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, 24 (2), 229-42

Renal Nerves Drive Interstitial Fibrogenesis in Obstructive Nephropathy

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Renal Nerves Drive Interstitial Fibrogenesis in Obstructive Nephropathy

Jinu Kim et al. J Am Soc Nephrol.

Abstract

The signals that drive fibrogenesis after an initiating insult to the kidney are incompletely understood. Here, we report that renal nerve stimulation after ureteral obstruction is the primary profibrotic signal and that renal denervation prevents both fibrogenesis and the inflammatory cascade. Local infusion of neural factors, norepinephrine, and calcitonin gene-related peptide (CGRP) in denervated kidneys mimicked the fibrotic response observed in innervated obstructed kidneys. Norepinephrine and CGRP act through the α(2)-adrenergic receptor and CGRP receptor, respectively, because blocking these receptors prevented fibrosis, the inflammatory response, and tubular cell death. In tubular epithelial cells, both norepinephrine and CGRP induced apoptosis and the release of profibrotic factors capable of stimulating the differentiation of fibroblasts to myofibroblasts. In conclusion, these data suggest that nerve-derived signaling molecules may drive renal fibrosis and that their suppression may be a therapeutic approach to fibrosis prevention.

Figures

Figure 1.
Figure 1.
Renal denervation (DNx) prevents tubulointerstitial fibrogenesis and inflammation during UUO. Renal denervation in left kidneys of male 129S1/SvImJ mice aged 8–10 weeks was carried out; 2 days after the onset, the left ureters were obstructed for 3 or 10 days. (A) Immunohistochemistry of TH and CGRP in renal denervation or intact kidneys (n=4 in each group). (B) TH expression in renal denervation or intact kidneys using Western blot analysis (n=4 in each group). (C and D) Norepinephrine (NE) and CGRP protein level in renal denervation or intact kidneys using ELISA. (E–G) Collagen deposition using Sirius red staining (E and F) and hydroxyproline measurement (G) after UUO or sham operation in renal denervation or intact kidneys (n=4 in each sham-group, n=6 in each UUO-group). (F) The Sirius red–positive area was measured in five randomly chosen high-power (×200) fields per kidney using NIH ImageJ software. (H and I) Immunohistochemistry of α-SMA in UUO or sham kidneys after renal denervation or intact kidneys (n=4 in each sham group, n=6 in each UUO group). The visible blue color indicates nuclei stained by DAPI. (I) The α-SMA–positive area was measured in five randomly chosen high-power (×200) fields per kidney using NIH ImageJ software. (J) α-SMA, fibronectin, TGF-β, and p-Smad3 expression using Western blot analysis in UUO or sham kidneys after renal denervation or intact kidneys (n=4 in each sham group, n=6 in each UUO group). Anti–β-actin antibody served as a loading control. (K–M) Immunohistochemistry of PMN and F4/80 in UUO or sham kidneys after renal denervation or intact kidneys. (L) The number of PMN-positive neutrophil was counted in five randomly chosen high-power (×200) fields per kidney (n=4 in each sham group, n=6 in each UUO group). (M) The F4/80-positive area was measured in five randomly chosen high-power (×200) fields per kidney using NIH ImageJ software (n=4 in each group). (N) ICAM-1, TNF-α, IL-1β, IL-6, MCP-1, KC, IP-10, and TLR4 expression using Western blot analysis in UUO or sham kidneys after renal denervation or intact kidneys (n=4 in each sham group, n=6 in each UUO group). Anti–β-actin antibody served as a loading control. Scale bars, 50 μm. *P<0.05, **P<0.01, ***P<0.001 versus intact kidneys. Error bars represent SDs.
Figure 2.
Figure 2.
