Growth factor ultrafiltration in experimental diabetic nephropathy contributes to interstitial fibrosis

Shi-Nong Wang and Raimund Hirschberg

Division of Nephrology and Hypertension, Harbor-UCLA Medical Center and University of California Los Angeles, Torrance, California 90509


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glomerular proteinuria is a risk factor for progression of chronic renal failure and contributes to renal interstitial fibrosis. In experimental diabetic glomerular sclerosis, there is translocation of high-molecular-weight growth factors, namely, hepatocyte growth factor (HGF) and transforming growth factor (TGF)-beta , from plasma into tubular fluid, both of which act on tubular cells through apical membrane receptors. In the present studies, the hypothesis is examined that ultrafiltered HGF and TGF-beta induce increased expression of extracellular matrix (ECM) proteins directly in tubular cells, or induce increased expression of cytokines that may act on interstitial myofibroblasts. Incubation of cultured tubular cells with recombinant human (rh) TGF-beta modestly raises expression of collagen type III, but rhHGF dose dependently blocks expression of this ECM protein. Both growth factors raise fibronectin expression up to fourfold and increase expression of platelet-derived growth factor (PDGF)-BB up to sixfold, but not of fibroblast growth factor-2. Pooled, diluted glomerular ultrafiltrate that had been collected by nephron micropuncture from rats with diabetic nephropathy (24-30 wk) also raises expression of fibronectin as well as PDGF-BB in proximal tubular cells. In the presence of neutralizing antibodies that block actions of HGF and TGF-beta , diabetic rat glomerular ultrafiltrate fails to increase tubular cell PDGF-BB expression. In NRK-49F renal interstitial myofibroblasts, rhPDGF-BB, in turn, raises the expression of collagen type III but not type I or fibronectin. The findings provide evidence for ultrafiltered HGF and TGF-beta to contribute to interstitial accumulation of ECM proteins by direct effects on tubular cells as well as indirect mechanisms, via PDGF-BB and its action on myofibroblasts. These events may be important mechanisms of proteinuria-induced renal interstitial fibrosis and accelerated progression of chronic renal failure in diabetic nephropathy and perhaps other proteinuric glomerular diseases.

collagen; fibronectin; renal interstitial fibrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GLOMERULAR PROTEINURIA IS a risk factor for progression of renal failure and is associated with progressive interstitial fibrosis (3, 5, 7, 10, 11, 16, 19-22). This relationship between glomerular protein ultrafiltration and interstitial fibrosis is universal and not limited to certain glomerular diseases. In fact, this rule applies to immunological as well as nonimmunological, primary, and secondary glomerular diseases as well as to diabetic nephropathy (4-6).

Previous studies from this laboratory have indicated that in the adriamycin-induced nephrotic syndrome and in streptozotocin-induced diabetic nephropathy in rats, glomerular ultrafiltration of proteins leads to translocation of high-molecular-weight growth factors into tubular fluid, including insulin-like growth factor (IGF)-I, transforming growth factor (TGF)-beta , and hepatocyte growth factor (HGF) (14, 28, 29). These growth factors become activated in tubular fluid and act on tubular cells through specific IGF-I, TGF-beta , and HGF receptors, respectively, that are expressed in apical membranes (1, 12, 14, 28, 29). Ultrafiltered TGF-beta and HGF "activate" tubular cells and raise the expression and basolateral secretion of monocyte chemoattractant protein (MCP)-1 and regulated on activation, normal T cell expressed and secreted (RANTES) (27). These C-C chemokines attract and activate macrophages, and macrophage-derived TGF-beta increases the expression of extracellular matrix proteins in interstitial myofibroblasts.

Because of the presence of ultrafiltered growth factors, diabetic rat proximal tubular fluid may "transform" tubular cells, i.e., may induce increased expression of extracellular matrix proteins in tubular cells. Growth factors that are present in glomerular ultrafiltrate may induce increased expression and basolateral secretion of cytokines in tubular cells that can directly interact with interstitial myofibroblasts without acting through macrophages as intermediaries.

