Renal Nerves Drive Interstitial Fibrogenesis in Obstructive Nephropathy

BASIC RESEARCH www.jasn.org

† Jinu Kim* and Babu J. Padanilam*

*Department of Cellular and Integrative Physiology and †Department of Internal Medicine, Section of Nephrology, University of Nebraska Medical Center, Omaha, Nebraska

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 a2-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.

J Am Soc Nephrol 24: 229–242, 2013. doi: 10.1681/ASN.2012070678

Fibrosis is responsible for chronic tissue injury in inflammation and the immune response.9 During tis- various organs. The histopathology of tubulointer- sue injury, neuropeptides are released by tissue pe- stitial fibrosis in CKD is characterized by loss of ripheral nerve terminals and have local functions, tubular cells, influx of leukocytes, and deposition of such as neurogenic inflammation.10 Although renal extracellular matrix consequent to myofibroblast sympathetic and sensory nerves are important ef- accumulation.1,2 During renal fibrosis, the tubular fectors of renal dysfunction in CKD,11,12 their role cell loss occurs mainly via apoptotic cell death.3 The in the development of renal injury and tubuloin- injured tubular cells release cytokines that generate terstitial fibrosis remains poorly defined. The kidney inflammatory and fibrotic responses.3 Although fi- is innervated by efferent sympathetic nerves as brogenesis is the final common pathway that leads well as peptidergic sensory afferent nerves, in which to ESRD, there is little information available regard- several neuroactive substances have been iden- ing the primary signals that drive fibrogenesis. Several tified.13 Given the pronounced effect of the renal profibrogenic factors, including TGF-b and connec- nerves on CKD, we sought to determine whether af- tive tissue growth factor (CTGF), are well established ferent and efferent nerve-derived neuropeptides/ inducers of fibrosis via stimulation of myofibroblast neurotransmitters and their signaling pathways are differentiation and extracellular matrix production in multiple organs.4–6 These factors are upregulated in both tubular and tubulointerstitial cells after kidney Received July 10, 2012. Accepted October 24, 2012. 7,8 injury, resulting in kidney fibrogenesis; however, Published online ahead of print. Publication date available at the primary signaling stimuli that induce the gener- www.jasn.org. fi ation of pro brogenic factors in the injured tissue Correspondence: Dr. Babu J. Padanilam, Department of Cellular have not been identified. and Integrative Physiology, 985850, Nebraska Medical Center, Sympathetic efferent and primary sensory nerves Omaha, Nebraska 68198-5850. Email: [email protected] are key players in the regulation of peripheral Copyright © 2013 by the American Society of Nephrology

J Am Soc Nephrol 24: 229–242, 2013 ISSN : 1046-6673/2402-229 229 BASIC RESEARCH www.jasn.org

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 (3200) fields per kidney using NIH ImageJ software. (H and I) Immunohistochemistry of a-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 a-SMA–positive area was measured in five randomly chosen high-power (3200) fields per kidney using NIH ImageJ software. (J) a-SMA, fibronectin, TGF-b, 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–b-actin antibody served as a loading control. (K–M) Immunohistochemistry of PMN and F4/80 in UUO or

230 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 229–242, 2013 www.jasn.org BASIC RESEARCH responsible for the fibrotic and inflammatory responses in neuropeptide Y (NY), CGRP,or substance P.Ten days after UUO, CKD. the denervated kidneys treated with either norepinephrine or CGRP showed a dose-dependent induction of collagen deposition; however, the collagen deposition was unaltered by NY RESULTS and substance P (Figure 2A and Supplemental Figure 2A). The a-SMA–positive area, a-SMA expression, and p-Smad3 level Renal Denervation Prevents Fibrogenesis were also increased by norepinephrine or CGRP treatment of fl and In ammation denervated UUO kidneys (Figure 2, B and C, and Supplemental fi We con rmed that renal denervation eliminated tyrosine hy- Figure 3B). Treatment of renal denervation kidney with norepi- – fi droxylase (TH) positive sympathetic nerve bers and calcitonin nephrine or CGRP, but not NYor substance P, led to neutrophil – fi gene-related peptide (CGRP) positive sensory nerve bers in and macrophage infiltration after UUO (Figure 2D). The expres- adventitia of intrarenal arteries (Figure 1A). Furthermore, sion of ICAM-1 and TNF-a was also increased by norepineph- renal denervation markedly decreased TH expression, norepi- rine or CGRP in denervated UUO kidneys (Figure 2E and nephrine, and CGRP levels in mouse whole kidneys (Figure 1, Supplemental Figure 2C). In contralateral uninjected kidneys, – B D). To determine the effect of renal denervation on renal however, neurotransmitters had no effect on fibrosis (Supple- fi brosis, we performed unilateral ureteral obstruction (UUO) mental Figure 2D). after renal denervation. Intact UUO kidneys showed a time- dependent increase of collagen deposition as measured by Sir- Norepinephrine and CGRP Induce Caspase-Dependent ius red–positive area and hydroxyproline level, whereas renal Tubular Cell Death denervation markedly reduced collagen deposition during In innervated kidneys, the norepinephrine level was not UUO (Figure 1, E–G). Immunohistochemistry demonstrated changed by UUO, but the CGRP level was significantly that tubulointerstitial expression of a-smooth muscle actin increased 1–24 hours after UUO (Figure 3A). Renal denerva- (a-SMA) was diminished by renal denervation compared tion persistently suppressed renal levels of norepinephrine and with that in intact UUO kidneys (Figure 1, H and I). The de- CGRP during UUO (Figure 3A). Twenty-four hours after nervated kidneys also reduced expressions of profibrotic UUO, immunohistochemical localization revealed CGRP proteins, including a-SMA, fibronectin, TGF-b,andphos- present at the apical and basolateral membranes of epithelial phorylated Smad3 (p-Smad3) during UUO (Figure 1J and Sup- cells in dilated or tubules positive on terminal deoxynucleo- plemental Figure 1A). Infiltration of polymorphonuclear tidyl transferase–mediated digoxigenin-deoxyuridine nick- neutrophil (PMN)–positive neutrophils and F4/80-positive end labeling (TUNEL), as well as on nerve fibers in the macrophages was increased after UUO in intact kidneys, adventitia of intrarenal arteries (Figure 3B). To assess whether whereas renal denervation inhibited the infiltration of neutro- neuropeptides/neurotransmitters are involved in the tubular phils and macrophages (Figure 1, K–M). In addition, denervated cell death, we assessed histologic damage and apoptosis. Renal kidneys showed decreased expression of proinflammatory fac- denervation lessened the histologic damage score based on tors, including intercellular adhesion molecule-1 (ICAM-1), periodic acid-Schiff (PAS) staining and the number of apo- TNF-a,IL-1b, IL-6, monocyte chemotactic protein-1 (MCP- ptotic cells assessed by TUNEL staining during UUO, com- 1), KC, interferon g–inducible protein of 10 kDa (IP-10), and pared with intact kidneys (Supplemental Figure 3, A and B). toll-like receptor 4 (TLR4) during UUO, compared with that in Additionally, renal denervation abolished the UUO-induced intact kidneys (Figure 1N and Supplemental Figure 1B). How- increase in expression of full-length poly(ADP-ribose) poly- ever, renal denervation after UUO was not renoprotective merase 1 (PARP1), an indicator of necrosis,14 as well as the against fibrosis and inflammation (Supplemental Figure 1, C expression of cleaved PARP1 and cleaved caspase-3, markers and D). These data suggest that renal nerve activation is required of apoptosis (Supplemental Figure 3C). In denervated kid- to instigate fibrogenesis, but it may not play a significant role neys, the treatment with norepinephrine or CGRP, but not during the progression of the injury. NY or substance P, evoked significant histologic damage and TUNEL-positive apoptotic cell death at 10 days after UUO Norepinephrine and CGRP Contribute to Fibrosis (Figure 3C). In UUO kidneys without renal denervation, and Inflammation treatment with norepinephrine or CGRP aggravated tubular Toidentify neurotransmitters that contributeto renal fibrosis, we dilation and TUNEL-positive apoptosis (Supplemental Figure treated denervated kidneys with exogenous norepinephrine, 3D). Consistent with the data of histologic damage and sham kidneys after renal denervation or intact kidneys. (L) The number of PMN-positive neutrophil was counted in five randomly chosen high-power (3200) 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 (3200) fields per kidney using NIH ImageJ software (n=4 in each group). (N) ICAM-1, TNF-a, IL-1b,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–b-actin antibody served as a loading control. Scale bars, 50 mm. *P,0.05, **P,0.01, ***P,0.001 versus intact kidneys. Error bars represent SDs.

