Proc. Natl. Acad. Sci. USA Vol. 88, pp. 9765-9769, November 1991 Medical Sciences Dietary protein restriction rapidly reduces transforming growth factor p1 expression in experimental glomerulonephritis (extraceliular matrix/transforming growth factor 8/glomerulonephritis) SEIYA OKUDA*, TAKAMICHI NAKAMURA*, TATSUO YAMAMOTO*, ERKKI RUOSLAHTIt, AND WAYNE A. BORDER*t *Division of Nephrology, University of Utah School of Medicine, Salt Lake City, UT 84132; and tCancer Research Center, La Jolla Cancer Research Foundation, La Jolla, CA 92037 Communicated by Eugene Roberts, August 19, 1991 (receivedfor review April 29, 1991) ABSTRACT Dietary protein restriction has been shown to TGF-/31 on both cell types is to regulate the synthesis of two slow the rate of loss of kidney function in humans with chondroitin/dermatan sulfate proteoglycans, biglycan and progressive glomerulosclerosis due to glomerulonephritis or decorin, both of which can bind TGF-P1 (23). In an experi- diabetes mellitus. A central feature of glomerulosclerosis is the mental model ofglomerulonephritis in the rat, we have found pathological accumulation of extracellular matrix within the a close association between elevated expression of the diseased glomeruli. Transforming growth factor j1 (TGF-.81) TGF-131 gene and the development of glomerulonephritis is known to have widespread regulatory effects on extracellular (10). Seven days after glomerular injury, at the time of matrix and has been implicated as a major cause of increased significant extracellular matrix accumulation, the glomeruli extracellular matrix synthesis and buildup of pathological showed a 5-fold increase in TGF-f31 mRNA and a nearly matrix within glomeruli in experimental glomerulonephritis. 50-fold increase in production ofbiglycan and decorin. Treat- In the present study, it is shown that administration of a low ment ofglomerulonephritic rats with an antiserum directed at protein diet to rats rapidly reduces the elevated expression of a synthetic peptide from mature human TGF-.81 blocked the TGF-.81 mRNA and TGF-pl1 protein that is known to occur induction of proteoglycan synthesis and prevented the his- within glomeruli after induction of glomerulonephritis. Com- tologic accumulation ofextracellular matrix in glomeruli (11). pared to a normal protein diet, glomerulonephritic rats receiv- Taken together the findings implicate TGF-,B1 as playing a ing the low protein diet did not develop an increase in glomer- central role in the pathogenesis of glomerulonephritis. ular extracellular matrix and showed significantly less protein- In humans with progressive glomerular disease, a reduc- uria. Glomeruli isolated from glomerulonephritic rats fed the tion in dietary protein intake has been advocated as being normal protein diet showed a marked increase in proteoglycan effective in slowing the rate ofloss ofrenal function (12). The synthesis on day 7 of disease and were demonstrated to be mechanism of the protein effect in preserving renal function secreting increased amounts of TGF-131 into the medium, has been suggested as being a reduction in glomerular pres- whereas glomeruli at the same point in time isolated from. rats sure and/or plasma flow (13, 14). In our model of experi- on a low protein diet showed no increase in proteoglycan mental glomerulonephritis, a low protein diet rapidly sup- production or TGF-.1 secretion. These results suggest that a pressed the expected increase in glomerular extracellular mechanism of'the rapid therapeutic effect of a low protein diet matrix. We now provide evidence that a mechanism of the on experimental glomerulonephritis is through suppression of therapeutic effect may be reduction of TGF-f31 gene expres- TGF-fl1 expression and prevention of the induction of extra- sion that results in suppression of the TGF-,81-induced in- cellular matrix synthesis within the injured glomeruli. crease in matrix synthesis in the injured glomeruli. Accumulation of extracellular matrix leading to glomerulo- METHODOLOGY sclerosis is the central pathological feature of progressive Experimental Diets. Two defined formulae protein diets kidney disease due to glomerulonephritis or diabetes mellitus were used (Teklad, Madison, WI). The normal protein diet (1). Morphometric analysis of glomeruli from diabetic pa- (22% protein; TD 86550) contained 25% casein and the low tients showed that the expanding volume of the glomerular protein diet (6% protein; TD 86551) contained 7% casein. mesangium (matrix and cells) correlated with the clinical Both diets were identical in fat and mineral content. Sucrose onset of proteinuria, hypertension, and a decreased glomer- and cornstarch provided 91% of the carbohydrate and the ular filtration rate (2). The factor(s) responsible for the diets were made isocaloric (3.5 kcal/g; 1 cal = 4.184 J) by buildup of extracellular matrix in human glomerular disease increasing the amount of sucrose in the low protein diet. To is largely unknown. equalize caloric intake per body weight