Norepinephrine and CGRP contribute to kidney fibrosis and inflammation during UUO. Renal denervation (DNx) in left kidneys of male 129S1/SvImJ mice aged 8–10 weeks was carried out; 2 days after the onset, norepinephrine (NE), NY, CGRP, or substance P (SP) (0, 1.2, 6, or 30 ng/kg per day) was continuously infused into kidneys via a mini-osmotic pump, and the left ureters were obstructed for 10 days. (A) Norepinephrine- and CGRP-induced collagen deposition using Sirius red staining in denervated UUO kidneys. The Sirius red–positive area was measured in five randomly chosen high-power (×200) fields per kidney using NIH ImageJ software. (B) Immunohistochemistry of α-SMA in denervated UUO kidneys after treatment. The visible blue color indicates nuclei stained by DAPI. The α-SMA–positive area was measured in five randomly chosen high-power (×200) fields per kidney using NIH ImageJ software. (C) α-SMA and p-Smad3 expression in norepinephrine- or CGRP-treated UUO kidneys using Western blot analysis. Anti–β-actin antibody served as a loading control. (D) Immunohistochemistry of PMN and F4/80 in norepinephrine- or CGRP-treated UUO kidneys. The number of PMN-positive neutrophils and the area of F4/80-positive macrophages were evaluated in five randomly chosen high-power (×200) fields per kidney. (E) ICAM-1 and TNF-α expression using Western blot analysis. Anti–β-actin antibody served as a loading control. n=6 in each group. Scale bar, 50 μm. *P<0.05, **P<0.01, ***P<0.001 versus 0 ng/kg per day. Error bars represent SDs.
Figure 3.
Figure 3.
Norepinephrine (NE) and CGRP contribute to tubular cell death in UUO kidneys. (A and B) Renal denervation (DNx) or intact in left kidneys of male 129S1/SvImJ mice aged 8–10 weeks was carried out; 2 days after the onset, the left ureters were obstructed for 0, 1, 3, 6, 12, 24, 72, or 240 hours. (A) The kidney norepinephrine and CGRP protein levels after UUO using ELISA (n=6 in each group). **P<0.01 versus 0 hours, ##P<0.01 versus intact kidneys. (B) Immunohistochemistry of CGRP and TUNEL assay of apoptotic cells in UUO kidneys after renal denervation or intact kidneys (n=6 in each group). The visible blue color indicates nuclei stained by DAPI. (C and D) Renal denervation in left kidneys of male 129S1/SvImJ mice aged 8–10 weeks was carried out; 2 days after the onset, norepinephrine, NY, CGRP, substance P (SP) (30 ng/kg per day), or vehicle (0.9% saline) was continuously infused into kidneys via a mini-osmotic pump, and the left ureters were obstructed for 10 d (n=6 in each group). (C) Kidneys were fixed for PAS staining to measure the histologic damage in tubules. TUNEL assay to detect apoptotic cells was done using In Situ Cell Death Detection kit. The visible blue color indicates nuclei stained by DAPI. The number of TUNEL-positive apoptotic cells in tubules was counted in five randomly chosen high-power (×200) fields per kidney. *P<0.05, **P<0.01 versus vehicle. (D) PARP1, cleaved PARP1, and cleaved caspase-3 expression using Western blot analysis. Anti–β-actin antibody served as a loading control. *P<0.05, **P<0.01 versus vehicle. (E–G) MCT cells were maintained in DMEM/high-glucose medium containing 10% FBS at 37°C with 5% CO2. The cells were grown until 70% confluence on culture plates and then changed to serum-free medium. After serum starvation for 6 hours, norepinephrine or CGRP was incubated to the culture at a final concentration of 1 nM in PBS (vehicle) (n=4 in each group). A pan-caspase inhibitor Q-VD-OPH was also added at the same time at a final concentration of 20 μM in DMSO (n=4 in each group). (E) TUNEL and PI staining in MCT cells treated with norepinephrine, CGRP, or vehicle. The visible blue color indicates