in PAN Discussion nephrosis with or without MPSL treatment. mRNA levels for Our studies indicated that the gene expression of basement the al (IV) chain and laminin A, BI and B2 chains and HSPG decreased with time in medulla of the control and PAN- membrane components in the glomeruli of PAN nephrosis was nephrotic rats with or without MPSL treatment (Fig. 4). There altered and that the mRNA expression of these genes in medulla were no differences in the mRNA levels of these genes in was similar in PAN nephrotic and control rats. Heparan sulfate medulla between PAN nephrotic and control rats. However, proteoglycan (HSPG), the major proteoglycan of the glomerular mRNA levels for the a! (I) chain and al (III) chain increased basement membrane, has been identified as one of the biochemsignificantly in the medulla of PAN nephrotic rats in the ical determinants of the charge-selective permeability of the

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medulla with methyiprednisolone treatment on days 0, 2, 8, 14 and 20, respectively. Lanes 11-15: PAN nephrotic medulla on days 0, 2, 8, 14 and 20, respectively. Data are expressed as a percentage of the control medulla on day 0. Symbols are: (0) control; (Li) PAN + MPSL; (•) PAN.

glomerular capillary [26]. Fishman and Karnovsky [27] suggested that glomerular epithelial cells are an immediate site of PAN toxicity, and showed decreased secretion of glucosamine and sulfate by cultured glomerular epithelial cells. Kasinath et al [281 demonstrated by a quantitative immunoperoxidase method that PAN caused a 28% reduction in the core protein of HSPG in epithelial cells. It is likely that glomerular epithelial cell damage by PAN leads to decreased glomerular HSPG synthesis. The lower mRNA levels for HSPG in glomeruli of PAN nephrosis in our studies may be associated with a reduced

changes occur slightly later. To determine whether mRNA changes are the effect of PAN or the result of proteinuria, we measured mRNA levels four hours after PAN injection (data not shown). mRNA levels for the basement membrane components were already altered in

rate of synthesis. As PAN did not affect HSPG mRNA levels in medulla, it is probable that the effect of PAN on HSPG mRNA is specific to glomeruli. Mahan et al [291 reported that glomer-

Laminin A chain mRNA expression was detected in the medulla of controls and PAN nephrotic rats with or without

characteristics are altered within four hours of PAN exposure,

and that foot process fusion and widespread epithelial cell

the early stage, suggesting that the alteration of basement membrane genes in glomeruli of PAN nephrosis is due to PAN rather than proteinuria. MPSL treatment, but not in glomeruli of PAN nephrosis except

ular capillary wall structure and epithelial cell morphologic for the eighth day, the peak of proteinuria. Mohan and Spiro

Nakamura et al: Effect of MPSL on ECM gene expression

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Fig. 5. Northern blot analysis of mRNA for cr1 chain of collagen I (A) and cxl chain of collagen III (B) in medulla. Lanes 1-5: control medulla on days 0, 2, 8, 14 and 20, respectively. Lanes 6-10: PAN nephrotic medulla with methylprednisolone treatment on days 0, 2, 8, 14 and 20, respectively. Lanes 11-IS: PAN nephrotic medulla on days 0, 2, 8, 14 and 20, respectively. Data are expressed as a percentage of the control medulla on day 0.

Symbols are: (0) control; () PAN + MPSL;

(•) PAN; * < 0.05; ** < 0.01; 0.001.

and HSPG in cultured mesangial cells (unpublished data). It is

laminin B chains. They considered that the small levels of therefore possible that intrinsic glomerular cells are responsible laminin A chain in tissues were probably due to preferential for type I collagen and type III collagen mRNA production. proteolytic degradation. Kleinman et at [7] reported that mRNA Further studies aimed at determining which cells synthesize for the laminin A chain was not expressed in the kidney. Our collagens I, III and IV, taminin chains and HSPG at different data indicated that mRNA for the laminin A chain was ex- time points in PAN nephrosis and controls would require a pressed predominantly in medulla, rather than in glomeruli, and that the mRNA levels of their laminin chains were not coordinately regulated in glomeruli. Our data also showed that mRNA

different approach, for example, in situ hybridization. Significant morphological changes in the renal interstitium of

PAN nephrosis have been reported [13]. Our results also levels for basement membrane components of control rats indicated that PAN affected the renal interstitium and mRNA decreased with age, a finding that was in agreement with levels for interstitial collagens. Moreover, we studied the effect of MPSL on the gene previous reports [7, 8, 31, 32].