J Am Soc Nephrol 24: 229–242, 2013 Renal Nerve in Fibrogenesis 231 BASIC RESEARCH www.jasn.org

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 (3200) fields per kidney using NIH ImageJ software. (B) Im- munohistochemistry of a-SMA in denervated UUO kidneys after treatment. The visible blue color indicates nuclei stained by DAPI. The a-SMA–positive area was measured in five randomly chosen high-power (3200) fields per kidney using NIH ImageJ software. (C) a-SMA and p-Smad3 expression in norepinephrine- or CGRP-treated UUO kidneys using Western blot analysis. Anti–b-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 (3200) fields per kidney. (E) ICAM-1 and TNF-a expression using Western blot analysis. Anti–b-actin antibody served as a loading control. n=6 in each group. Scale bar, 50 mm. *P,0.05, **P,0.01, ***P,0.001 versus 0 ng/kg per day. Error bars represent SDs.

232 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 229–242, 2013 www.jasn.org BASIC RESEARCH

TUNEL assay, exogenous norepinephrine or CGRP increased protective effects (Figure 5, A–F, and Supplemental Figure 4A). the expression of PARP1, cleaved PARP1, and cleaved caspase- The treatment with a2-AR antagonists also decreased the influx 3 in denervated UUO kidneys (Figure 3D). Infusion of nor- of PMN-positive neutrophils and the levels of ICAM-1, TNF-a, epinephrine or CGRP, however, had no effect on systolic BP, MCP-1, and KC protein expression in UUO kidneys (Figure 5, indicating that the UUO injury in renal denervation kidneys G–I, and Supplemental Figure 4A). In addition, the histologic treated with norepinephrine or CGRP is not a consequence of damage score on PAS-stained sections and the number of apo- hemodynamic alterations (Supplemental Figure 3E). ptotic cells on TUNEL-stained sections were decreased in UUO The in vivo dataontheroleofnorepinephrineandCGRP kidneys treated with a2-AR antagonists (Figure 6, A–D). in tubular cell death are complemented by our studies in Consistent with the data of histologic damage and apoptosis, 15 mouse renal proximal tubular (MCT) cells. Treatment treatment with a2-AR antagonists diminished the expres- with norepinephrine or CGRP time-dependently induced sion of PARP1, cleaved PARP1, and cleaved caspase-3 both TUNEL-positive apoptotic cell death and propidium proteins in UUO kidneys (Supplemental Figure 4B). Treat- iodide (PI)–positive necrotic cell death; however, the increase ment with a2A-ora2C-AR antagonist was more protective in the number of TUNEL-positive cells occurred earlier than than a2B-AR antagonist (Figures 5 and 6). Norepinephrine did the increase in the number of PI-positive cells (Figure 3E). levels in kidneys from mice treated with a2-AR antagonists Cleaved PARP1 and cleaved caspase-3 levels were also in- were not altered (Supplemental Figure 4C). Furthermore, creased by norepinephrine or CGRP treatment, but PARP1 a2-ARs were upregulated and expressed on tubular cells expression was not significantly altered (Figure 3F). Cotreat- in UUO kidneys (Figure 6, E–G). In cultured renal tubular ment with pan-caspase inhibitor significantly attenuated cells, exogenous norepinephrine-induced TUNEL-positive both TUNEL-positive apoptotic and PI-positive necrotic apoptotic and PI-positive necrotic cell death was also reduced cell death induced by norepinephrine or CGRP (Figure after treatment with a2A-, a2B-, and a2C-AR antagonists 3G), indicating secondary necrosis induced by norepineph- (Supplemental Figure 5A). Furthermore, the tubular cells rine and CGRP. secreted less TGF-b1 and CTGF protein after treatment with a2A-ora2C-AR antagonists, but not a2B-AR antagonist Norepinephrine and CGRP Upregulate Profibrogenic (Supplemental Figure 5B). Growth Factors in Tubular Cells Next, we tested whether norepinephrine- or CGRP-treated CGRP Receptor Is Responsible for CGRP-Mediated tubular cells can produce profibrogenic factors. The treatment Fibrosis, Inflammation, and Tubular Cell Death with norepinephrine or CGRP for 48 hours significantly To determine whether CGRP-mediated tubulointerstitial fi- induced the expression of TGF-b, CTGF, and fibronectin in brosis is dependent on its receptor, we used a specificCGRP tubular cells, but not p-Smad3 and a-SMA expression (Figure receptor antagonist. CGRP receptor antagonist treatment 4A). TGF-b1 and CTGF levels in the incubation media were markedly inhibited collagen deposition in mouse kidneys after elevated after 48-hour exposure to norepinephrine or CGRP, 10 days of UUO, as shown by reduced Sirius red–positive area indicating release of these proteins from the cells (Figure 4B). and hydroxyproline content (Figure 7, A and B). The a-SMA– To determine whether norepinephrine- or CGRP-induced re- positive area was significantly reduced in CGRP receptor lease of profibrogenic factors from tubular cells induces dif- antagonist–treated kidneys after UUO, compared with that ferentiation of interstitial fibroblasts, we isolated conditioned in vehicle-treated UUO kidneys (Figure 7C). The a-SMA medium from the tubular cell culture and treated rat kidney and p-Smad3 expression was also lessened by treatment with interstitial fibroblasts (NRK-49F) with the conditioned me- CGRP receptor antagonist (Figure 7D). UUO kidneys treated dium. Exposure to the conditioned medium from norepi- with the CGRP antagonist also showed reduction in proin- nephrine- or CGRP-treated tubular cells significantly induced flammatory responses, as demonstrated by decreased PMN- a-SMA and p-Smad3 protein levels in interstitial fibroblasts positive neutrophil influx into kidney, ICAM-1, and TNF-a (Figure 4C). expression (Figure 7, E and F). Additionally, the CGRP receptor antagonist treatment significantly diminished induced a2-Adrenergic Receptor Is Responsible for histologic damage assessed from PAS-stained sections and Norepinephrine-Mediated Fibrosis, Inflammation, and TUNEL-positive apoptotic cell death in UUO kidneys (Figure Tubular Cell Death 7G). The effects of UUO on PARP1, cleaved PARP1, and To determine the role of adrenergic receptor (AR) in norepi- cleaved caspase-3 protein levels were reduced by CGRP recep- nephrine-mediated tubulointerstitial fibrosis, we treated mice tor antagonist treatment (Figure 7H). In tubular cell culture, undergoing UUO with a panel of AR-subtype selective the treatment with the CGRP receptor antagonist reduced the antagonists. Ten days after UUO, mice treated with a2-AR percentages of TUNEL-positive apoptotic and PI-positive ne- subtype antagonists displayed significantly reduced Sirius crotic cell death (Supplemental Figure 5C). Furthermore, the red–positive area, hydroxyproline content, a-SMA–positive CGRP receptor antagonist reduced the release of TGF-b1and area, and a-SMA and p-Smad3 expression in whole kidneys, CTGF protein from CGRP-treated tubular cells (Supplemen- whereas treatment with a1and b-AR antagonists yielded no tal Figure 5D).

