Epidermal growth factor deficiency predisposes to progressive renal disease

Abstract

Epidermal growth factor (EGF) is produced in the kidney by thick ascending limbs of the loop of Henle and by distal convoluted tubules (DCTs). Reduced urinary EGF levels have been associated with chronic kidney disease but it is not known whether physiological levels of EGF protect the kidney from progressive renal disease. Here, we show that EGF-deficient mice on a mixed genetic background had increased urinary microalbumin, and a subset of these mice developed severe progressive renal disease with azotemia that was not seen in WT or TGFα-deficient littermates with this mixed genetic background. These azotemic EGF-deficient mice developed crescentic glomerulonephritis linked to HB-EGF/EGFR hyperactivation in glomeruli, as well as attenuation of the proximal tubule brush border, distal convoluted tubule (DCT) dilatation, and kidney fibrosis associated with renal β-catenin/mTOR hyperactivation. The observation of these severe renal pathologies only in a subset of EGF-deficient mice suggests that independent segregation of strain-specific modifier alleles contributes to the severity of the renal abnormalities that only manifest when EGF is lacking. These findings link the lack of EGF to renal pathologies in the adult mammalian kidney, in support of a role of physiological levels of EGF for maintaining the function of glomeruli, proximal tubules, and DCTs. These observations suggest that diminished EGF levels predispose kidneys to progressive renal disease.

Keywords: epidermal growth factor; glomerulonephritis; kidney; renal failure; renal fibrosis.

Figure 1:. EGF deficiency leads to highly increased BUN levels in a subset of adult mice.

A. Mendelian ratios in 4-weeks old offspring from indicated crosses. Absolute numbers and percentiles are shown. Parentheses show expected Mendelian ratios. The deficiency of Egf or Tgfa does not affect the viability of these mice. B. BUN serum levels were measured in different age groups (up to 12 months of age) in WT, Egf^−/− mice, Egf^−/−Tgfa^−/WT mice, and Tgfa^−/− mice derived from the same intercrosses of Egf^−/WTTgfa^−/WT mice. Every measurement represents a different mouse (not serial measurements on the same mice). A subset of Egf^−/− mice and Egf^−/−Tgfa^−/WT mice show increased BUN levels relative to their age-matched WT or Tgfa^−/− control mice (circles with red margin). BUN levels in Tgfa^−/− mice were within the same normal range as seen in WT mice. An increased BUN value was defined as being higher than the highest BUN value in the age-matched WT and Tgfa^−/− group. For each column of Egf^−/− mice and Egf^−/−Tgfa^−/WT mice, the percentile of mice with an increased BUN is shown (%), as well as the total numbers of mice with an increased BUN of the total numbers of mice in that group (number of mice with increased BUN/total number of mice). Graph shows mean ± SEM. Below the graph, the summary indicates for Egf^−/− mice and Egf^−/−Tgfa^−/WT mice across all age groups the percentile and the total number of mice with an increased BUN relative to the total number of mice of these groups. C. Graph shows BUN values (mg/dl) in black and urinary microalbumin concentrations (mAlb in mg/l) in blue. For the Egf^−/− mouse group the percentile of mice with increased urinary microalbumin is shown (%), as well as the total numbers of mice with increased urinary microalbumin of the total numbers of mice in that group (number of mice with increased urinary microalbumin/total number of mice). An increased value was defined to be higher than the highest value in the WT and Tgfa^−/− group. A relative increase in urinary microalbumin is present in Egf^−/− mice with normal BUN serum levels. Age-matched Tgfa^−/− mice derived from the same intercrosses do not show increased urinary microalbumin compared to WT controls. All mice are ~10–13 months of age. P-values are shown for microalbumin measurements compared to the WT group (Mann-Whitney test). Graph shows mean ± SEM.

Figure 2:. Azotemic EGF-deficient mice show loss of DCT and PT differentiation markers and increased renal β-catenin/mTOR signaling activity.

Western blotting of whole kidney lysates from the following mice: WT mice with normal kidney function (1-, 2- and 11-months-old), 1 months-old Egf^−/− mice with normal kidney function, 4 months-old Egf^−/− mouse with renal failure and fibrosis (BUN 497; shown in Figures 2 and 3), 11-months-old Egf^−/− mouse with a moderate increase in BUN (56) and no renal fibrosis, 2-months-old Egf^−/WT mouse (BUN 29) and 4-months-old Egf^−/WTTgfa^−/− mouse with normal kidney function (BUN 34). The 4 months-old Egf^−/− mouse kidney with renal failure and fibrosis shows diminished protein levels of NCC and Slc3a1 (but not of NKCC2), increased active β-catenin levels and mTOR activation, with an increase in phosphorylation of 4E-BP1 at Thr37/46, p70 S6 kinase and TFEB. In contrast, the kidney of the 11-months-old Egf^−/− mouse with a moderate increase in BUN (56) and no renal fibrosis did not show these changes. Black arrowheads indicate protein size (based on protein ladder, indicated in kDa). Values show densitometric values for Western blot bands normalized to β-actin.

Figure 3:. Crescentic glomerulonephritis, renal fibrosis, and dilatation of PTs and distal nephron segments in azotemic Egf^−/− mice.

