Sustained Activation of EGFR Triggers Renal Fibrogenesis after Acute Kidney Injury

Antibodies and Reagents

Antibodies against p-EGFR, p-STAT3, STAT3, p-Akt, Akt, and vimentin were purchased from Cell Signaling Technology (Dancers, MA). Antibody to Pax-2 was purchased from Invitrogen (Carlsbad, CA). Antibodies to EGFR, proliferating cell nuclear antigen (PCNA), GAPDH, collagen 1(A2), and fibronectin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against p-histone H3 was purchased from Abcam (Cambridge, MA). Antibody to neutrophil gelatinase-associated lipocalin (NGAL) was purchased from R&D Systems (Minneapolis, MN). Antibody to α-smooth muscle actin (α-SMA) and α-tubulin, all secondary antibodies, and all other chemicals were from Sigma-Aldrich (St. Louis, MO).

Wa-2 Mice and Unilateral I/R Model

Wa-2 mice were maintained on a C57BL/6Jei×C3H/HeSnJ background (Jackson Laboratory, Bar Harbor, ME).²⁰ The unilateral I/R model was established in 6- to 8-week-old male Wa-2 mice and their wild-type (WT) littermates, with six mice in each group. Mice were anesthetized with 75 mg/kg ketamine (i.p.) and 50 mg/kg dexmedetomidine (Dexdomitor) (i.m.). A flank incision was made on the left side; the renal arteries and veins were isolated from the surrounding tissue by blunt dissection and then occluded with a nontraumatic vascular clamp (85-g pressure; Roboz Surgical Instruments, Gaithersburg, MD) for 45 minutes at 37°C. The sham-operated kidney was used as the control, in which the renal pedicles were isolated, but no clamps were applied. At days 2, 7, 14, or 28 after surgery, animals were sacrificed and the kidneys were collected and then horizontally cut into two pieces. One piece of the kidney was fixed in formaldehyde and used for immunofluorescence, immunochemistry, and Masson's trichrome staining; another piece was snap-frozen in liquid nitrogen and used for immunoblot analysis. Because the healthy contralateral kidney can complement the function of the injured kidney, there is usually no azotemia in this model. Thus, renal function was not measured.

Tubular Injury

Tubular injury was scored on a scale from 0 to 3, where 0 = normal, 1 = injury <30%, 2 = injury 30% to 60%, 3 = injury >60%. To detect cell death, kidneys were removed at day 2 after I/R injury. The TUNEL staining was performed according to the protocol provided by Roche Molecular System (Branchburg, NJ).

Immunoblot Analysis

Immunoblot analysis for tissue samples was performed according to our previous protocols.²¹ The densitometry analysis of immunoblot results was conducted with ImageJ software version 1.45 (NIH, Bethesda, MD).

Immunohistochemical and Immunofluorescent Staining

Immunofluorescence staining was performed according to the procedure described in our previous studies.²¹ Renal tissues were fixed in 4.5% buffered formalin, dehydrated, and then embedded in paraffin. For immunofluorescent staining, the tissue sections were rehydrated and labeled with antibodies, including primary antibodies Pax-2 (1:50), PCNA (1:50), p-EGFR (1:50), α-SMA (1:200), p-histone H3 (1:250), p-STAT3 (1:100), and p-Akt (1:100), and then exposed to fluorescein isothiocyanate or Texas Red–labeled secondary antibodies (Invitrogen). PAS staining was performed in the Department of Pathology at Rhode Island Hospital. Masson's trichrome staining was performed according to the procedure described by the manufacturer (Sigma-Aldrich). For quantitative assessment of renal fibrosis, the collagen tissue area (blue color) was measured using Image Pro-Plus software version 7 (Media-Cybernetics, Silver Spring, MD) by drawing a line around the perimeter of positive staining area, and the average ratio to each microscopic field (×100) was calculated and graphed.

Statistical Analysis

All data were presented as means ± SEM for each group. Comparisons between intergroups were made using one-way analysis of variance followed by Tukey's test. Statistically significant differences were considered at P < 0.05.