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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,

234 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 229–242, 2013 www.jasn.org BASIC RESEARCH

DISCUSSION suggesting that TGF-b induced by norepinephrine or CGRP in tubular epithelial cells triggers fibrosis by driving phosphor- Interstitial fibrosis is the hallmark of CKD regardless of its ylation of Smad3 in adjacent fibroblasts. cause.1 During the past two decades, causative roles for Our data revealed that renal CGRP levels were increased inflammation, fibroblast activation, tubular and microvas- soon after UUO, with maximal levels observed during the first cular injury, and apoptosis have been established in the day. CGRP protein was evident at apical or basolateral development of tubulointerstitial fibrogenesis.16 Several membranes of epithelial cells in dilated or TUNEL-positive molecules are implicated in the progression of renal fibrosis, tubules, as well as in sensory nerve fibers. Inhibition of CGRP including various cytokines, chemokines, angiogenic fac- receptor prevented injury, whereas long-term administration tors, and growth factors and the renin-angiotensin sys- of CGRP in denervated UUO kidneys induced tubular cell tem.1,17 These molecular responses and signaling events apoptosis, inflammatory cascade, as well as fibrogenesis. In indicate the importance of a cytokine milieu and an inflam- vitro, treatment of tubular cells with CGRP also induced apo- matory cascade, including infiltration of leukocytes during ptosis, suggesting that the mechanism by which CGRP induces fibrogenesis. Despite these great strides, there remains a gap apoptosis may be due to its direct effect in tubular cells and not in the our understanding of the processes that induce these due to indirect effects, such as fibrosis or inflammation. CGRP myriad signaling events after an initial insult to the kidney.18 enhances apoptosis in thymocytes22 and inhibits apoptosis in Therefore, identification of the primary signal or the core retina cells,23 suggesting that CGRP-mediated apoptosis de- signaling pathway that instigate renal fibrogenesis after an pends on cell types. initial stimulus is essential for better understanding of the Sensory activity has been shown to be associated with syndrome and in developing effective therapeutic strategies inflammation.24 The autonomic nervous system has been im- for preventing, reversing, or limiting progression of fibro- plicated in acute and chronic inflammation of various or- genesis.19 gans.25,26 Because CGRP has potent vasodilator activity, it In cultured renal tubular cells, presence of CGRP or has long been considered to be involved in aggravation of norepinephrine induced the generation and release of TGF- inflammation by increasing the blood flow, the number of b1 and CTGF. Furthermore, CGRP and norepinephrine circulating cells, and chemotactic factors. This possibility is induced the production of extracellular matrix, including supported by in vitro data demonstrating that exposure of fibronectin, but did not induce epithelial-mesenchymal tran- endothelial cells to CGRP increases the expression of adhesion sition because no expression of a-SMA or p-Smad3 was ob- molecules for binding to neutrophils.27 Consistent with this served. One of the main mediators of fibronectin production finding, our data demonstrate that CGRP induces ICAM-1 is through TGF-b signaling;20 however, p-Smad3 was not in- expression after UUO. Although the kidney encounters vari- duced by CGRP or norepinephrine in cultured renal proximal ous immune-mediated diseases, such as GN, the role of neu- tubular cells, suggesting that autocrine signaling through roimmune interactions has not been well studied. Our data TGF-b was not activated in renal tubular cells. Fibronectin showed that inhibition of CGRP receptor prevented the ex- can probably be produced by alternate pathways, such as ac- pression of inflammatory molecules as well as infiltration of tivation of c-Jun N-terminal kinase signaling pathway.21 On leukocytes into the renal parenchyma, suggesting that neuro- the other hand, culturing of fibroblasts in the presence of the immune interactions can occur in UUO kidneys. supernatants from CGRP- or norepinephrine-treated tubular Afferent nerve activation due to an initial insult could ac- cells induced myofibroblast differentiation via p-Smad3, tivate renal efferent sympathoexcitation. Our data implicating

norepinephrine, NY, CGRP, substance P (SP) (30 ng/kg per day), or vehicle (0.9% saline) was continuously infused into kidneys via amini- 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 (3200) 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–b-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 mMinDMSO(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 (3200) 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–b-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 (3200) fields per plate. **P,0.01, ***P,0.001 versus DMSO. Scale bar, 50 mm. Error bars represent SDs.

