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. 2012 Apr;180(4):1441-53.
doi: 10.1016/j.ajpath.2011.12.039. Epub 2012 Feb 17.

Hedgehog-Gli pathway activation during kidney fibrosis

Steven L Fabian  1 , Radostin R Penchev, Benoit St-Jacques, Anjali N Rao, Petra Sipilä, Kip A West, Andrew P McMahon, Benjamin D Humphreys
Affiliations

Hedgehog-Gli pathway activation during kidney fibrosis

Steven L Fabian et al. Am J Pathol. 2012 Apr.

Abstract

The Hedgehog (Hh) signaling pathway regulates tissue patterning during development, including patterning and growth of limbs and face, but whether Hh signaling plays a role in adult kidney remains undefined. In this study, using a panel of hedgehog-reporter mice, we show that the two Hh ligands (Indian hedgehog and sonic hedgehog ligands) are expressed in tubular epithelial cells. We report that the Hh effectors (Gli1 and Gli2) are expressed exclusively in adjacent platelet-derived growth factor receptor-β-positive interstitial pericytes and perivascular fibroblasts, suggesting a paracrine signaling loop. In two models of renal fibrosis, Indian Hh ligand was upregulated with a dramatic activation of downstream Gli effector expression. Hh-responsive Gli1-positive interstitial cells underwent 11-fold proliferative expansion during fibrosis, and both Gli1- and Gli2-positive cells differentiated into α-smooth muscle actin-positive myofibroblasts. In the pericyte-like cell line 10T1/2, hedgehog ligand triggered cell proliferation, suggesting a possible role for this pathway in the regulation of cell cycle progression of myofibroblast progenitors during the development of renal fibrosis. The hedgehog antagonist IPI-926 abolished Gli1 induction in vivo but did not decrease kidney fibrosis. However, the transcriptional induction of Gli2 was unaffected by IPI-926, suggesting the existence of smoothened-independent Gli activation in this model. This study is the first detailed description of paracrine hedgehog signaling in adult kidney, which indicates a possible role for hedgehog-Gli signaling in fibrotic chronic kidney disease.