Phosphorylation and Expression of EGFR in the Kidney after I/R Injury in WT and Wa-2 Mice

As an initial step toward understanding the role of EGFR in renal repair and renal fibrosis, we examined the phosphorylation and expression of EGFR in the kidney after I/R injury in Wa-2 mice and their WT littermates. To induce the severe kidney injury that is able to trigger fibrogenesis and to ensure enough survival animals (at least six) for analysis, a model of unilateral renal ischemia for 45 minutes was used. As shown in Figure 1, A–C, I/R injury induced phosphorylation of EGFR in the kidneys of WT mice, which was slightly increased at day 2, remained at similar levels at day 7, peaked at day 14, and remained elevated at 28 days. Total EGFR was also increased at day 2 and then gradually increased until 28 days after ischemia. However, the up-regulation of phosphorylated EGFR (p-EGFR) was not only due to the increased level of total EGFR because the ratio of p-EGFR to total EGFR was still increased in a time-dependent manner during the course of I/R injury (Figure 1B). In Wa-2 mice, renal p-EGFR levels were significantly inhibited at both the early (day 2) (Figure 1, D and E) and the late phase (day 28) of the injury (Supplemental Figure S1). The renal EGFR level in Wa-2 mice was not altered at day 2 (Figure 1, D and F), but was slightly reduced at day 28 after ischemic injury (Supplemental Figure S1). Immunofluorescent staining showed that p-EGFR was primarily located in renal tubular cells in the early phase (day 2) of I/R injury (Figure 1G), but it was also observed in interstitial myofibroblasts as indicated by co-localization of α-SMA and p-EGFR (Supplemental Figure S2C). Further, myofibroblasts are mostly localized in the area with severely damaged renal tubules, but rare to find in areas with less tubular damage (Supplemental Figure S2, A–C) at 2 days after I/R injury. The number of cells expressing both α-SMA and p-EGFR or α-SMA or p-EGFR alone were markedly reduced in the kidneys of Wa-2 mice (Supplemental Figure S2D). These data illustrated that the kidney exposed to severe ischemic injury led to EGFR activation, which occurs predominately in tubular cells and also in myofibroblasts in the early phase of AKI. Further, EGFR activation is required for activation of renal interstitial fibroblasts.

Reduction of EGFR Kinase Activity Potentiates the Kidney Damage in the Early Phase of I/R-Induced AKI

To determine the role of EGFR activation in I/R-induced AKI, changes in kidney morphology, apoptosis, and expression of the injury marker neutrophil gelatinase associated lipocalin (NGAL),²² were assessed at 2 days after ischemia. Tubular dilation, swelling, necrosis, luminal congestion, and hemorrhage were present in the kidneys of WT mice subjected to I/R injury, but these changes were more severe in Wa-2 mice (Figure 2A). Scoring of kidney sections for histopathological damage to the tubules showed an increase in renal damage in Wa-2 mice as compared with WT mice (Figure 2B). Sham-operated WT and Wa-2 mice incurred no tubular injury (Figure 2, A and B). The TUNEL staining showed more positivity in renal tubular cells of Wa-2 mice compared with WT mice after I/R injury (Figure 2, C and D). NGAL was not detected in control kidneys by immunoblot analysis, but its expression level was increased in the kidney of WT mice after I/R injury and further increased in the injured kidneys of Wa-2 mice (Figure 2, E and F). Immunofluorescent staining showed that NGAL expression is limited to renal proximal tubular cells (data not shown). Taken together, these data indicate that EGFR activation is necessary for renoprotection in the early phase of AKI.

EGFR Activation Is Required for Renal Tubular Cell Dedifferentiation and Proliferation in the Early Phase of I/R-Induced AKI

Fate mapping studies indicated that renal repair after AKI is carried out mainly by proliferation of dedifferentiated intrinsic renal tubular cells.²³ Although EGFR has been shown to be involved in renal tubular cell proliferation after AKI,⁷ little is currently known about the signaling mechanism that regulates renal tubular cell dedifferentiation in response to injury in vivo. Our previous studies showed that EGFR activation plays a regulatory role in primary cultures of renal tubular cell dedifferentiation after oxidant injury.²⁴ Here, we further examined whether EGFR also mediates this process in injured kidney at 2 days after ischemia. As shown in Figure 3, A and B, Pax2, a hallmark of dedifferentiation of renal epithelial cells located in nuclei,²⁵ is absent in healthy adult tubules, but was re-expressed in the kidneys of WT mice after I/R injury. Its expression was markedly decreased in the kidneys of Wa-2 mice. Similarly, vimentin, another marker of renal tubular cell dedifferentiation, was also re-expressed in the kidneys of WT mice subjected to I/R injury and significantly decreased in Wa-2 mice (Figure 3, C and D).