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Figure 4. Norepinephrine (NE) and CGRP upregulate TGF-b 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-b,CTGF, p-Smad3, a-SMA, and fibronectin expression in MCT cells treated with norepinephrine, CGRP, or vehicle using Western blot analysis. Anti–b-actin antibody served as a loading control. (B) The released level of TGF-b1 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 a-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–b-actin antibody served as a loading control. *P,0.05, **P,0.01, ***P,0.001 versus vehicle. Error bars represent SDs.

norepinephrine signaling through upregulated a2-AR sub- expressed. Further studies are needed to determine whether types to induce fibrogenesis in UUO kidneys are intriguing such heterodimerization, and the resulting signaling events, because a2-AR subtypes were initially identified as feedback may explain the similar renoprotective effects of inhibiting inhibitors of neurotransmitter release in adrenergic and other a2A-ora2C-AR subtypes during UUO. neurons.28 In the mouse heart-failure model, deletion of sym- Elevated plasma norepinephrine is observed in patients 31 pathoinhibitory a2-ARs results in increased norepinephrine with CKD and ESRD. In the present study, UUO did not levels, which elicit typical cardiovascular consequences, in- significantly increase renal norepinephrine levels; however, cluding aggressive remodeling of the left ventricle, cardiac hy- inhibition of ARs prevented UUO-induced injury, whereas 29,30 pertrophy, and fibrosis. Furthermore, a2A-anda2C-ARs long-term administration of norepinephrine prompted seem to play essential roles in the prevention of heart failure injury in denervated UUO kidneys. Norepinephrine has pre- progression. In contrast, our data indicate that activation of viously been shown to induce apoptosis in neonatal cardio- the a2-AR subtypes, especially a2A-anda2C-ARs, are detri- myocytes through a reactive oxygen species/TNF-a/caspase mental in the progression of renal injury, inflammation, and signaling pathway.32 In our study, exogenous norepinephrine fibrogenesis. Inhibition of a2-ARs with their antagonists had induced TNF-a and caspase activation via the a2-AR-mediated no effect on norepinephrine levels but decreased chemokine process. On the other hand, exogenous norepinephrine has been expression in the UUO kidney, suggesting a possible mecha- reported to induce proximal tubule necrotic cell death in dogs.33 nism by which norepinephrine may promote leukocyte re- Our in vivo data indicate that norepinephrine infusion increased cruitment and inflammation. Further, activation of the PARP1 expression in the denervated kidney, supporting a pos- a2-AR subtypes on tubular cells in the UUO kidney may trig- sible explanation for the necrotic cell death observed after UUO. ger signaling events other than sympathoinhibition, such as The signaling pathway by which norepinephrine induces apo- activation of TGF-b and CTGF signaling pathways to instigate ptosis and necrosis was not delineated in this study and requires fibrogenesis. The reason for the inhibitory effects of multiple further investigation. a2-AR subtypes on UUO injury measures is not clear. It has In conclusion, this study provides incontrovertible evidence been shown that the a2C-AR can alter a2A-AR signaling by that renal nerve activation is a primary mechanism instigating heterodimerization, with these complexes acting as a func- fibrogenesis in the UUO kidney. Our data implicate both renal tional signaling unit in cells in which both subtypes are afferent nerve–derived CGRP and efferent nerve–derived

236 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 229–242, 2013 www.jasn.org BASIC RESEARCH norepinephrine in triggering tubular damage, the inflamma- Collagen Deposition tory cascade, and fibrogenesis. Inhibiting the actions CGRP or Collagen deposition was assessed by both Sirius red staining and norepinephrine, alone or in combination, might represent a hydroxyproline assay as previously described.14 The area of positive novel effective therapeutic strategy to prevent or limit progres- Sirius red staining was measured in five randomly chosen high-power sion of renal fibrogenesis at its onset in CKD. (3200) fields per kidney using National Institutes of Health (NIH) ImageJ software (http://rsb.info.nih.gov/ij/).

Kidney Norepinephrine and CGRP Levels CONCISE METHODS For measurement of kidney norepinephrine contents, the kidneys werehomogenized using 1 mM EDTAand 4mM sodium metabisulfite Mice and Surgical Preparation in 0.1 N HCl. After centrifugation of the homogenates, norepineph- Male 129S1/SvImJ mice aged 8–10 weeks were purchased from Jack- rine concentration in the supernatants was determined by ELISA son Laboratories. All mouse experiments were performed in accor- (ALPCO Diagnostics, Windham, NH), as suggested by the manufac- dance with the animal protocols approved by the Institutional Animal turer. For measurement of kidney CGRP contents, the kidneys were Care and Use Committee of the University of Nebraska Medical Cen- homogenized in 2 N acetic acid. The homogenates were then incubated ter. UUO was conducted as previously reported.14,34 Briefly, the mice at 90°C for 10 minutes and centrifuged. After extraction of the super- were anesthetized with an intraperitoneal injection of a cocktail con- natants using solid phase extraction cartridges (Waters, Milford, MA), taining ketamine (200 mg/kg body weight) and xylazine (16 mg/kg CGRP concentration in the samples was determined by ELISA (Cay- body weight). After the left kidney was exposed through the left man, Ann Arbor, MI) as suggested by the manufacturer. flank incision, the left ureter was ligated completely near the kidney pelvis using a 5-0 silk tie. Sham-operated mice underwent the same Cell Culture and Treatment surgical procedure without the ureter ligation. Renal denervation MCT cells were maintained in RPMI-1640 medium (Gibco, Grand was carried out 2 days before UUO or sham operation, as described Island, NY) supplemented with 10% FBS (Invitrogen, Carlsbad, CA) 35 fl previously. Brie y, the left kidney artery and vein were exposed at 37°C with 5% CO2. The cells were grown until 70% confluence on through the abdominal incision and isolated from the surrounding culture plates and then changed to serum-free medium. After serum connective tissue under anesthesia with ketamine and xylazine. For starvation for 6 hours, norepinephrine or CGRP was incubated for 48 strippingthenerves,thekidneyvessels were painted for 2 minutes hours to the culture at a final concentration of 1 nM in PBS (vehicle). with 95% ethanol and for 2 minutes with PBS. Intact mice under- This concentration and time were chosen after TUNEL and PI stain- went the same surgical procedure without stripping of the kidney ing in dose- and time-dependent experiments (0.1, 1, 10, or 100 nM nerves. For local treatment of neurotransmitters, norepinephrine norepinephrine or CGRP for 0, 1, 3, 6, 12, 24, or 48 hours). Pan- (Sigma, St. Louis; 1.2, 6, or 30 ng/kg body weight per day), NY caspase inhibitor Q-VD-OPh (Sigma, final concentration of 20 mM