In the present studies, the hypothesis was tested that HGF and TGF-beta in glomerular ultrafiltrate from rats with diabetic nephropathy act directly on urinary space-lining epithelial cells of the nephron causing increased expression of interstitial extracellular matrix proteins such as collagen (col) alpha 2I, col alpha 1III, and fibronectin. Moreover, the interaction of tubular fluid growth factors may increase the expression of cytokines such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF)-2 that may stimulate interstitial fibroblasts to express increased amounts of interstitial matrix proteins.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To test this hypothesis, experiments were performed in cultured tubular cells that were exposed to glomerular ultrafiltrate that was collected extensively from rats with diabetic nephropathy. Glomerular ultrafiltrate from rats with diabetic nephropathy (24-30 wk) but not from control rats contains active HGF and TGF-beta (26, 28).

In Vivo Studies

Early proximal tubular fluid was collected from Spraque-Dawley rats at 24-30 wk after induction of diabetes with streptozotocin (85 mg/kg). Animals were given subcutaneous insulin to maintain blood glucose at 300-350 mg/dl. The micropuncture techniques to collect intensively glomerular ultrafiltrate from diabetic and control rats have been described previously (14). Glomerular ultrafiltrate from rats with diabetic nephropathy at 24-30 wk of diabetes contains HGF as demonstrated previously by Western blot analysis in this laboratory, and glomerular ultrafiltrate from normal control rats does not contain HGF at levels recognized by this method (26-28). Levels of total and active TGF-beta in early proximal tubular fluid from rats with diabetic nephropathy are 729 ± 72 and 76 ± 31 pg/ml, respectively. In normal rat early proximal tubular fluid, active TGF-beta is undetectable, and total TGF-beta levels have been estimated at 34 ± 9 pg/ml (25, 26, 28).

In Vitro Studies

Cell cultures. In vitro studies of effects of proximal tubular fluid, recombinant human (rh) HGF, and rhTGF-beta were performed in cultured cells originated from mouse proximal tubules and medullary collecting ducts. Mouse proximal tubular cells (mPTC) had been obtained as a kind gift from Dr. Carolyn Kelly (Univ. of California San Diego). These cells were originally outgrown from proximal tubule S3 segments and immortalized by transfection with the simian virus 40 T antigen (13). Mouse inner medullary collecting duct cells (mIMCD-3) and NRK-49F cells, which are rat kidney-derived myofibroblasts, were obtained from a commercial source (ATCC, Rockville, MD). These latter cells express alpha -smooth muscle actin as tested in this laboratory. All cells were grown in DMEM/Ham's F-12 containing normal culture medium glucose levels (100 mg/dl) and supplemented with 10% FCS.

Effect of diabetic rat glomerular ultrafiltrate and recombinant growth factors on the expression of collagen type I and III and fibronectin in proximal tubular and collecting duct cells. Proximal tubular cells were grown in 96-well plates to ~80% confluence and then growth arrested by incubation with serum-free medium containing BSA (25 µg/ml). Cells were then exposed for 24 h to serum-free medium (control) or medium containing pooled, diluted (1:5) proximal tubular fluid (25 µl/well) that had been collected by micropuncture from rats with diabetic nephropathy (n = 4). In separate experiments, proximal tubular cells were also incubated in 24-well plates with serum-free medium (control) or medium containing either rhHGF (1 nM, R&D Systems, Minneapolis, MN) or rhTGF-beta 1 (1 nM, R&D Systems) (n = 6 each). Media were removed, and RNA was extracted using the RNA-Stat-60 reagent and the method recommended by the manufacturer (Tel-Test, Friendswood, TX).