Dedifferentiated renal tubular cells acquire the ability to proliferate and migrate, and proliferation of intrinsic renal epithelial cells has been recognized as the predominant mechanism of renal regeneration.²⁶ We further examined the role of EGFR in renal tubular proliferation in the injured kidney by assessing expression of PCNA, a marker of cell proliferation, at 2 days after ischemia. Immunofluorescence staining showed that PCNA was mainly expressed in the nucleus of proximal tubular cells, and the number of PCNA-positive cells was increased in the injured kidneys of WT mice but significantly decreased in Wa-2 mice (Figure 3, E and F). A very small number of PCNA-positive cells was also observed in the kidneys of WT control mice, reflecting the minimal turnover rate of renal tubular cells under physiological conditions. Western blot analysis confirmed such changes in the kidneys of WT and Wa-2 mice after I/R injury (Figure 3, G and H). Therefore, these results indicated that EGFR activation is required for renal tubular cell dedifferentiation and proliferation following I/R injury.

EGFR Activation Is Required for STAT3 and Akt Phosphorylation in the Early Phase of I/R-Induced AKI

Because activation of STAT3 and Akt signaling pathways has been reported to be associated with cell survival and proliferation,^27–30 we examined the phosphorylation status of renal STAT3 and Akt in WT and Wa-2 mice in the early time course of I/R-induced AKI. Phospho-Akt (p-Akt) and phospho-STAT3 (p-STAT3) were not detectable in sham-operated kidneys, and their levels were increased in the kidney of WT mice subjected to I/R injury. However, I/R-induced expression of p-Akt and p-STAT3 was significantly suppressed in the kidneys of Wa-2 mice (Figure 4, A, B, and D). Total STAT3 and Akt were also increased in the injured kidneys of WT mice, but remained at the same levels in the kidneys of Wa-2 mice (Figure 4, A, C, and E). These data suggest that EGFR may regulate renal regenerative responses through activation of, at least, the STAT3 and Akt signaling pathways.

EGFR Activation Contributes to Renal Fibrogenesis at the Late Phase of I/R Injury

AKI can progress to CKD,^3–5 which is characterized by activation of renal interstitial fibroblasts (as indicated by expression of α-SMA) and expression of extracellular matrix proteins (ie, fibronectin and collagen I).^31–33 We investigated whether EGFR activation is associated with renal fibrogenesis after AKI. As shown in Figure 5, A–D, basal levels of collagen I and α-SMA were detected in the kidneys of WT mice. After I/R injury, their expression levels were slightly elevated at 2 days, further increased at 7 days, and reached the maximum at 14 and 28 days (Figure 5, A–C). Fibronectin was not detectable in control kidneys. After I/R injury, its expression was induced at 14 days and continuously elevated up to 28 days after I/R injury in the kidney of WT mice (Figure 5, A and D). By contrast, the expression level of fibronectin was significantly reduced, and collagen I and α-SMA were suppressed to basal levels at 28 days in Wa-2 mice subjected to chronic I/R injury (Figure 5, E–H). Consistent with those observations, I/R injury also induced expression of interstitial collagen fibrils in the kidney of WT mice at 14 and 28 days, which was reduced in Wa-2 mice as demonstrated by Masson's trichrome staining (Figure 6, A and B, and Supplemental Figure S3).

Transformation of interstitial fibroblasts into α-SMA–positive myofibroblasts is the main mechanism for production of extracellular matrix proteins in the evolution of CKD.^32,34 We therefore examined localization and expression of α-SMA and p-EGFR in the kidneys of both WT and Wa-2 mice at 28 days after I/R injury. p-EGFR was colocalized with most of the α-SMA-positive cells (Figure 6, C and D) and also expressed in the renal tubular cells of the injured kidney (Supplemental Figure S1), indicating that p-EGFR is expressed in both renal interstitial myofibroblasts and tubular epithelial cells in the late phase of ischemic injury. Taken together with diminished expression of α-SMA, collagen I, and fibronectin in the injured kidneys of Wa-2 mice, this suggests that EGFR plays a critical role in mediating activation of renal interstitial myofibroblasts and renal fibrogenesis after prolonged I/R injury.