(R&D Systems, Minneapolis, MN; 1.2, 6, or 30 ng/kg body weight in DMSO), a2A-AR antagonist BRL44408 (R&D System, final con- per day), CGRP (R&D Systems; 1.2, 6, or 30 ng/kg body weight per centration of 10 mM in DMSO), a2B-AR antagonist imiloxan (R&D day),orsubstanceP(R&DSystems;1.2,6,or30ng/kgbodyweight System, final concentration of 1 mM in DMSO), a2C-AR spiroxatrine per day) was continuously infused into the cortical region of the (R&D System, final concentration of 100 nM in DMSO), or CGRP denervated kidney via an intrathecal catheter attached to a mini- receptor antagonist CGRP8–37 (R&D System, final concentration of osmotic pump (Alzet, Palo Alto, CA) that was inserted at the same 100 nM in PBS) was also added at the same time of norepinephrine or time as UUO. The catheter was anchored to the obstructed ureter, CGRP treatment. This concentration was chosen after TUNEL and PI and the osmotic pump was placed toward the subcutaneous site. For staining in dose-dependent experiments (10 or 100 nM and 1, 10, 20, treatment with respective antagonists of AR subtypes, doxazosin or 50 mM). NRK-49F cells (American Type Culture Collection, Man-

(R&D Systems, 12 mg/kg body weight per day) against a1-AR, assas, VA) were maintained in DMEM/F-12 medium supplemented atipamezole (R&D Systems, 2.4 mg/kg body weight per day) against with 10% FBS (Invitrogen) at 37°C with 5% CO2.After6hoursof a2-AR, metoprolol (R&D Systems, 12 mg/kg body weight per day) treatment with norepinephrine or CGRP in MCT cells at a ﬁnal con- against b1-AR, ICI118551 (R&D Systems, 2.4 mg/kg body weight centration of 1 nM in PBS (vehicle), the culture plate was washed per day) against b2-AR, L748337 (R&D Systems, 2.4 mg/kg body using PBS, changed to serum-free media, and continued for 48 hours. weight per day) against b3-AR, BRL44408 (R&D Systems, 12 mg/kg The conditioned medium was then collected and added to NRK-49F body weight per day) against a2A-AR, imiloxan (R&D Systems, 12 cells starved for 24 hours. In the conditioned medium, norepineph- mg/kg body weight per day) against a2B-AR, spiroxatrine (R&D rine and CGRP levels were not detected by ELISA (ALPCO Diagnos-

Systems, 12 mg/kg body weight per day) against a2C-AR, or vehicle tics and Cayman, respectively). The dose-dependent treatment (0.1, (10% DMSO in PBS) was intraperitoneally administered via the 1, 10, or 100 nM) with norepinephrine or CGRP alone did not change mini-osmotic pump (Alzet) beginning 24 hours before UUO. For a-SMA expression in NRF-49F cells starved for 24 hours (data not inhibition of CGRP, mice were given CGRP8–37 (R&D Systems, 120 shown). The released level of TGF-b1 and CTGF in the supernatant mg/kg body weight per day) or 0.9% saline via intraperitoneal im- of MCT cells treated with norepinephrine, CGRP, or vehicle for 48 plantation of the mini-osmotic pump (Alzet) beginning 24 hours hours was examined by ELISA (BioLegend, San Diego, CA) and West- before UUO or sham operation. ern blot analysis, respectively.

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Figure 5. a2-AR antagonists reduce tubulointerstitial fibrosis and inflammation after UUO. Male 129S1/SvImJ mice aged 8–10 weeks was continuously treated with doxazosin (a1-AR antagonist, 12 mg/kg per day), atipamezole (a2-AR antagonist, 2.4 mg/kg per day), metoprolol (b1-AR antagonist, 12 mg/kg per day), ICI118551 (b2-AR antagonist, 2.4 mg/kg per day), L748337 (b3-AR antagonist, 2.4 mg/kg per day), BRL44408 (a2A-AR antagonist, 12 mg/kg per day), imiloxan (a2B-AR antagonist, 12 mg/kg per day), spiroxatrine (a2C- 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 (3200) fields per kidney using NIH ImageJ software. (D) a-SMA expression using immunohistochemistry in UUO kidneys treated with AR antagonists. The visible blue color indicates nuclei stained by DAPI. (E) The a-SMA–positive area was measured in five randomly chosen high-power (3200) fields per kidney using NIH ImageJ software. (F) a-SMA and p-Smad3 expression in UUO kidneys treated

238 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 229–242, 2013 www.jasn.org BASIC RESEARCH

Figure 6. a2-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 (a1-AR antagonist, 12 mg/kg per day), atipamezole (a2-AR antagonist, 2.4 mg/kg per day), metoprolol (b1-AR antagonist, 12 mg/kg per day), ICI118551 (b2-AR antagonist, 2.4 mg/kg per day), L748337 (b3-AR antagonist, 2.4 mg/kg per day), BRL44408 (a2A-AR antagonist, 12 mg/kg per day), imiloxan (a2B-AR antagonist, 12 mg/kg per day), spiroxatrine (a2C- 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 (3200) 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) a2A-AR, a2B-AR, and a2C-AR expression using Western blot analysis. Anti–b-actin antibody served as a loading control. *P,0.05, **P,0.01 versus 0 days. (G) Immunohistochemistry of a2A-AR, a2B-AR, and a2C-AR in UUO or sham kidneys. Scale bar, 50 mm. Error bars represent SDs.

with AR antagonists using Western blot analysis. Anti–b-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 (3200) ﬁelds per kidney. (I) ICAM-1, TNF-a, MCP-1, and KC expression in UUO kidneys treated with AR antagonists using Western blot analysis. Anti–b-actin antibody served as a loading control. Scale bar, 50 mm. *P,0.05, **P,0.01, ***P,0.001 versus vehicle; #P,0.05 versus imiloxan. Error bars represent SDs.