In each sample, col alpha 2I, col alpha 1III, and fibronectin mRNA as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were measured by RT-PCR. For RT-PCR, random-primed first-strand cDNA was synthesized from RNA from each well using a commercially available kit and the recommended procedure (Boehringer Mannheim, Indianapolis, IN). Aliquots of cDNA were then amplified using specific primers: 5'-TGTTCGTGGTTCTCAGGGRTAG-3' (sense) and 5'-TTGTCGTAGCAGGGTTCTTTC-3' (antisense) for col alpha 2I, 5'-CGAGGTAACAGAGGTGAAAGA-3' (sense) and 5'-AACCCAGTATTCTCCGCTCTT-3' (antisense) for col alpha 1III, 5'-TTTTGACAACGGGAAGCATTATCAGATAA-3' (sense) and 5'-TGATCAAAACATTTCTCAGCTATTGG-3' (antisense) for fibronectin, and 5'-CCATGGAGAAGGCCGGGG-3' (sense) and 5'-CAAAGTTGTCATGGATGACC-3' (antisense) for mouse GAPDH.

The PCR was performed in 50 µl reaction mixtures. For amplification of cDNA, the reaction mixture contained 5 µl of 10× PCR buffer (500 mM KCl, 100 mM Tris · HCl, 25 mM MgCl2), 4 µl dNTP mixture (dATP, dTTP, dCTP, and dGTP; 2.5 mM each), 0.25 µl Taq polymerase (5 U/µl), 200 ng oligonucleotide primers, and 2 µl cDNA.

For quantitative analysis, 50 µl of PCR products were electrophoresed in 4% agarose gels containing 0.5 µg/ml ethidium bromide. Resolved gels were photographed under ultraviolet light with Polaroid negative film (Polaroid, Cambridge, MA). Bands were scanned by densitometry, and densitometric units for fibronectin, col alpha 2I, and col alpha 1III mRNA were expressed relative to GAPDH mRNA of the same sample.

In similar experiments, mIMCD-3 cells that were grown to ~80% confluence in 24-well plates were also growth arrested and then incubated for 24 h with serum-free medium (control, n = 6) or serum-free medium containing rhHGF at 1-4 nM (n = 6 each) or rhTGF-beta 1 at 0.4-2 nM (n = 6 each). Subsequently, mRNA was extracted from each well, and mRNA levels encoding mouse GAPDH and fibronectin were examined by RT-PCR as described above.

Effect of diabetic rat glomerular ultrafiltrate and rhHGF and rhTGF-beta on tubular cell expression of cytokine growth factors (PDGF-B and FGF-2). Previous studies from this laboratory had shown that HGF and TGF-beta , which are both present in tubular fluid in rats with glomerular proteinuria, activate tubular cells through apical membrane receptors causing basolateral secretion of the two C-C chemokines MCP-1 and RANTES (manuscript in press). These chemokines cause peritubular attraction and activation of macrophages that express growth factors, primarily TGF-beta , which in turn raise the expression of extracellular matrix proteins by interstitial fibroblasts. However, ultrafiltered HGF and TGF-beta may also induce increased expression of cytokine growth factors in tubular cells that may then directly interact with interstitial fibroblasts raising the expression of extracellular matrix proteins without macrophages acting as intermediaries.

To examine this latter question, studies were performed in proximal tubular and mIMCD-3 cells that were incubated with HGF or TGF-beta or with pooled proximal tubular fluid from diabetic rats (which contains both of these growth factors), and the expression of FGF-2 and PDGF-B was examined.

Both mPTC and mIMCD-3 cells were grown to ~80% confluence in 24-well plates. Cells were growth arrested and then incubated with serum-free medium (control, n = 6) or medium containing rhHGF (1 nM) or rhTGF-beta 1 (1 nM). After 24 h, media were removed and RNA was extracted from each well. FGF-2, PDGF-B, and GAPDH mRNA were examined by RT-PCR using the following mouse-specific primers: FGF-2, 5'-ACACGTCAAACTACAACTCCA-3' (sense) and 5'-TCAGCTCTTAGCAGACATTGG-3' (antisense); PDGF-B, 5'-GTGTGAGACAATAGTGACCC-3' (sense) and 5'-TTGGAGTCAAGAGAAGCC-3' (antisense); and the GAPDH primers as described above.