mRNA levels for interstitial collagens including cxl (I) chain expression of basement membrane components and interstitial and a! (III) chain were detected in both the medulla and collagens in PAN nephrosis. It is well known that morphologglomeruli of controls and PAN nephrotic rats. PAN did not ical changes in glomerular epithelial cells in PAN rats treated affect glomerular mRNA levels for cr1 (I) chain or cr1 (III) chain.

with glucocorticoid improve markedly and proteinuria de-

Several immunofluorescence studies have shown that type III creases significantly [12]. Glucocorticoid has been reported to collagen is present in the interstitium and blood vessels of the induce qualitative and quantitative changes in glycosaminoglynormal kidney, but not in any structures of the glomeruli can synthesis in nonrenal tissues [38, 39]. Recently, Kasinath et [33—351. A possible explanation for these conflicting results at [14] showed that dexamethasone increased the HSPG core could be low immunofluorescence sensitivity, posttranscrip- protein content of cultured glomerular epithelial cells in vitro. tional inhibition or increased turnover. Cultured mesangial cells Our results indicate that MPSL probably ameliorated the damhave been demonstrated to produce not only basement mem- aged glomerular epithelial cells and increased HSPG mRNA brane components but also type I and type III collagens [36, levels in glomeruli of PAN nephrosis. However, MPSL did not 37]. Recently Ohyama et al [15] showed that cultured mesangial affect HSPG mRNA levels in glomeruli of control rats or in cells expressed mRNA for at (I) chain, cr1 (III) chain and cr1 medulla of PAN treated and control rats in vivo. The mechanism by which glucocorticoid inhibits collagen (IV) chain. We also recognized mRNA expression for cxl (I) chain and cxl (III) chain as well as at (IV) chain, laminin chains mRNA levels appears to be complicated. Sterling et at [40]

880

Nakamura et a!: Effect of MPSL on ECM gene expression

suggested that the decline in procollagen synthesis in cells or tissues treated with glucocorticoids may be a reflection of the decrease in the total cellular content of procollagen-specific mRNA sequences and that glucocorticoids may regulate procollagen production primarily at the level of mRNA. Studies of the molecular effects of dexamethasone on collagen synthesis have shown that dexamethasone decreases type I collagen mRNA [41, 421. In dermal fibroblasts, hydrocortisone reduced the concentration of mRNAs for a! (I), a2 (I), a! (III) and a2 (V)

abnormal mRNA levels persisted to day 20. This abnormal gene expression was partially ameliorated by MPSL treatment. Acknowledgments This work was supported in part by a Grant-in-Aid for Intractable Diseases from the Ministry of Health and Welfare, Japan. The authors thank Drs. Y. Yamada and D.M. Noonan, Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes of Health, Bethesda, MD, and Dr. G.R. Martin, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA, for generously providing cloned DNAs.

collagen genes [43]. Weiner et al demonstrated that in an in vivo model of fibrosis dexamethasone produced transcriptional inhibition of type I collagen gene expression [44], and cells treated Reprint requests to Dr. Hikaru Koide, Division of Nephrology, with dexamethasone had decreased type I and type IV collagen Department of Medicine, Jun tendo University School of Medicine, mRNA steady-state levels due in part to diminished transcrip- 2-I-I, Hongo, Tokyo 113, Japan. tion rates of the gene 145]. Our data indicate that MPSL reduced References glomerular mRNA levels for al (IV) chain and laminin chains as

well as medullary mRNA levels for a! (I) chain and al (III) chain in PAN nephrosis, while MPSL did not affect these mRNA levels in control glomeruli and medulla. Although the mechanism by which glucocorticoid reduces collagen mRNA levels remains unclear, a decrease in the transcription [45, 46] and lability of collagen mRNAs [41, 42] have been reported. We previously reported that there is little difference in the pattern, distribution and intensity of staining of type IV collagen, laminin and HSPG between PAN nephrosis and controls

[11]. We were unable to show by the immunofluorescence method that changes in mRNA reflected altered protein expres-

sion. In the present study, MPSL did not alter the pattern, distribution or intensity of these proteins as determined by the immunofluorescence method (data not shown). This may reflect a low turnover rate of existing ECM proteins which could mask subtle protein changes, low immunofluorescence sensitivity or abnormal posttranscriptional control. Further studies are also needed to examine the possible effects of MPSL on posttranscriptional control of matrix biosynthesis, including message stability and protein degradation, in PAN nephrosis. PAN nephrosis is a complex disease which exhibits a biphasic pattern of protein excretion [47, 48]. Minimal glomerular lesions and interstitial cellular infiltrates are observed during