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Figures

Figure 1
Figure 1
Expression pattern of the Hedgehog (Hh) pathway in the mouse kidney. Reporter mice sonic hedgehog (Shh)-GFPCre; R26-LacZ Ihh-nLacZ, Ptch1-nLacZ, Gli1-nLacZ, and Gli2-nLacZ adult mice), in situ hybridization, and quantitative PCR were used to measure and localize the Hh pathway expression. In situ results are from P1 kidney; reporter mouse and quantitative PCR are from the adult. A: Shh is observed only in the papilla (p) and ureter (arrow). Ihh is expressed in inner cortex and medulla (m), and Ptch1 is expressed in cortex (c), medulla, and papilla. Both Indian hedgehog (Ihh) and Patched1 (Ptch1) exhibit prominent staining at the corticomedullary junction. B: Gli1 and Gli2 are expressed in cortex, medulla, and papilla though Gli1, similar to Ihh and Ptch1, was highest at the corticomedullary junction, whereas Gli2 was highest in the inner medulla and papilla. C–E: Quantitative PCR confirms the differences in regional expression for all Hh pathway members: Shh, Ihh, desert hedgehog (Dhh) (C); Ptch1 (D); Gli1, GLi2, Gli3 (E). N = 3 mice throughout. *P < 0.05, **P < 0.005, and ***P < 0.001 by analysis of variance followed by Tukey's post test. Dotted lines mark corticomedullary and medullopapillary junctions. Scale bar = 50 μm.
Figure 2
Figure 2
Hedgehog (Hh) ligands are expressed in tubular epithelial cells, and intersititial cells respond to Hh ligands. High-powered images of LacZ expression and in situ hybridization reveal that sonic hedgehog (Shh) and Indian hedgehog (Ihh) expression is restricted to tubular epithelial cells. A: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining of Shh-GFPCre; R26-LacZ kidney with subsequent immunofluorescent detection of aquaporin 2 (AQP2) demonstrates that Shh is expressed in the collecting duct. B: In the outer medulla, Ihh-positive cells have a cuboidal morphology (arrows) and co-stain with the proximal tubular marker Lotus tetragonolobus lectin (LTL). C: Patched1 (Ptch1)-nLacZ expression is present in the glomerulus (g), tubules (t), endothelium (arrows), and interstitium (arrowheads), whereas Gli1-nLacZ and Gli2-nLacZ expression was restricted to interstitial cells. Ptch1, Gli1 and Gli2-positive cells were enriched in the perivascular space around arteries and arterioles (lower three panels). Scale bar = 50 μm. nLacZ, nuclear lacZ.
Figure 3
Figure 3
Expression of Gli2, but not Gli1, occurs in the epithelial cells of the ureteric bud in the nephrogenic zone of developing kidneys. A: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining of kidneys from Gli1-nLacZ mice is exclusively interstitial and does not reveal staining of ureteric bud epithelium (arrow) in P0 and P7 mice. B: X-gal staining of kidneys from Gli2-nLacZ mice reveals staining of ureteric bud epithelium (arrows) in the outer cortex in newborn mice (P0). X-gal staining in epithelial cells is decreased (arrows) in kidneys from 7-day-old mice (P7) and is almost completely absent in kidneys from 14-day-old mice (P14). Scale bar = 50 μm. nLacz, nuclear LacZ.
Figure 4
Figure 4
The Hedgehog (Hh) pathway is activated in two different models of kidney fibrosis. A–D: Corticomedullary lysates were obtained on days 3, 7, and 14 after unilateral ureteral obstruction (UUO) or sham surgery, and mRNA expression was normalized to glyceraldehydes-3-phosphate dehydrogenase (GADPH). Collagen 1α1 (Col1α1) and α-smooth muscle actin (α-SMA) (A) increased during UUO, confirming fibrosis. Patched1 (Ptch1) (B) increased slightly by day 14 of UUO. Gli1 and Gli3 levels (C) progressively rose throughout UUO, and Gli2 levels increased on days 7 and 14. Indian hedgehog (Ihh) (D) significantly increased after UUO, peaking at day 3, whereas sonic hedgehog (Shh) and desert hedgehog (Dhh) were minimally expressed. N = 5 UUO and 5 sham mice per time point. E–H: Medullary lysates for fibrotic markers, Gli1, Gli2, Gli3, and Ptch1, and cortical lysates for Ihh were obtained on days 3, 7, 14, and 28 after ischemia reperfusion injury (UIRI), or day 3 after sham surgery. mRNA expression was normalized to GAPDH. Similar to the peak increase of Col1α1 and α-SMA at day 7 (E), Gli1, Gli2, Gli3 (G), Ihh (H), and to a lesser degree Ptch1 (F), increase after UIRI with a peak at day 7. Gli1 also exhibits a second peak on day 28. N = 4 mice for UIRI days 1, 3, 14, and 28, and N = 3 mice for UIRI day 7 and sham. *P < 0.05, **P < 0.005, and ***P < 0.001 by analysis of variance followed by Dunnett's post test.
Figure 5
Figure 5
During unilateral ureteral obstruction (UUO) the number of Gli1, Gli2, and Patched1 (Ptch1)-expressing cells increases. A: Low-power images of cortex from 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining of kidney sections from Gli1-nLacZ mice show a progressive increase in X-gal staining with increasing duration of UUO. B: Higher-power images of boxed areas from A demonstrate that Gli-nLacZ-positive cells remain exclusively interstitial. C: Quantification of the number of Gli1-nLacZ-positive cells in the kidney cortex shows a substantial increase during UUO. D: X-gal staining for Gli2 and Ptch1 shows an increase in the number of X-gal-positive interstitial cells in 7-day UUO kidneys compared to uninjured kidneys. X-gal staining for Indian hedgehog (Ihh) expression remains restricted to tubules in both uninjured and UUO kidneys. E: The number of Gli2-expressing cells per 200× field of view increases in the cortex and medulla, although the increase in the cortex is not as dramatic as that seen for Gli1. Ptch1-expressing cells per 100× field of view of the cortex increase in the interstitium, but not the tubules. Ihh-nLacZ mice do not show an increase in the number of X-gal-positive cells per 100× field of view in the cortex during UUO. N = 4 mice per time point for Gli1 and Ptch1, and N = 3 mice per time point for Gli2 and Ihh. Scale bar = 50 μm. *P < 0.05, **P < 0.005, and ***P < 0.001 by analysis of variance followed by Dunnett's post test. CLK, contralateral kidney; nLacZ, nuclear LacZ.
