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Prevention of renal vascular and glomerular fibrosis by epidermal growth factor receptor inhibition ¹

INSERM U.489, Hôpital Tenon, and
^* AP-HP, Laboratoire de Physiologie, UFR St. Antoine, Paris, France

²Correspondence: INSERM U.489, Paris 75020, France. E-mail: christos.chatziantoniou{at}tnn.ap-hop-paris.fr

SPECIFIC AIMS

Hypertension is often associated with the development of renal vascular and glomerular fibrosis, a pathological process characterized by abnormal accumulation of extracellular matrix (mainly collagens) within the renal vasculature. The purpose of the present study was to investigate whether epidermal growth factor receptor (EGFR) activation participates in the development of renal fibrosis and to test whether blockade of EGFR activation would have therapeutic effects.

PRINCIPAL FINDINGS

1. EGFR activation in renal cortex during NO deficiency
These experiments were performed in a model of hypertension-induced chronic renal failure, the NO deficiency model. NO synthesis was inhibited by administrating a NO synthase inhibitor, L-NAME. In this model, 4–6 wk of L-NAME treatment are enough to develop renal vascular and glomerular fibrosis in rats.

Systolic blood pressure rose after 4 wk of L-NAME treatment (P<0.01, Fig. 1 , upper panel). To test the status of activation of the EGFR, Western blot was performed in renal cortical slices freshly isolated from control and L-NAME-treated rats. As shown in Fig. 1 , lower panel, the level of phosphorylation of the EGFR is negligible in control animals but clearly appears during NO deficiency.

View larger version (43K): [in this window] [in a new window]   Figure 1. Top: systolic blood pressure from controls and rats treated with L-NAME, with L-NAME + gefitinib, or gefitinib alone for 4 wk. Values are mean ± SE of 15 animals in all groups except gefitinib alone (n=10) . *P < 0.01 vs. control. Bottom: EGFR activation during NO inhibition, evaluated by Western blot, in renal cortical slices.

Hypertension induced by NO deficiency is accompanied by severe fibrotic lesions in glomeruli and renal vessels shown by the intense green staining indicative of abnormal extracellular matrix accumulation (Fig. 2 B) and an increased index of glomerulosclerosis (2.80±0.37 vs. 0.20±0.12 in L-NAME 4 wk and control, respectively, P<0.001). Renal function deteriorated as indicated by the abnormal increase of urinary protein excretion (1.65±0.24 vs. 0.40±0.09 mg/mol, P<0.01) and elevation of plasma creatinine concentration, (72±5 vs. 40±2 µmol/L, P<0.01).

View larger version (122K): [in this window] [in a new window]   Figure 2. Representative examples of extracellular matrix staining by Masson trichromic solution in controls (A) and rats treated with either L-NAME (B), L-NAME + gefitinib (C), or gefitinib alone (D) for 4 wk. Note intense green staining indicative of extracellular matrix during inhibition of NO (B); development of glomerular fibrosis was prevented during inhibition of the EGFR (C). Bar = 10 µm.

2. Effect of EGFR inhibition on the L-NAME-induced alterations of renal function and structure
To investigate whether EGFR activation was involved in the development of this fibrotic process, an orally active EGFR inhibitor, gefitinib (Iressa), was administered concomitant with L-NAME treatment. Gefitinib administration blocked phosphorylation of EGFR in renal cortical slices (Fig. 1 , lower panel) without affecting the L-NAME-induced increase of systolic blood pressure (172±7 mmHg, Fig. 1 , upper panel). Treatment with the EGFR inhibitor prevented the development of glomerulosclerosis (Fig. 2C ; sclerotic index 1.22±0.28, P<0.01 vs. the L-NAME group). This preservation of the renal structure was accompanied by an improvement in renal function as indicated by the excretion of proteins in urine and the plasma concentration of creatinine (0.72±0.29 mg/mol, and 43±5 µmol/L, respectively, P<0.01 vs. L-NAME for both parameters).

3. EGFR-induced activation of the MAPK/ERK pathway during NO deficiency
We verified whether the MAPK/ERK cascade was activated in renal tissue during L-NAME-induced fibrosis and, if so, whether this activation was due to EGFR. Western blot analysis performed in freshly renal cortical tissue indicated a significant increase of MAPK activity in L-NAME-treated animals (167±15% of control, P<0.05 vs. control). This activation was totally blocked by concomitant treatment with the EGFR inhibitor gefitinib (96±9% of control).

4. Effect of EGFR inhibition on the increased collagen I gene expression induced by NO deficiency
The abnormal accumulation of extracellular matrix during renal fibrosis is mainly due to increased collagen synthesis (particularly collagen type I). We verified the effect of EGFR inhibition on collagen I activation. L-NAME treatment resulted in a significant increase of collage I gene expression in glomeruli (170±25% of control, P<0.01) that was completely inhibited when the EGFR inhibitor was coadministered with L-NAME (82±18%, P<0.01 vs. L-NAME).

5. EGFR as mediator of the ET-1-induced activation of collage I gene: experiments in mice
We and other investigators have demonstrated that ET-1 is a major inducer of renal fibrosis in the L-NAME model by stimulating collagen I gene in glomeruli and renal vessels. It is difficult, however, to demonstrate a straightforward interaction between ET-1, EGFR activation and establishment of fibrosis in protocols that last 28 days. For this reason, we used a transgenic strain of mice in which early changes in collagen I gene activity can be easily detected to test whether EGFR is a mediator of the fibrogenic action of ET-1 in acute experiments. These mice were chosen based on previous studies showing that the expression pattern of the luciferase reporter gene in embryos and adult animals closely correlates with tissue distribution of collagen I under physiological and/or pathological conditions.