EGFR Activation Is Required for Expression of PCNA in Renal Myofibroblasts after I/R Injury

Another feature of renal fibrogenesis is proliferation of renal interstitial myofibroblasts. Because EGFR activation has been demonstrated to be associated with proliferation of cultured renal fibroblasts in vitro,¹³ we here examined whether EGFR activation also contributes to their proliferation in vivo after I/R injury by measuring expression of PCNA. Western blot analysis indicated that PCNA was barely detectable in the kidney of the sham-operated WT mice; I/R injury induced its expression, and none was detected in Wa-2 mice (Figure 7, A and B). Consistent with this observation, immunostaining showed a large number of PCNA-positive cells in the kidneys of WT mice after I/R injury, and a smaller number of PCNA-positive cells was in the kidney of Wa-2 mice. In the early phase of AKI, we observed that PCNA is mostly expressed in the nucleus of tubular cells (Figure 3E). In contrast to this, PCNA was mainly expressed in the cytoplasm of interstitial renal myofibroblasts 28 days after I/R injury, which is indicated by its colocalization with α-SMA (Figure 7, C and D). To confirm the localization of PCNA in renal interstitial myofibroblasts, cultured NRK-49F cells were also stained with antibodies against PCNA, α-SMA, and DAPI. As shown in Supplemental Figure S4, PCNA is localized in the cytoplasm of NRK-49F cells. Because it has been reported that PCNA expressed in the cytoplasm of neutrophils promotes cell survival,^35,36 the same location of PCNA in renal interstitial fibroblasts may also play a protective role in addition to its growth promoting effects (see Discussion).

EGFR Activity Contributes to Renal Epithelial Cell Arrest in the G2/M Phase of Cell Cycle after I/R Injury

It has been shown that renal tubular cells arrested in the G2/M phase demonstrate a prominent profibrotic phenotype and result in increased production of profibrotic growth factors/cytokines.³⁷ Phosphorylation of histone H3 (p-H3) at serine 10 is a hallmark of cells in G2/M phase.³⁸ To understand the role of EGFR in this process, we examined the expression of p-H3 by immunoblot analysis and immunostaining in WT and Wa-2 mice subjected to I/R injury. As shown in Figure 8, A and B, a large number of renal tubular cells labeled with p-H3 were detected in WT mice at 28 days after I/R injury; the number of p-H3–positive cells was significantly decreased in the injured kidney of Wa-2 mice. Consistent with this observation, immunoblot analysis showed that p-H3 was barely observed in sham-operated mice, but its expression was increased in the kidney of WT mice after I/R injury and decreased in Wa-2 mice (Figure 8, C and D). Thus, activation of EGFR is essential for driving renal epithelial cell cycle to arrest at G2/M phase after I/R injury.

EGFR Mediates the Phosphorylation of STAT3 and Akt after Prolonged I/R Injury

STAT3 has been shown to be activated in the obstructed kidney and is associated with the development and progression of renal fibrosis in a murine model of UUO.^21,34 The Akt pathway was also shown to mediate HB-EGF–induced heart fibrosis and proliferation of cardiac fibroblasts.³⁹ Thus, we examined whether EGFR also mediates the activation of these signaling pathways at the later stage of I/R injury. As shown in Figures 9 and 10, phosphorylated levels of STAT3 and Akt were significantly increased at 28 days after I/R injury in WT mice. By contrast, phosphorylated STAT3 and Akt levels were diminished in the injured kidney of Wa-2 mice. Immunofluorescent co-staining of p-STAT3 and α-SMA indicated that p-STAT3 was mainly expressed in renal interstitial myofibroblasts (Figure 9D), which was consistent with its localization in UUO-injured kidneys.³⁴ Further, co-staining of p-Akt with α-SMA was also observed in the injured kidneys of WT mice (Figure 10D). Expression levels of both were markedly reduced in the kidneys of Wa-2 mice (Figures 9 and 10). These data, combined with localization of p-EGFR in renal myofibroblasts at the later stage of AKI, suggest that EGFR may mediate activation and proliferation of renal myofibroblasts through activation of STAT3 and Akt signaling pathways.