Figure 6
Figure 6
Gli1, Gli2, and Patched1 (Ptch1)-positive interstitial cells co-express the pericyte marker platelet-derived growth factor receptor-β (PDGFR-β) and acquire expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in unilateral ureteral obstruction (UUO) kidneys. To determine which interstitial cell-types express Gli1, Gli2, and Ptch1 in uninjured and injured kidneys, day 7 UUO and contralateral (CLK) kidney sections from Gli1-nLacZ, Gli2-nLacZ, and Ptch1-nLacZ mice were immunofluorescently co-labeled for nLacZ and various cell-type specific markers and pictures were obtained from the inner renal cortex. nLacZ in interstitial cells from all three reporter mice colocalized with the pericyte marker PDGFR-β in both CLK (arrows) and UUO kidneys. nLacZ in interstitial cells for all three reporter mice colocalized with the myofibroblast marker α-SMA in the UUO kidneys. Gli1, Gli2, and Ptch1-expressing cells often come into close contact with cells expressing the macrophage marker F4/80 and endothelial marker CD31, although the vast majority of these cells do not co-express these markers (insets) for CLK and UUO. nLacZ, nuclear LacZ.
Figure 7
Figure 7
Gli1-expressing cells undergo increased proliferation early in unilateral ureteral obstruction (UUO), and stimulation of a pericyte-like cell line with Hedgehog (Hh) agonists induces cell proliferation in vitro. A: To determine whether proliferation of Gli1-expressing cells increases during UUO, sections from days 0, 3, and 7 UUO kidneys from Gli1-nLacZ mice were co-labeled for nLacZ and Ki-67. B: The percentage of nLacZ-positive cells co-staining for Ki-67 was determined by dividing the number of nLacZ+; Ki-67+ double positive cells (arrows) by the total number of nLacZ-positive cells in the outer medulla. Scale bar = 25 μm. N = 3 mice for days 0 and 7; N = 4 mice for day 3. A greater percentage of Gli1-nLacZ cells costained with Ki-67 at day 3 compared to days 0 and 7. C: Stimulation of 10T1/2 cells with sonic hedgehog (Shh) ligand or smoothened agonist (SAG) induces a large upregulation of Gli1 as assessed by quantitative PCR. Platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β had no effect. D, E: 10T1/2 cells labeled with propidium iodine and analyzed by fluorescence-activated cell sorting after stimulation with either Shh (D), SAG (E), or 10% fetal bovine serum (FBS) show an increase in the percentage of cells in S+G2M and a decrease in the percentage of cells in G1 compared to Cos7 control media for Shh and water vehicle control for SAG. F, G: The percentage of 10T1/2 cells positive for 5-bromo-2-deoxyuridine (BrdU) uptake increases with stimulation with Shh (F), SAG (G), or 10% FBS compared to Cos7 control media or vehicle control. N = 3 per condition. *P < 0.05, ** P < 0.005, and ***P < 0.001 by analysis of variance followed by Dunnett's post test (B and D–G) and by two-tailed Student's t-test (C). nLacZ, nuclear LacZ.
Figure 8
Figure 8
IPI-926 inhibits Hedgehog (Hh) signaling in unilateral ureteral obstruction (UUO) kidneys. A: To determine whether IPI-926 inhibits Hh signaling, IPI-926 (40 mg/kg) (N = 5) or vehicle (N = 5) was given daily by gastric lavage for 8 days to Gli1-nLacZ mice starting the day before UUO. Mice were sacrificed on UUO day 7. B: The increase in cortical 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining seen in UUO kidney sections from vehicle-treated Gli1-nLacZ mice compared to contralateral kidney (CLK) is completely abolished in IPI-926-treated mice. C: mRNA expression by quantitative PCR of Gli1, Gli2, Gli3, and Patched1 (Ptch1) from UUO and CLK kidney lysates from BALB/c mice treated in a blinded fashion with IPI-926 (N = 6) or vehicle (N = 8) shows inhibition of Gli1 expression in CLK and UUO. The increase in Gli2 and Gli3 expression in UUO compared to CLK was not inhibited by IPI-926. BALB/c mice were sacrificed on UUO day 10 and treated daily with IPI-926 40 mg/kg or vehicle for 12 days starting 2 days before surgery. *P < 0.05, **P < 0.005, and ***P < 0.001 by two-tailed Student's t-test.
Figure 9
Figure 9
IPI-926 does not decrease renal fibrosis in unilateral ureteral obstruction (UUO). A: mRNA expression by quantitative PCR of fibrotic markers in day 10 UUO corticomedullary kidney lysates from BALB/c mice did not decrease with 12 days of IPI-926 treatment (N = 6) initiated 2 days before surgery compared to vehicle treatment (N = 8). B: The increase in α-smooth muscle actin (α-SMA) protein expression in UUO compared to contralateral kidney (CLK) as determined by Western blot and quantified by integrated optical density (IOD) α-SMA/IOD glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was not inhibited by IPI-926-treatment. C and D: Trichome stains (C) of UUO day 10 kidney sections from C57BL/6 mice and blindly scored (D) for interstitial fibrosis/tublar atrophy percentage (IF/TA%) by a pathologist did not show a difference between IPI-926-treated (N = 6) and vehicle-treated groups (N = 7). C57BL/6 mice also received daily IPI-926 (40 mg/kg) or vehicle for 12 days beginning 2 days before UUO. ns, nonsignificant by two-tailed Student's t-test.
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