nuclei stained by DAPI. The number of TUNEL-positive apoptotic or PI-positive necrotic cells was counted in five randomly chosen high-power (×200) fields per plate. *P<0.05, **P<0.01, ***P<0.001 versus vehicle. (F) PARP1, cleaved PARP1, and cleaved caspase-3 expression in MCT cells treated with norepinephrine, CGRP, or vehicle using Western blot analysis. Anti–β-actin antibody served as a loading control. **P<0.01, ***P<0.001 versus vehicle. (G) The number of TUNEL-positive apoptotic or PI-positive necrotic cells cotreated with Q-VD-OPh plus norepinephrine, CGRP, or vehicle was counted in five randomly chosen high-power (×200) fields per plate. **P<0.01, ***P<0.001 versus DMSO. Scale bar, 50 μm. Error bars represent SDs.
Figure 4.
Figure 4.
Norepinephrine (NE) and CGRP upregulate TGF-β and CTGF in tubule epithelial cells. (A and B) MCT cells were grown until 70% confluence on culture plates and then changed to serum-free medium. After serum starvation for 6 hours, norepinephrine or CGRP was incubated to the culture at a final concentration of 1 nM in PBS (vehicle) for 48 hours (n=4 in each group). (A) TGF-β, CTGF, p-Smad3, α-SMA, and fibronectin expression in MCT cells treated with norepinephrine, CGRP, or vehicle using Western blot analysis. Anti–β-actin antibody served as a loading control. (B) The released level of TGF-β1 and CTGF in the supernatant of MCT cells treated with norepinephrine, CGRP, or vehicle for 48 hours using ELISA and Western blot analysis, respectively. (C) After 6 hours of treatment with norepinephrine, CGRP, or vehicle in MCT cells, the culture plate was washed using PBS, changed to serum-free media, and continued for 48 hours. The conditioned medium was then collected and added to normal rat kidney interstitial fibroblast NRK-49F cells starved for 24 hours. Shown is expression of p-Smad3 and α-SMA protein in MCT cells incubated for 48 hours with the conditioned medium from norepinephrine-, CGRP- or vehicle-treated MCT cells using Western blot analysis (n=4 in each group). Anti–β-actin antibody served as a loading control. *P<0.05, **P<0.01, ***P<0.001 versus vehicle. Error bars represent SDs.
Figure 5.
Figure 5.
α2-AR antagonists reduce tubulointerstitial fibrosis and inflammation after UUO. Male 129S1/SvImJ mice aged 8–10 weeks was continuously treated with doxazosin (α1-AR antagonist, 12 mg/kg per day), atipamezole (α2-AR antagonist, 2.4 mg/kg per day), metoprolol (β1-AR antagonist, 12 mg/kg per day), ICI118551 (β2-AR antagonist, 2.4 mg/kg per day), L748337 (β3-AR antagonist, 2.4 mg/kg per day), BRL44408 (α2A-AR antagonist, 12 mg/kg per day), imiloxan (α2B-AR antagonist, 12 mg/kg per day), spiroxatrine (α2C-AR antagonist, 12 mg/kg per day), or vehicle (10% DMSO in PBS) via an intraperitoneal implantation of a mini-osmotic pump 24 hours before UUO (n=5). (A–C) Kidney collagen deposition was evaluated by Sirius red staining (A and B) and hydroxyproline measurement (C) in UUO kidneys treated with AR antagonists. (B) The Sirius red–positive area was measured in five randomly chosen high-power (×200) fields per kidney using NIH ImageJ software. (D) α-SMA expression using immunohistochemistry in UUO kidneys treated with AR antagonists. The visible blue color indicates nuclei stained by DAPI. (E) The α-SMA–positive area was measured in five randomly chosen high-power (×200) fields per kidney using NIH ImageJ software. (F) α-SMA and p-Smad3 expression in UUO kidneys treated with AR antagonists using Western blot analysis. Anti–β-actin antibody served as a loading control. (G) Immunohistochemistry of PMN in UUO kidneys treated with AR antagonists. (H) The number of PMN-positive neutrophil was counted in five randomly chosen high-power (×200) fields per kidney. (I) ICAM-1, TNF-α, MCP-1, and KC expression in UUO kidneys treated with AR antagonists using Western blot analysis. Anti–β-actin antibody served as a loading control. Scale bar, 50 μm. *P<0.05, **P<0.01, ***P<0.001 versus vehicle; #P<0.05 versus imiloxan. Error bars represent SDs.