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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 (CGRP8–37 or C8–37;120mg/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) a-SMA expression in UUO kidneys treated with CGRP receptor antagonist or vehicle using immunohistochemistry. The visible blue color indicates nuclei stained by DAPI. (D) a-SMA and p-Smad3 expression using Western blot analysis. Anti–b-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-a expression in UUO kidneys treated with CGRP receptor antagonist or vehicle using Western blot analysis. Anti–b-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– b-actin antibody served as a loading control. Scale bar, 50 mm. *P,0.05, **P,0.01, ***P,0.001 versus vehicle. Error bars represent SDs.

Immunohistochemistry, Histology, TUNEL Assay (Santa Cruz Biotechnology, Santa Cruz, CA), a-SMA (Sigma), F4/80

Immunohistochemical staining of the kidneys was performed on (Abcam), a2A-AR (Cell Applications, San Diego, CA), a2B-AR (Santa 36–38 paraffin sections or cryosections as previously described. Briefly, Cruz Biotechnology), a2C-AR (Santa Cruz Biotechnology), or PMN 4% paraformaldehyde-fixed kidney sections were rehydrated and la- (Accurate, Westbury, NY). The sections were then incubated to fluo- beled with antibodies against TH (Abcam, Cambridge, MA), CGRP rescein or peroxidase-conjugated secondary antibodies (Vector

240 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 229–242, 2013 www.jasn.org BASIC RESEARCH

Laboratories, Burlingame, CA). The a-SMA–positive area was mea- Carmines (UNMC) for editing the manuscript. The authors also thank sured in five randomly chosen high-power (3200 magnification) fields Kelly E. Long (UNMC) for mouse care and Youngsu Cho (UNMC) for per kidney using NIH Image J software. The number of PMN-positive technical assistance with Western blot and cell culture. cells was counted in five randomly chosen high-power (3200 magni- B.J.P. is supported by a UNMC Deans fund, American Heart fication) fields per kidney. PAS-stained sections were used for histologic Association-Grant in Aid, Nebraska Research Initiative fund, and damage score as described previously.39 Histologic damage of tubular National Institutes of Health grants DK-083291 and DK-090332. injury was scored by percentage of tubules that displayed tubular necrosis, cast formation, and tubular dilation as follows: 0 = normal, 1 = ,10%, 2=10%–25%, 3=26%–50%, 4=51%–75%, and 5 = .75%. 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242 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 229–242, 2013 A Sham UUO 3 d α-SMA Fibronectin TGF-β p-Smad3 Intact DNx Intact DNx 25 Intact 12 12 25 α-SMA 20 DNx 10 10 20 8 8 15 15 Fibronectin 6 6 10 10 4 *** 4 ** ** TGF-β Expression *** ** vs. intact in sham) 5 ** 2 2 5 * d *** p-Smad3 0 0 0 0 (fol Sham 3 10 Sham 3 10 Sham 3 10 Sham 3 10 β-Actin Days during UUO

B Sham UUO 3 d ICAM-1 TNF-α IL-1β IL-6 Intact DNx Intact DNx 10 10 10 5 ICAM-1 8 8 8 4 TNF-α 6 6 6 3 4 4 ** 4 2 IL-1β ** ** * 2 * 2 * 2 1 IL-6 0 0 0 0 MCP-1 MCP-1 KC IP-10 TLR4 4 4 80 8 KC Expression

ld vs. intact in sham) 3 3 60 6 IP-10 o (f 2 2 40 * 4 TLR4 * * ** 1 1 20 2 * β-Actin *** 0 0 0 0 Sham 3 10 Sham 3 10 Sham 3 10 Sham 3 10 Days during UUO

C D UUO 15 d α-SMA Fibronec tin 25 Days of DNx after UUO 1.5 1.2 1.0 20 Intact-2 1 3 5 1.0 15 0.8 α-SMA 0.6 10 * 0.5 0.4 g/mg kidney) g/mg Fibronectin ** μ 5 ** ( Hydroxyproline 0.2 0 TGF-β 0.0 0.0 Intact-2 1 3 5 Days of DNx after UUO p-Smad3 TGF-β p-Smad3 1.5 1.5 UUO 15 d ICAM-1 1.0 1.0 TNF-α 0.5 0.5 TLR4 * * TH 0.0 0.0

β-Actin ICAM-1 TNF-α vs. intact) pression x d 121.2 151.5 E

(fol 1.0 0.8 1.0 0.6 0.4 0.5 * 0.2 * 0.0 0.0 TLR4 TH 1.5 1.2 101.0 1.0 0.8 0.6 0.5 * 0.4 0.2 ** ** ** ** 0.0 0.0 Intact -2 1 35 Intact -2 1 35 Days of DNx after UUO Days of DNx after UUO Supplemental Figure 1 UUO 15 d UUO 15 d A B α-SMA p-Smad3 ** 20 ** 10 ** 4 * ** y) g/d) * n ine kk ee 15 *** 8 3 * 6 10 * * 2 4 Expressio g/mg kidn 5 1

ld vs. 0 ng/ 2 μ ( Hydroxyprol oo

0 (f 0 0

NENY CGRP SP ICAM-1 TNF-α DNx + UUO 10 d C 5 ** 4 * * /d) ** gg 4 * ** 3 0 ng/kg/d * 3 1.2 ng/kg/d 2 2 6 ng/kg/d vs. 0 ng/k

Expression 1 30 ng/kg/d dd 1

(fol 0 0 NENY CGRP SP NENY CGRP SP DNx + UUO 10 d DNx + UUO 10 d D Contralateral uninjected kidneys 1.5 after 10 d of UUO icle) n hh Vehicle NE NY CGRP SP oo 1.0 Fibronectin Fibronectin 0.5 α-SMA