I. KEFALIDES NA: Structure and biosynthesis of basement membranes. mt Rev Connect Tissue Res 6:63—104, 1973 2. VRACKO R: Basal lamina scaffold-anatomy and significance for maintenance of orderly tissue structure. Am J Patho! 77:314—338, 1974

3. TIMPL R, DZIADEK M: Structure, development and molecular pathology of basement membrane. mt Rev Exp Patho! 29:1—112, 1986 4. ENGEL JE, ODERMATT E, ENGEL A, MADRI J, FURTHMAYR H, ROHDE H, TIMPL R: Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. J Mo! Biol 150:97—120, 1981 5. LAURIE GW, IN0uE S, BING JT, HA5SELL R: Visualization of the large heparan sulfate proteoglycan from basement membrane. Am J

Anat 181:320—326, 1988 6.

of the levels of type 111 and type I collagen mRNA in most but not all mouse fibroblasts. J Biol Chem 260:531—536, 1985 7. KLEINMAN ilK, EBIHARA I, KILLEN PD, SASAKI M, CANNON FB,

YAMADA Y, MARTIN GR: Genes for basement membrane proteins

are coordinately expressed in differentiating F9 cells but not in normal adult murine tissues. Dev Biol 122:373—378, 8.

1987

BOOT-HANDFORD RP, KURKINEN M, PROCKOP DJ: Steady-state

levels of mRNAs coding for the type IV collagen and laminin polypeptide chains of basement membranes exhibit marked tissuespecific stoichiometric variations in the rat. J Biol Chem 262:12475— 12478, 1987 9.

the first phase, whereas glomerulosclerosis is evident later during the second phase [47, 48]. However, the effects of MPSL on glomerular or medullary mRNA for ECM components in the second phase of PAN nephrosis remain unknown.

LIAU G, YAMADA Y, DE CROMBRLJGGHE B: Coordinate regulation

FRENK 5, ANroNowlcz 1, CRAIG JM, METCOFF J: Experimental nephrotic syndrome in rats by aminonucleoside: Renal lesions and body electrolyte compositions. Proc Soc Exp Biol Med 89:424—427,

I955 10. VERNIER RL, PAPERMASTER BW, GOOD RA: Aminonucleoside nephrosis. I. Electron microscopic study of the renal lesion in rats.

Med 109:115—126, 1959 Diamond et a! [49] demonstrated by double immunohisto- II. JExp NAKAMURA T, EBIHARA 1, SHIRATO I, TOMINO Y, KOIDE H: chemical labeling studies that the source of the antigenically Modulation of basement membrane component gene expression in expressed tumor necrosis factor and interleukin I in the gbglomeruli of aminonucleoside nephrosis. Lab Invest (in press) meruli of acute PAN nephrosis is the glomerular macrophage. 12. EDDY AA, MICHAEL AF: Acute tububointerstitial nephritis associated with aminonucleoside nephrosis. Kidney mt 33:14—23, 1988 Therefore, the beneficial effect of MPSL on proteinuria during 13. FUJIWARA Y: An ultrastructure study of the effect of the steroid in the early phase of the disease may be related to the inhibition of puromycin aminonucleoside nephrosis rats. Virchows Arch A 405:

inflammatory cellular infiltrates and of cytokine generation. We

showed that MPSL did not affect mRNA levels for ECM components in control glomeruli and medulla. This suggests that MPSL may prevent the synthesis of cytokines that regulate the expression of genes for ECM components. In summary, we demonstrated that glomerular mRNA levels for a! (IV) chain and laminin chains increased while those for HSPG decreased significantly in PAN nephrosis, that medullary

mRNA levels for a! (I) chain and al (III) chain increased markedly at the peak of proteinuria in PAN nephrosis and that

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KASINATH BS, SINGI-I AK, KANWAR YS, LEVIS EJ: Dexametha-

sane increases hepat-an sulfate proteoglycan core protein content of glomerular epithelial cells. J Lab C/in Med 115:196—202, 1990 15. OHYAMA K, SEYER JM, RAGHOW R, KANG AH: Extracellular matrix phenotype of rat mesangial cells in culture. Biosynthesis of

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AM: "Pulse" methylprednisolone therapy in the treatment of severe glomerulonephritis. J Pediatr 88:307—314, 1976

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