Figure 6.
Figure 6.
α2-AR antagonists reduce tubular cell death and are upregulated after UUO. (A–D) Male 129S1/SvImJ mice aged 8–10 weeks were continuously treated with doxazosin (α1-AR antagonist, 12 mg/kg per day), atipamezole (α2-AR antagonist, 2.4 mg/kg per day), metoprolol (β1-AR antagonist, 12 mg/kg per day), ICI118551 (β2-AR antagonist, 2.4 mg/kg per day), L748337 (β3-AR antagonist, 2.4 mg/kg per day), BRL44408 (α2A-AR antagonist, 12 mg/kg per day), imiloxan (α2B-AR antagonist, 12 mg/kg per day), spiroxatrine (α2C-AR antagonist, 12 mg/kg per day), or vehicle (10% DMSO in PBS) via an intraperitoneal implantation of a mini-osmotic pump 24 hours before UUO (n=5). (A) Histologic damage indicated by PAS staining in UUO kidneys treated with AR antagonists. (B) The histologic damage was scored by counting the percentage of tubules. *P<0.05, **P<0.01 versus vehicle; #P<0.05 versus imiloxan. (C) TUNEL assay to detect apoptotic cells in UUO kidneys treated with AR antagonists using In Situ Cell Death Detection kit. The visible blue color indicates nuclei stained by DAPI. (D) The number of TUNEL-positive apoptotic cells in tubules was counted in five randomly chosen high-power (×200) fields per kidney. *P<0.05, **P<0.01 versus vehicle; #P<0.05 versus imiloxan. (E–G) Left ureters of male 129S1/SvImJ mice aged 8–10 weeks were obstructed for 0, 1, 3, or 10 days (n=4). (E and F) α2A-AR, α2B-AR, and α2C-AR expression using Western blot analysis. Anti–β-actin antibody served as a loading control. *P<0.05, **P<0.01 versus 0 days. (G) Immunohistochemistry of α2A-AR, α2B-AR, and α2C-AR in UUO or sham kidneys. Scale bar, 50 μm. Error bars represent SDs.
Figure 7.
Figure 7.
CGRP inhibition reduces tubulointerstitial fibrosis, inflammation, and tubular cell death during UUO. Male 129S1/SvImJ mice aged 8–10 weeks were given a CGRP receptor antagonist (CGRP or C; 120 μg/kg per day) or 0.9% saline (vehicle) via an intraperitoneal implantation of the mini-osmotic pump from 24 hours before UUO or sham operation (n=5). (A and B) Collagen deposition was evaluated by Sirius red staining (A) and hydroxyproline measurement (B) in UUO kidneys treated with CGRP receptor antagonist or vehicle. (C) α-SMA expression in UUO kidneys treated with CGRP receptor antagonist or vehicle using immunohistochemistry. The visible blue color indicates nuclei stained by DAPI. (D) α-SMA and p-Smad3 expression using Western blot analysis. Anti–β-actin antibody served as a loading control. (E) Immunohistochemistry of PMN in UUO kidneys treated with CGRP receptor antagonist or vehicle. (F) ICAM-1 and TNF-α expression in UUO kidneys treated with CGRP receptor antagonist or vehicle using Western blot analysis. Anti–β-actin antibody served as a loading control. (G) Histologic damage in UUO kidneys treated with CGRP receptor antagonist or vehicle indicated by PAS staining. TUNEL assay in UUO kidneys treated with CGRP receptor antagonist or vehicle using In Situ Cell Death Detection kit. The visible blue color indicates nuclei stained by DAPI. (H) PARP1, cleaved PARP1, and cleaved caspase-3 expression in UUO kidneys treated with CGRP receptor antagonist or vehicle using Western blot analysis. Anti–β-actin antibody served as a loading control. Scale bar, 50 μm. *P<0.05, **P<0.01, ***P<0.001 versus vehicle. Error bars represent SDs.

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