among the experi- Recently, transforming growth factor /1 (TGF-p1) has mental groups, the amount of normal protein chow fed to the been shown to have widespread effects on extracellular rats was adjusted at 3-day intervals to equal the amount of matrix (3). In various cell lines, TGF-f31 is known to (i) chow consumed by rats fed the low protein diet. All animals increase production of proteoglycans, fibronectin, and col- had free access to tap water during the study. lagens (4, 5), (ii) decrease secretion ofproteases and increase Experimental Model. Glomerulonephritis was induced in levels ofprotease inhibitors (6), and (iiM) stimulate expression Sprague-Dawley rats (4-6 wk old) by intravenous injection of extracellular matrix receptors on cells (7). We have found of anti-thymocyte antiserum as described (10). Control rats that TGF-f31 has dramatic effects on the production of received an equivalent injection of preimmune serum. To extracellular matrix components by cultured rat glomerular determine the effects of dietary protein on development of mesangial (8) and epithelial cells (9). A principal action of glomerulonephritis, repeat experiments were conducted by using the following four groups: (i) six normal control rats fed The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: TGF-f31, transforming growth factor (1. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 9765 Downloaded by guest on October 2, 2021 9766 Medical Sciences: Okuda et al. Proc. Natl. Acad Sci. USA 88 (1991) the normal protein diet, (ii) six normal control rats fed the low increase of the extracellular matrix and cell number on days protein diet, (iii) six glomerulonephritic rats fed the normal 7 and 14, and then resolution over a 3-month period. The low protein diet, and (iv) six glomerulonephritic rats fed the low protein diet suppressed the expansion of the glomerular protein diet. Both the normal and low protein diets were extracellular matrix that occurred in glomerulonephritic rats started on day 1, at the time ofanti-thymocyte or preimmune fed the normal protein diet but did not interfere with healing serum administration, and maintained until the time of sac- of the areas of mesangial cell lysis, the net effect being that rifice. Kidneys for immunohistologic examination and isola- the glomneruli appeared nearly normal on day 7 in glomeru- tion of glomeruli were harvested from normal control rats lonephritic rats receiving the low protein diet. The therapeu- before (day 0) and 7 days after preimmune serum injection tic effect of the low protein diet on glomerular histology is and from glomerulonephritic rats on days 1, 4, 7, 14, and 28 in terms in Fig. 1A. The low protein diet after injection of anti-thymocyte antiserum. At each time shown quantitative point the following measurements were also made: serum also reduced the amount ofproteinuria in glomerulonephritic creatinine, 24-h urinary protein excretion, systolic blood rats (Fig. 1B). Control rats did not show any changes in pressure, and body weight as described (10). Kidney tissue glomerular matrix nor did they manifest proteinuria. Renal was prepared for light and immunofluorescence microscopy function as measured by serum creatinine levels did not by standard published methods (10). Glomerular extracellu- change in this model and no differences were noted between lar matrix was evaluated by an observer unaware of the control and glomerulonephritic groups (data not shown). source of the tissue. Thirty glomeruli were selected at ran- Normal control and glomerulonephritic rats receiving the dom and the percentage of the glomerular area occupied by normal protein diet tended to have higher body weights (Fig. extracellular matrix was estimated by using a quantitative 2A) and blood pressures (Fig. 2B) than did the groups being scale as described (10, 11). fed the low protein diets; these differences became significant Bioassay of TGF-131. The ability of TGF-(31 to induce on days 14 and 28. production of proteoglycans by normal rat mesangial cells in culture was used as a bioassay to detect TGF-.31 in condi- A 80 1 I tioned medium from glomerular cultures as described (10). To include measurement of both mature and precursor TGF-f31 activity in the bioassay, aliquots of the conditioned medium were acidified to pH 3.2 for 1 h by addition of 1 M HCl. The acidified medium was brought to pH 7.4 with 1 M w transiently 0cr NaOH and dialyzed against serum-free RPMI medium for 24 Cx, h at 40C. Growth Factors and Antibodies. Porcine platelet TGF-p1 was obtained from R & D Systems (Minneapolis). The polyclonal TGF-P1 neutralizing antibody was prepared as described (10). A second polyclonal antibody (anti-LC) made against a synthetic peptide corresponding to the first 30 amino acids ofmature TGF-f81 was kindly provided by K. C. Flanders and M. B. Sporn (National Institutes of Health). Rabbit antibodies produced against synthetic peptides from the core proteins of human biglycan and decorin were a DAYS generous gift from L.