In separate experiments, growth-arrested mPTC cells were incubated for 24 h with serum-free medium (control); medium containing a mixture of neutralizing antibodies against HGF, TGF-beta and their receptors (anti-TGF-beta , 5 µg/ml, R&D Systems; anti-TGF-beta receptor type II, 3 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA; anti-HGF, 3 µg/ml, Santa Cruz Biotechnology; and anti-HGF receptor, 3 µg/ml, Santa Cruz Biotechnology); medium containing pooled proximal tubular fluid that had been collected from rats with diabetic nephropathy at 1:5; or medium containing pooled proximal tubular fluid in the presence of the above mixture of neutralizing antibodies. Cells were incubated for 24 h, and PDGF-B and GAPDH mRNA were measured by RT-PCR as described above.

Effect of rhPDGF-B on expression of extracellular matrix proteins in cultured rat renal myofibroblasts. PDGF-B, which is derived from tubular cells and augmented by HGF- and TGF-beta -containing diabetic rat tubular fluid, may act on interstitial myofibroblasts and induce increased expression of extracellular matrix proteins by these latter cells.

To test this question, rat renal interstitial myofibroblasts (NRK-49F cells) were incubated, for 24 h, with rhPDGF-B (1 or 10 nM; R&D Systems), rhTGF-beta 1 (400 pM), or serum-free medium as control (n = 6 each). Media were removed, RNA was extracted from each sample as above, and col alpha 2I, col alpha 1III, fibronectin, and GAPDH mRNA were measured by RT-PCR using the following rat specific primers: col alpha 2I, 5'-TGTTCGTGGTTCTCAGGGTAG-3' (sense) and 5'-TTGTCGTAGCAGGGTTCTTTC-3' (antisense); col alpha 1III, 5'-CGAGGTAACAGAGGTGAAAGA-3' (sense) and 5'-AACCCAGTATTCTCCGCTCTT-3' (antisense); fibronectin, 5'-TTTTGACAACGGGAAGCATTATCAGATAA-3' (sense) and 5'-TGATCAAAACATTTCTCAGCTATTGG-3' (antisense); rat GAPDH, 5'-GACAAGATGGTGAAGGTCGG-3' (sense) and 5'-CATGGACTGTGGATCATGAGC-3' (antisense).

Statistical Analysis

PCR-derived mRNA levels are expressed as ratio over GAPDH mRNA. Data are means ± SE. Comparisons were made by ANOVA followed by Student-Newman-Keuls multicomparison tests.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

At the time of early proximal tubular fluid collection by nephron micropuncture (24-30 wk after induction of diabetes), renal histology was examined qualitatively by light microscopy of periodic acid-Schiff-stained slides. Glomeruli were minimally abnormal and showed mild mesangial widening. The renal interstitium was not expanded. However, rats were proteinuric, and the total urinary protein excretion was 145 ± 19 µg/min as measured in bladder urine that was collected during the micropuncture procedures.

Glomerular Ultrafiltrate From Diabetic Rats and HGF and TGF-beta Raise the Expression of Extracellular Matrix Proteins in Tubular Cells

At baseline, the expression of col alpha 2I mRNA in proximal tubular cells is extremely low (at least 2-3 orders of magnitude lower than in NRK-49F fibroblasts), and neither HGF nor TGF-beta increases col alpha 2I mRNA levels in these cells. In mIMCD-3 cells, col alpha 2I mRNA could not be detected at all with the RT-PCR method used, not even after incubation with HGF or TGF-beta .

Col alpha 1III is expressed in proximal tubular cells. Incubation of the cells with TGF-beta (2 nM) moderately increases col alpha 1III mRNA levels by ~40% (Fig. 1A). In contrast, incubation with rhHGF does not increase col alpha 1III mRNA levels, and high concentrations of this growth factor (which likely exceed the in vivo levels of HGF in glomerular ultrafiltrate) lower col alpha 1III expression in proximal tubular cells (Fig. 1B).