α-SMA Expressi p-Smad3

(Fold vs. ve 000.0 p-Smad3 Vehicle NE NY CGRP SP β-Actin Contralateral uninjected kidneys after 10 d of UUO Supplemental Figure 2 A PAS stain B TUNEL; DAPI stain Intact DNx 5 Intact DNx 30 4 20

l damage 3 sitive cells field aa oo 2 per 10 Sham Sham 1 ** * * * ** *** Histologic 0 TUNEL-p 0 Sham 1310 Sham 1310 Days during UUO Days during UUO

Intact DNx UUO 10 d UUO 10 d UUO 10

C Sham UUO 3 d Sham UUO 10 d PARP1 Cleaved PARP1 Cleaved caspase-3 Intact DNx Intact DNx Intact DNx Intact DNx 6 25 30 5 20 25 PARP1 4 20 Cleaved PARP1 15 3 15 10 xpression Cleaved caspase-3 . intact in sham) 2 10 ss EE ** 1 * 5 5 ** β-Actin ** ** ** 0 0 0 (fold v Sham 3 10 Sham 3 10 Sham 3 10 Days during UUO

D PAS stain E Vehicle

VhilVehicle NE CGRP ee NE 200 CGRP 20 * * 150 15 100

10 (mmHg) 50 5 UUO 10 d UUO 10 0 ystolic blood pressur ystolic Tubular dilation Tubular (% lumen area)

0 SS -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 Days after UUO 40 DNx UUO * * ± NE or CGRP 30 20 per field O 10 d O 10 10 EL-positive cells UU U 0 TUN Vehicle NE CGRP UUO 10 d Vehicle NE CGRP TUNEL; DAPI stain Supplemental Figure 3 A α-SMA p-Smad3 ICAM-1 1.5 1.5 1.5

1.0 1.0 1.0 # * # * # * 0.5 ** # 0.5 # 0.5 * # ) ** * ** e *** ** ** *** 0.0 0.0 0.0

TNF-α MCP-1 KC

Expression 1.5 1.5 1.5 (fold vs. vehicl 1.0 1.0 # 1.0 * * * * # # # 050.5 ** ** 050.5 ** 050.5 * # # *** *** *** 0.0 0.0 0.0

UUO 10 d UUO 10 d UUO 10 d

B UUO 10 d

PARP1 Cleaved PARP1 Cleaved caspase-3

β-Actin

PARP1 Cleaved PARP1 Cleaved caspase-3 1.5 1.5 1.5 hicle) ion 1.0 1.0 1.0 e s * * # * 0.5 # 0.5 # # 0.5 ** ** # # Expres ** *** *** *** ***

(fold vs. v *** 0.0 0.0 0.0

UUO 10 d UUO 10 d UUO 10 d C 4 3 2 g kidney) ney NE ney d m

Ki 1 (pg/ 0

UUO 10 d Supplemental Figure 4 A TUNEL; DAPI stain NE + vehicle NE + BRL44408 NE + Imiloxan NE + Spiroxatrine 40 30 20 * (%) -positive cells L 10 ** ** 0 TUNE 30

(%) * reatment for 48 h ositive cells 10 T p **

PI- ** 0

NE + vehicle NE + BRL44408 NE + Imiloxan NE + Spiroxatrine Treatment with NE for 48 h PI; DAPI stain B

1 0.8 1.2 β 0.6 1.0 * * 0.8 0.4

sed TGF- 0.4

(ng/ml) ** ased CTGF vs. vehicle)

a ** e 020.2 d 0.2 (fol Rel Rele 0.0 0.0 Supplemental Figure 5

Treatment with NE for 48 h Treatment with NE for 48 h

C TUNEL; DAPI stain D 8-37 CGRP CGRP + CGRP 50 1 1.0 β 40 0.8 30 0.6 * (%) 20 0.4 (ng/ml) ased TGF- L-positive cells e E 10 ** 020.2

0 Rel 0.0 TUN

40 30 1.5 20 (%) Treatment for 48 h Treatment

CTGF 1.0 positive cells RP alone) RP

- 10 d G

PI ** 0 0.5 − +−+CGRP8-37 **

− −++CGRP Release 0.0 Treatment for 48 h (fold vs. C − +−+CGRP8-37 − −++CGRP CGRP CGRP + CGRP8-37 Treatment for 48 h PI; DAPI stain SUPPLEMENTAL FIGURE LEGENDS

Supplemental Figure 1: DNx preconditioning prevents expressions in pro-fibrotic proteins and pro-inflammatory proteins. (A and B) DNx in left kidneys of male 129S1/SvImJ mice aged 8 to 10 weeks was carried out; then 2 d after the onset, the left ureters were obstructed for 3 or 10 d (n=4 in each sham-group, n=6 mice in each UUO-group). (A) Kidney levels of α-SMA, fibronectin, TGF-β and p-Smad3 protein were measured by Western blot analysis after UUO or sham-operation in DNx or intact kidneys. Anti-β-actin antibody served as a loading control. The bands were quantified using Lab Works analysis software. (B) Kidney levels of ICAM-1, TNF-α, IL-1β, IL-6, MCP-1, KC, IP-10 and TLR4 protein expression were measured by Western blot analysis after UUO or sham-operation in DNx or intact kidneys. Anti-β-actin antibody served as a loading control. The bands were quantified using Lab Works analysis software. (C and D) UUO in left ureters of male 129S1/SvImJ mice aged 8 to 10 weeks were obstructed for 15 d, and DNx was carried out 1, 3 or 5 d after UUO (n=4 in each group). (C) Collagen deposition using hydroxyproline measurement after UUO in DNx or intact kidneys (n=4 in each sham-group, n=6 in each UUO-group). (D) The Sirius red- positive area was measured in 5 randomly chosen high-power (x 200) fields per kidney using NIH ImageJ software. *P < 0.05, **P < 0.01, ***P < 0.001 versus intact. Error bars represent SD.