ET-1 alone or mixed with PD153035 (another specific inhibitor of EGFR tyrosine kinase that is chemically different from gefitinib) was administered in renal cortical slices freshly isolated from transgenic mice harboring the luciferase gene under the control of collagen I promoter. ET-1 produced an almost twofold increase of luciferase activity (177±29% vs. baseline, n=8, P<0.01). PD153035 completely blocked the increase of luciferase activity induced by ET-1 (111±12% for ET-1+PD153035, P<0.05 vs. ET-1, n=8).

CONCLUSIONS AND SIGNIFICANCE

In the present study we investigated the hypothesis that the EGFR participates in the development of renal vascular fibrosis and looked at the mechanisms participating in this phenomenon. We used an experimental model of hypertension associated with renal vascular and glomerular fibrosis (model of inhibition of NO synthesis or L-NAME model). We earlier showed that in this model endothelin plays an important role in the development of renal vascular damage. Experiments were performed in two species: rats and a strain of transgenic mice harboring the luciferase gene under the control of the collagen I-2 chain promoter. We used rats to demonstrate that 1) activation of EGFR, MAPK pathway and collagen I expression accompanied the development of renal structural and functional alterations leading to renal failure, and 2) pharmacological treatment with a specific inhibitor of EGFR canceled MAPK pathway and collagen I activation, inhibited renal functional and structural alterations, and protected the animals against the development of renal vascular fibrosis and failure. Then we used the strain of transgenic animals to establish that endothelin activates collagen I gene through EGFR activation in the renal cortex. To our knowledge, this is among the first studies reporting that EGFR activation plays an important role in the development of renal vascular fibrosis and that use of its inhibitors can have a beneficial, anti-fibrotic action in vivo.

EGFR activation is considered a key event in epithelial cell proliferation. Increased activation of EGFR is involved in tumor genesis in a variety of organs. It has been proposed that EGFR could induce cellular proliferation in acute tubular necrosis and that EGFR activation is involved in the cyst formation in polycystic renal disease. Transgenic mice expressing a truncated EGFR at the basolateral membrane of proximal tubular cells displayed a reduced degree of tubulo-interstitial lesions after 5/6 nephrectomy. Our data clearly indicate that EGFR is activated in the renal cortical tissue concomitant with the development of sclerotic lesions and impairment of renal function. The cause-effect relationship is indicated by the fact that inhibition of EGFR activation prevented development of renal fibrosis and the degradation of renal function. This protection occurred independent of the elevation of systolic blood pressure. This finding is not surprising, because we recently demonstrated that angiotensin or endothelin receptor blockade canceled the activation of collagen I gene promoter in the renal vasculature and prevented the development of fibrosis independent of blood pressure levels in the L-NAME model.

The development of vascular fibrosis has been attributed to the fibrogenic action of vasoactive peptides such as angiotensin II or endothelin. Several investigators have described the efficiency of angiotensin II blockers to prevent the development of renal, vascular, and cardiac fibrosis; we recently demonstrated that these drugs induce regression of renal vascular fibrosis by inhibiting collagen synthesis. Similarly, endothelin antagonism was accompanied by reversal of vascular and renal fibrosis in the DOCA salt-, angiotensin II-, or L-NAME-induced hypertension in rodents in vivo. Although the efficiency of these treatments is well documented, the underlying mechanisms are still a matter for debate. A novel hypothesis has emerged suggesting that the trophic effects of vasoactive peptides can be mediated by activation of growth factor receptors. In earlier studies we demonstrated that the development of renal fibrosis in the L-NAME model was due to increased synthesis and/or action of endothelin locally in the renal vessels and glomeruli. To integrate our previous results with the new data, we propose that the development of renal vascular and glomerular fibrosis, at least in the L-NAME model, is due to a mechanism that starts at endothelin and leads to increased collagen I synthesis through activation of EGFR and the MAPK pathway.

In conclusion, we investigated whether activation of the EGFR is involved in the mechanism(s) controlling the development of renal vascular fibrosis. We have found that EGFR, possibly activated by endothelin, participates in the activation of collagen I and abnormal formation of extracellular matrix through a MAPK-mediated mechanism. An important novel finding is that the development of renal fibrosis was prevented by in vivo treatment with an EGF receptor inhibitor. To our knowledge this is the first in vivo study showing that blockade of the EGFR pathway displays therapeutic effects in renal fibrosis, a disease considered to be incurable. Until now, EGFR inhibitors have been considered promising anti-cancer drugs; our results suggest that they could be used to treat fibrotic diseases as well.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the proposed pivotal role EGF receptor plays in mediating renal vascular fibrosis. During chronic absence of NO, ET-1 is activated and stimulates the EGF receptor. Once the EGF receptor is phosphorylated, it activates the MAPK/ERK pathway, which in turn induces fibrosis by activating synthesis of extracellular matrix proteins such as collagen I. Use of EGF receptor inhibitors blocks this pathological process and preserves the decline of renal function and structure.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0702fje;

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