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Fig. 1.   Effects of transforming growth factor (TGF)-beta (A) and hepatocyte growth factor (HGF) (B) on expression of collagen type III in proximal tubular cells. TGF-beta 1 (2 nM) moderately increases collagen (col) alpha 1III mRNA levels (A). In contrast, HGF dose dependently reduces the expression of col alpha 1III (B). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. * P < 0.05 (n = 6 each).

Both proximal tubular as well as mIMCD-3 cells express fibronectin under control conditions, and interstitial accumulation of fibronectin contributes to interstitial fibrosis. Both growth factors, HGF and TGF-beta , which are present in glomerular ultrafiltrate from rats with diabetic nephropathy but not from control animals, increase the expression of this extracellular matrix protein directly in tubular cells (Fig. 2). At equimolar concentrations, the effects of TGF-beta on fibronectin expression are more potent compared with HGF. In proximal tubular cells, incubation with HGF or TGF-beta (2 nM) for 48 h increases fibronectin mRNA levels about two- and threefold, respectively (Fig. 2). Incubation of the cells with rhHGF at 0.2 or 2 nM for only 24 h fails to increase fibronectin mRNA levels in proximal tubular cells, and 4 nM HGF raises fibronectin within 24 h of incubation to 165 ± 9% of control values (P < 0.05). This suggests that the action of HGF in diabetic rat tubular fluid on expression of fibronectin is relatively weak compared with TGF-beta . Nevertheless, direct actions of ultrafiltered HGF and TGF-beta contribute to increased expression of ECM proteins, specifically collagen type III and fibronectin, in proximal tubular cells.


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Fig. 2.   Effect of recombinant human (rh) HGF (2 nM) and rhTGF-beta 1 (2 nM) on the expression of fibronectin in proximal tubular cells. * P < 0.05 (n = 6 each).

In mIMCD-3 cells, both HGF as well as TGF-beta raise the expression of fibronectin within 24 h of incubation up to 3.5-fold (Fig. 3). Although these cells seem to react more sensitively to HGF compared with proximal tubular cells, the effect of equimolar TGF-beta on fibronectin expression in mIMCD-3 cells appears to be greater compared with rhHGF (Fig. 3).


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Fig. 3.   Effect of rhHGF and rhTGF-beta 1 on expression of fibronectin in mIMCD-3 cells. * P < 0.05 (n = 6 each).

Incubation of proximal tubular cells with pooled diabetic rat glomerular ultrafiltrate also increases the expression of fibronectin (Fig. 4). Although the increase appears to be moderate in magnitude, on average ~51%, it should be taken into account that the tubular fluid was fivefold diluted with serum-free medium. Given that in vivo accumulation of extracellular matrix in renal interstitial fibrosis is a very slow but progressive process, this rise in fibronectin expression, although moderate, is likely to make important contributions.


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Fig. 4.   Pooled, 1:5-diluted early proximal tubular fluid that had been collected from rats with diabetic nephropathy (DM-PTF) tends to increase fibronectin mRNA levels in cultured proximal tubular cells. * P = 0.0705 (n = 4).

HGF, TGF-beta , and Diabetic Rat Glomerular Ultrafiltrate Increase the Expression of PDGF-B But Not of FGF-2 in Tubular Cells

Incubation of proximal tubular cells with pooled proximal tubular fluid that was collected from rats with diabetic nephropathy and contains HGF as well as TGF-beta (28) increases the expression of the cytokine PDGF-B about twofold (Fig. 5). Incubation with HGF or TGF-beta 1 (1 nM) each raises the PDGF-B mRNA levels 3.2- and 6.1-fold, respectively (Fig. 6). Moreover, incubation of proximal tubular cells with tubular fluid in the presence of neutralizing antibodies that block the bioactivity of HGF and TGF-beta abolishes this effect of diabetic tubular fluid on the expression of PDGF-B (Fig. 5).