Supplemental Figure 2: NE and CGRP contribute to fibrosis and inflammation in UUO kidneys. (A-C) DNx in left kidneys of male 129S1/SvImJ mice aged 8 to 10 weeks was carried out; 2 d after the onset, NE, NY, CGRP or SP (0, 1.2, 6 or 30 ng/kg/d) was continuously infused into kidneys via a mini-osmotic pump; and the left ureters were obstructed for 10 d (n=6 in each group). (A) NE- and CGRP-induced collagen deposition using hydroxyproline measurement in denervated UUO kidneys. (B and C) α-SMA, p-Smad3, ICAM-1 and TNF-α expression in either NE-, NY-, CGRP- or SP-treated UUO kidneys using Western blot analysis. The bands were quantified using Lab Works analysis software. (D) Fibronectin, α-SMA and p- Smad3 expression in either NE-, NY-, CGRP- or SP-uninjected contralateral kidneys after 10 d of UUO using Western blot analysis (n=6 in each group). The bands were quantified using Lab Works analysis software. *P<0.05, **P <0.01 versus 0 ng/kg/d. Error bars represent SD.

Supplemental Figure 3: NE and CGRP contribute to tubular cell death in UUO kidneys. (A-C) DNx in left kidneys of male 129S1/SvImJ mice aged 8 to 10 weeks was carried out; then 2 d after the onset, the left ureters were obstructed for 1, 3 or 10 d (n=4 in each sham-group, n=6 in each UUO-group). (A) The kidneys were fixed for PAS staining to measure the histological damage in tubules. The histological damage in tubules on PAS-stained kidney sections was scored by counting the percentage of tubules. Five randomly chosen high-power (x 200) fields per kidney were used for the counting. (B) The kidneys were fixed for TUNEL assay to detect apoptotic cells using In Situ Cell Death Detection kit. The visible blue color 1 indicates nuclei stained by DAPI. The number of TUNEL-positive apoptotic cells in tubules was counted in 5 randomly chosen high-power (x 200) fields per kidney. (C) Kidney expression levels of PARP1, cleaved PARP1 and cleaved caspase-3 protein were measured by Western blot analysis. Anti-β-actin antibody served as a loading control. *P<0.01, **P<0.05, ***P<0.001 versus intact. (D) NE or CGRP (30 ng/kg/d) was continuously infused into kidneys via a mini- osmotic pump, and the left ureters were obstructed for 10 d (n=4 in each group). The kidneys were fixed for PAS and TUNEL staining to measure the tubular dilation and detect apoptotic cells in tubules, respectively. The visible blue color indicates nuclei stained by DAPI. The lumen area of tubular dilation on PAS-stained kidney sections was measured in 5 randomly chosen high-power (x 200) fields per kidney using NIH ImageJ software. The number of TUNEL-positive apoptotic cells in tubules was counted in 5 randomly chosen high-power (x 200) fields per kidney. *P<0.05 versus vehicle. (E) Systolic blood pressure of mice was measured by a noninvasive taill cuff method. Mice were placed on a heated platform (30°C) in an isolated chamber, and systolic blood pressure levels were obtained. Mice were trained for 5 d before DNx followed by daily recording for experimental days (n=5 in each group). Scale bar, 50 μm. Error bars represent SD.

Supplemental Figure 4: α2-AR antagonists reduce tubulointerstitial fibrosis and inflammation after UUO. Male 129S1/SvImJ mice aged 8 to 10 weeks was continuously treated with doxazosin (α1-AR antagonist, 12 mg/kg/d), atipamezole (α2-AR antagonist, 2.4 mg/kg/d), metoprolol (β1-AR antagonist, 12 mg/kg/d), ICI118551 (β2-AR antagonist, 2.4 mg/kg/d), L748337 (β3-AR antagonist, 2.4 mg/kg/d), BRL44408 (α2A-AR antagonist, 12 mg/kg/d), imiloxan (α2B-AR antagonist, 12 mg/kg/d), spiroxatrine (α2C-AR antagonist, 12 mg/kg/d) or vehicle (10% DMSO in PBS) via an intraperitoneal implantation of a mini-osmotic pump 24 h before UUO (n=5). (A) α-SMA, p-Smad3, ICAM-1, TNF-α, MCP-1 and KC expression in UUO kidneys treated with α2-AR antagonists using Western blot analysis. The bands were quantified using Lab Works analysis software. (B) PARP1, cleaved PARP1 and cleaved caspase-3 expression in UUO kidneys treated with α2-AR antagonists using Western blot analysis. Anti-β-actin antibody served as a loading control. The bands were quantified using Lab Works analysis software. (C) NE level in UUO kidneys using ELISA assay. *P<0.05, **P<0.01, ***P<0.001 versus vehicle; #P < 0.05 versus imiloxan. Error bars represent SD.

Supplemental Figure 5: NE and CGRP induce renal tubular apoptosis via α2-ARs and CGRP receptor, respectively. (A and B) Mouse renal proximal tubular cell line (MCT) was 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 h, 1 nM of NE and either 10 μM of α2A-AR antagonist

BRL44408, 1 μM of α2B-AR antagonist imiloxan, 100 nM of α2C-AR antagonist spiroxatrine, or vehicle (10% DMSO in PBS) was co-treated to the culture (n=4). (A) The MCT cells co- treated with NE and either α2A-AR antagonist, α2B-AR antagonist, α2C-AR antagonist, or vehicle 2 for 48 h were fixed for TUNEL and PI staining to detect apoptotic and necrotic cells, respectively. The visible blue color indicates nuclei stained by DAPI. The number of TUNEL- positive apoptotic or PI-positive necrotic MCT cells was counted in 5 randomly chosen high- power (x 200) fields per plate. (B) The released level of TGF-β1 and CTGF in the supernatant of MCT cells co-treated with NE- and respective antagonists of α2-AR subtypes for 48 h was examined by ELISA assay and Western blot analysis. The protein bands were quantified using Lab Works analysis software. *P < 0.05, **P < 0.001 versus vehicle. (C and D) MCT cells were grown until 70% confluence on culture plates and then changed to serum-free medium. After serum starvation for 6 h, 1 nM of CGRP plus/minus either 10 nM of CGRP antagonist (CGRP8-37) was incubated for 48 h (n=4). (C) The MCT cells co-treated with NE and CGRP8-37 for 48 h were fixed for TUNEL and PI staining to detect apoptotic and necrotic cells, respectively. The visible blue color indicates nuclei stained by DAPI. The number of TUNEL- positive apoptotic or PI-positive necrotic MCT cells was counted in 5 randomly chosen high- power (x 200) fields per plate. (B) The released level of TGF-β1 and CTGF in the supernatant of MCT cells co-treated with CGRP and CGRP8-37 for 48 h was examined by ELISA assay and Western blot analysis. The protein bands were quantified using Lab Works analysis software. *P < 0.05, **P < 0.001 versus no treatment. Error bars represent SD.