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. 2008 Feb;19(2):281-9.
doi: 10.1681/ASN.2007030290. Epub 2008 Jan 9.

PDGF-C is a proinflammatory cytokine that mediates renal interstitial fibrosis

Frank Eitner  1 , Eva Bücher, Claudia van Roeyen, Uta Kunter, Song Rong, Claudia Seikrit, Luigi Villa, Peter Boor, Linda Fredriksson, Gudrun Bäckström, Ulf Eriksson, Arne Ostman, Jürgen Floege, Tammo Ostendorf
Affiliations

PDGF-C is a proinflammatory cytokine that mediates renal interstitial fibrosis

Frank Eitner et al. J Am Soc Nephrol. 2008 Feb.

Abstract

PDGF-C is a potent mitogen for fibroblasts in vitro. Transgenic PDGF-C overexpression in the heart or liver induces organ fibrosis, and PDGF-C expression is upregulated at sites of interstitial fibrosis in human and rat kidneys; however, the effect of inhibiting PDGF-C on the development of renal fibrosis in vivo is unknown. Renal fibrosis was induced in C57BL/6 mice by unilateral ureteral obstruction (UUO), and then mice were treated with neutralizing anti–PDGF-C antiserum or nonspecific IgG. An increase in PDGF-C expression was observed in fibrotic areas after UUO, contributed in large part by infiltrating macrophages. Treatment with anti–PDGF-C reduced renal fibrosis by 30% at day 5 and reduced interstitial myofibroblast accumulation by 57%. In vitro, PDGF-C was a potent mitogen for renal fibroblasts and induced chemokine expression. In vivo, anti–PDGF-C treatment produced a decrease in the expression of the renal chemokines CCL2 and CCL5 (85 and 67% reductions, respectively), accompanied by a significant decrease in leukocyte infiltration and CCR2 mRNA expression. Further supporting a role of PDGF-C in renal fibrosis, PDGF-C−/− mice demonstrated a reduction in fibrosis and leukocyte infiltration in response to UUO compared with wild-type littermates. In conclusion, specific neutralization or lack of PDGF-C reduces the development of renal inflammation and fibrosis in obstructed mouse kidneys. Leukocyte-derived PDGF-C induces chemokine expression, which may lead to the recruitment of additional leukocytes, creating an amplification loop for renal inflammation and fibrosis.