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Fig. 5.   Pooled, 1:5-diluted early proximal tubular fluid from rats with diabetic nephropathy (DM-PTF) increases expression of platelet-derived growth factor (PDGF)-B about twofold in proximal tubular cells. Incubation of cells with diabetic rat early proximal tubular fluid in the presence of a mixture of antibodies that neutralize HGF, TGF-beta , and their respective receptors (DM-PTF+AB) blocks the DM-PTF-induced increase in PDGF-B expression. CO, control; AB, mixture of neutralizing antibodies. * P < 0.05 (n = 4 each).



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Fig. 6.   Effect of recombinant HGF (1 nM) and TGF-beta 1 (1 nM) on expression of PDGF-B in proximal tubular cells. * P < 0.05 (n = 6 each).

In mIMCD-3 cells, the effect of each of the two growth factors appears weaker compared with proximal tubular cells. In mIMCD-3 cells, 1 nM HGF does not significantly increase PDGF-B mRNA, and equimolar TGF-beta raises PDGF-B mRNA levels 3.4 ± 0.3-fold (P < 0.05).

In both proximal tubular as well as mIMCD-3 cells, FGF-2 mRNA is not detectable in the baseline state with the RT-PCR method used. Incubation with either HGF (1 nM) or TGF-beta (1 nM) does not increase FGF-2 mRNA, at least not to the extent measurable with the current method.

Effects of PDGF-B on the Expression of Extracellular Matrix Proteins in Rat Renal Interstitial Myofibroblasts

In vivo, tubular cell-derived PDGF-B may interact with interstitial fibroblasts. In the kidney, interstitial fibroblasts are believed to be the major source for extracellular matrix proteins that accumulate in renal interstitial fibrosis. Hence, PDGF-B may be an important tubule-derived mediator of proteinuria-induced interstitial fibrosis.

In the present studies, NRK-49F rat renal fibroblasts were incubated with rhPDGF-B (1 nM) or, for comparison, with TGF-beta 1 (400 pM). As shown in Fig. 7, incubation of fibroblasts with PDGF-BB failed to increase col alpha 2I or fibronectin mRNA (Fig. 7, A and C) but increased col alpha 1III mRNA levels significantly (P < 0.05, Fig. 7B). In contrast, TGF-beta , which may be primarily derived from macrophages in vivo, increased the expression of each of the three extracellular matrix proteins in rat renal myofibroblasts about 2.5- to 4-fold (Fig. 7).


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Fig. 7.   Effects of PDGF-BB (1 nM) and TGF-beta 1 (0.4 nM) on col alpha 2I (A), col alpha 1III (B), and fibronectin (C) in rat renal interstitial myofibroblasts (NRK-49F cells). * P < 0.05 (n = 6 each).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Clinical trials and clinicopathological studies have indicated that there are correlations between the degree of glomerular proteinuria, interstitial fibrosis, and the rate of progression of chronic renal failure (2, 5, 20, 22). These relationships have been shown to occur in diabetic nephropathy as well as in other (primary or secondary) glomerular diseases.

Glomerular proteinuria in experimental diabetic nephropathy causes glomerular ultrafiltration of HGF and TGF-beta into tubular fluid (25, 26, 28). Both growth factors interact with their specific receptors that are present in apical membranes in various tubular segments (12, 25, 26). Ultrafiltered HGF and TGF-beta may each act on tubular cells separately and independently, or may act synergistically. Synergistic interactions of TGF-beta and HGF signaling pathways have recently been described (9).