A. H&E, PAS, and Trichrome stained kidney sections from a 6-months-old WT mouse, a 4-months-old Egf^−/− mouse with renal failure (BUN 497), a 6-months-old Egf^−/−Tgfa^−/WT mouse with renal failure (BUN 224), and a 6-months-old Tgfa^−/− mouse that has normal kidney function (BUN 19). Lack of EGF leads to dilatation of distal nephron segments in the renal cortex with protein casts (green arrows), as well as atrophic tubular structures with a diminished lumen (black arrows) in Egf^−/− and Egf^−/−Tgfa^−/WT mice. Glomeruli in kidneys of azotemic Egf^−/− mice or Egf^−/−Tgfa^−/WT mice show cellular crescents in Bowman’s space (red arrows). Trichrome staining shows areas of tubulointerstitial cortical fibrosis (blue; yellow arrows) and glomerulosclerosis (blue; blue arrow) in Egf^−/− and Egf^−/−Tgfa^−/WT mice as well. These changes are not seen in WT or Tgfa^−/− mice. Scale bars 100 μm. B. Lower magnification H&E images of same Egf^−/− mouse kidney as shown in (A). Wedge-shaped cortical areas with atrophic tubules that have a diminished lumen are seen (black arrows), as well as dilated tubular segments (green arrow). Scale bar 500 μm (middle) and 250 μm (right). C. Higher magnification images of glomeruli of kidneys stained with PAS of WT, Egf^−/− and Egf^−/−Tgfa^−/WT mice shown in (A). EGF deficiency is associated with lobulated glomeruli and the formation of cellular crescents in Bowman’s space (green arrow). PAS⁺ material is also seen in glomerular capillaries in these mice (black arrows). Scale bars 50 μm. D. Toluidine blue staining of semithin plastic-embedded kidneys of WT mice and Egf^−/− mice with azotemia. Dilated distal nephron tubules are seen in Egf^−/− mice (red arrow). PTs show dilatation and a diminished apical brush border membrane in Egf^−/− mice as well (black arrow), which is not seen in WT littermates (orange arrow). Scale bars 50 μm.

Figure 4:. Crescentic glomerulonephritis associated with HB-EGF/EGFR hyperactivation in azotemic EGF-deficient mice.

A. Left: Co-immunolabeling for smooth muscle actin (SMA) and the endothelial cell marker CD31 (left panel) shows glomerulosclerosis (yellow arrow) and cellular crescents with strongly SMA⁺ parietal Bowman’s capsule in azotemic Egf^−/−Tgfa^−/WT mice (white arrow). Bowman’s capsule does not show SMA immunoreactivity in age-matched WT mice. Right: F4/80⁺ macrophages strongly accumulate at sites of periglomerular fibrosis and the tubulointerstitial space in these mice as well, whereas this macrophage accumulation is not seen in the kidneys of WT mice (NCC in green labels DCTs). B. Left: C3 immunolabeling (green; yellow arrow) is detected in dilated glomerular capillaries in azotemic Egf^−/−Tgfa^−/WT mice but not in WT mice. Right: Glomerular IgG deposits (yellow arrows) are seen in azotemic Egf^−/−Tgfa^−/WT mice. C. CD45 immunolabeling shows an extensive leukocytic cellular infiltrate in the tubulointerstitial space, the periglomerular area and also within glomeruli in azotemic Egf^−/− mice and Egf^−/−Tgfa^−/WT mice but not in WT controls (yellow arrows). D. Left: CD3⁺ T-cells (yellow arrows) are seen in glomeruli of azotemic Egf^−/−Tgfa^−/WT mice but not in glomeruli of WT mice. Right: No cleaved caspase 3⁺ cells (apoptosis marker) are observed in glomeruli of WT or Egf^−/−Tgfa^−/WT mice. E. Left: HB-EGF immunolabeling shows increased HB-EGF in glomerular cells adjacent to crescents (yellow arrows) in EGF-deficient mice. Right: An antibody against phosphorylated EGFR (Tyr1068) that is associated with activation of EGFR signaling activity shows strongly increased EGFR activation in crescents of glomeruli in EGF-deficient mice (yellow arrows). WT controls do not show this increase in HB-EGF and EGFR (Tyr1068) phosphorylation. Scale bars 50 μm. Kidney sections from a 6-months-old WT mouse, a 4-months-old Egf^−/− mouse with renal failure (BUN 497), and a 6-months-old Egf^−/−Tgfa^−/WT mouse with renal failure (BUN 224), as shown in Figure 2.

Figure 5:. EGF-deficient mice with renal insufficiency show PT and DCT defects.

A. Lotus Tetragonolobus Lectin (LTL, in green) and phalloidin (in white) label the apical brush border membrane of PTs (red arrow). Dilatation of PTs and an attenuation of the brush border membrane are observed in azotemic EGF-deficient mice (yellow arrows). Scale bars 50 μm. B. Immunolabeling of the 4 months-old Egf^−/− mouse kidney with renal failure and fibrosis (shown in A and B) demonstrates increased cytoplasmic TFEB labeling in distal nephron segments (yellow arrow) compared to EGF-expressing distal nephron segments (white arrow) in WT littermates. Scale bars 50 μm.

Figure 6:. EGF-deficient mice with renal insufficiency show dilatation of DCTs and CTs and periglomerular fibrosis.

Immunolabeling of a 4 months-old Egf^−/− mouse kidney with renal failure and fibrosis shows dilated DCTs (Pvalb⁺NCC⁺) (yellow arrows) and CTs/cortical CDs (Aqp2⁺) (blue arrow), increased macrophage infiltration (F4/80⁺), and fibrosis with SMA⁺ myofibroblasts particularly in a periglomerular distribution (orange arrows), whereas TALs (NKCC2⁺) are not dilated (red arrow). These changes are not seen in a WT age-matched littermate. PNA (rhodamine-conjugated peanut agglutinin) labels distal nephron epithelial cells, whereas contiguous red labeling is seen in PTs. Scale bars 100 μm.