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Figures

Figure 1.
Figure 1.
Neutralizing activity and specificity of the affinity-purified sheep anti–PDGF-C IgG. Porcine aortic endothelial cells stably expressing transfected relevant PDGF receptor isoforms were stimulated with various PDGF isoforms in the presence or absence of anti–PDGF-C. After lysis of the cells, receptors were enriched, and the receptor phosphorylation level was determined by immunoblotting with phosphotyrosine antibodies. (A) Immunoblot detects significant reduction of phosphotyrosine only in cells treated with anti–PDGF-C. No effects are noted in the case of PDGF-AA–or PDGF-BB–induced receptor phosphorylation. (B) Dosage-dependent effects of PDGF-CC–induced receptor phosphorylation without affecting the PDGF-DD–induced phosphorylation.
Figure 2.
Figure 2.
PDGF-C is overexpressed in murine renal fibrosis. (A) In healthy normal mice, PDGF-C is localized by immunohistochemistry within arteriolar smooth muscle cells and within individual tubular epithelial cells. (B and C) After UUO, PDGF-C is overexpressed within the injured kidneys. In addition to constitutive arteriolar and tubular expression, PDGF-C is expressed within individual interstitial cells. (D) Western blot analysis confirms the increased renal expression of PDGF-C. Compared with healthy normal mice, PDGF-C expression increases approximately three-fold in renal lysates from mice at day 10 after UUO (top). Labeling of the same blot with an anti-actin antibody confirms equal protein loading in each lane (bottom). (E through G). Confocal microscopy demonstrates that PDGF-C is expressed within infiltrating macrophages. ER-HR3–positive macrophages are labeled with a green color (E) and PDGF-C with a red color (F). The merged image identifies the yellow color of double-positive, PDGF-C–expressing macrophages (G). Note the PDGF-C–expressing cell that is not a macrophage in the top part of the image. This cell is likely a tubular cell or a vascular smooth muscle cell.
Figure 3.
Figure 3.
PDGF-C antagonism reduces murine renal fibrosis. Histologic staining for Sirius red (A) and immunohistochemistry for collagen type I (B), collagen type IV (C) and α-SMA (D) reveals a significant decrease of all four parameters of renal fibrosis/fibrogenesis in mice that were treated with anti–PDGF-C as compared with control IgG in the course of UUO. Representative illustrations of left obstructed kidneys of mice treated with control IgG (left) and mice treated with anti–PDGF-C (middle). The results of the computer-assisted morphometry measurements are summarized in the right. □, mice treated with control IgG; ▪, mice treated with anti–PDGF-C. Data are means ± SD.
Figure 4.
Figure 4.
PDGF-C is a mitogen for renal fibroblasts in vitro and induces chemokine production. (A) Renal fibroblasts were stimulated with different concentrations of PDGF-AA and PDGF-CC. Both isoforms act as mitogens for these cells in vitro. PDGF-C is a more potent pro-proliferative stimulus resulting in significant cell proliferation already at lower concentrations. Combination of both PDGF-A and PDGF-C does have an additive effect on renal fibroblast proliferation. *P < 0.05 versus PDGF-AA (100 ng/ml) and PDGF-CC (5 ng/ml); **P < 0.05 versus PDGF-AA (100 ng/ml) and PDGF-CC (5 and 10 ng/ml). (B) In addition to its mitogenic properties, stimulation of renal fibroblasts with PDGF-C also induces cellular chemokine production. After PDGF-C stimulation, the chemokine CCL2 is increased both on the mRNA level (□) and on the protein level (•) within a few hours.
Figure 5.
Figure 5.
PDGF-C antagonism reduces renal leukocyte infiltration and renal chemokine expression in murine renal fibrosis. (A) Treatment of UUO-induced murine renal fibrosis with neutralizing PDGF-C results in a significant reduction of renal chemokine and chemokine receptor mRNA expression. Graphics illustrate the mean relative chemokine mRNA expression compared with the contralateral control kidney of each mouse. (B) Treatment with neutralizing anti–PDGF-C results in decreased renal infiltration of macrophages (top) and T lymphocytes (bottom). Representative images of left obstructed kidneys of mice treated with control IgG (left) or anti–PDGF-C (middle). The results of the computer-assisted morphometry measurements are summarized in the right. □, mice treated with control IgG; ▪, mice treated with anti–PDGF-C. Data are means ± SD.
Figure 6.
Figure 6.
PDGF-C deficiency protects from renal fibrosis. At day 5 after UUO, immunohistologic staining for α-SMA, macrophages (ER-HR3–positive cells), and T lymphocytes (CD3-positive cells) in left obstructed kidneys reveals significant reductions of all three parameters in PDGF-C−/− mice (n = 7) compared with wild-type littermates (n = 7). At day 10 after UUO, labeling of Sirius red and immunohistochemical staining for collagen I and IV in left obstructed kidneys reveal significant reductions of renal fibrosis in PDGF-C−/− mice (n = 6) compared with wild-type littermates (n = 6). Data are means ± SD.
Figure 7.
Figure 7.
Hypothesis of mechanisms involved in PDGF-C–induced renal fibrosis. Increased PDGF-C expression acts directly on PDGF-α receptor–expressing renal myofibroblasts. In addition, PDGF-C induces local chemokine production, resulting in leukocyte attraction. PDGF-C–containing macrophages result in an autoamplification feedback by locally increasing the amount of renal PDGF-C.
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