There are essentially three types of pathways through which ultrafiltered HGF and TGF-beta may contribute to the accumulation of extracellular matrix proteins in the peritubular interstitium and, hence, to progressive interstitial fibrosis. First, ultrafiltered HGF and TGF-beta induce increased extracellular matrix production directly in tubular cells as shown in the present studies. Indeed, as shown in Fig. 4, early proximal tubular fluid obtained from rats with diabetic nephropathy by micropuncture which contains the two growth factors raises fibronectin expression in tubular cells. Equimolar comparisons between the effects of TGF-beta and HGF on tubular cell fibronectin expression indicate that the effects of ultrafiltered TGF-beta are greater in this respect than those of HGF (Figs. 2 and 3). TGF-beta increases collagen type III mRNA levels (Fig. 1A), but HGF fails to increase the expression of this extracellular matrix protein in proximal tubular cells (Fig. 1B). At high concentrations, HGF actually reduces the expression of collagen type III. Although actual levels of HGF in proximal tubular fluid from rats with diabetic nephropathy are not known since the presence of this growth factor had been demonstrated with a nonquantitative method (Western blot analysis), it is fair to estimate that levels are most likely lower than is required for reducing collagen type III expression. To this end, it is noteworthy that transgenic mice overexpressing HGF develop renal fibrosis (23). Hence, TGF-beta and perhaps HGF, which are both present in tubular fluid in diabetic nephropathy (and likely in other nephrotic glomerular diseases), may contribute to interstitial fibrosis by directly raising expression of certain extracellular matrix proteins such as collagen type III and fibronectin in tubular cells.

Second, ultrafiltered HGF and TGF-beta increase tubular cell expression of cytokine growth factors, specifically, PDGF-B as shown in the present studies. Although it is difficult to quantitatively translate in vitro findings into in vivo pathobiological events, it appears from respective experimental findings in the current studies that this indirect pathway involving tubular cell PDGF-B may contribute only moderately, since only one out of three extracellular matrix proteins, namely, collagen type III, increased upon incubation of myofibroblasts with PDGF-BB. However, this latter pathway is macrophage independent. Tang et al. (24) demonstrated in vivo that actions of PDGF-BB on renal fibroblasts contribute to the development of interstitial fibrosis, and several other investigators (8, 15, 17, 18) demonstrated that PDGF raises extracellular matrix expression and activates renal and nonrenal fibroblasts.

Third, ultrafiltered HGF and TGF-beta interact with apical tubular cell receptors and raise the basolateral secretion of MCP-1 and RANTES and perhaps other chemokines as recently shown in this laboratory (manuscript in press; 28). These C-C chemokines attract macrophages that secrete TGF-beta and may, in fact, be the dominant source for renal interstitial TGF-beta activity (10). Even though diabetic nephropathy is generally not perceived as an inflammatory disease, a renal interstitial macrophage infiltrate does occur, and its onset precedes measurable interstitial fibrosis (5). Macrophage-derived TGF-beta can act on interstitial myofibroblasts inducing increased production of extracellular matrix proteins. Experimental evidence supporting this indirect pathway has been previously demonstrated in this laboratory (manuscript in press; 27).

Findings from this study point toward the importance of tubular epithelial cells as the initial targets of glomerular proteinuria and ultrafiltered HGF and TGF-beta as the signals that initiate cell-cell interactions that contribute to interstitial fibrosis. Current therapeutic strategies to reduce the effects of glomerular proteinuria on tubulointerstitial injury and progression of renal failure aim at reducing the rate of glomerular protein ultrafiltration with antihypertensives, specifically with angiotensin-converting enzyme inhibitors. However, interruption of activation of tubular cells by ultrafiltered proteins, specifically high-molecular-weight growth factors (TGF-beta , HGF), could open additional avenues of treatment and prevention.


    ACKNOWLEDGEMENTS

This work was supported by Juvenile Diabetes Foundation Grants 196050 and 1-1998-350. S.-N. Wang was supported by a grant from the National Kidney Foundation of Southern California.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. Hirschberg, Div. of Nephrology and Hypertension, Harbor-UCLA Medical Center, Box 406, 1000 West Carson St., Torrance, CA 90509 (E-mail: rhirschberg{at}rei.edu).

Received 5 August 1999; accepted in final form 2 November 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 278(4):F554-F560
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