Adrenomedullin Inhibits Connective Tissue Growth Factor Expression

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector original article http://www.kidney-international.org & 2008 International Society of Nephrology

Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis T Nagae1, K Mori1, M Mukoyama1, M Kasahara1, H Yokoi1, T Suganami1, K Sawai1, T Yoshioka1, M Koshikawa1, Y Saito1, Y Ogawa1, T Kuwabara1, I Tanaka1, A Sugawara1, T Kuwahara2 and K Nakao1

1Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan and 2Department of Nephrology, Osaka Saiseikai Nakatsu Hospital, Osaka, Japan

Systemic administration of the potent vasodilating peptide Tubulointerstitial fibrosis is a common feature of progressive adrenomedullin reduces cardiac and renal fibrosis in renal injury and predicts the long-term outcome of renal hypertensive animals. Here, we investigated the effects function.1 Among proposed mechanisms by which interstitial of kidney-specific adrenomedullin gene delivery in fibrosis progresses, transforming growth factor-b (TGFB1) normotensive rats after unilateral ureteral obstruction, plays a central role in the pathogenesis.2–4 TGFB1 promotes an established model of renal tubulointerstitial fibrosis. the accumulation of extracellular matrix, through the enhan- Overexpression of exogenous adrenomedullin in the renal ced synthesis of extracellular matrix proteins, such as fibro- interstitium following ureteral obstruction significantly nectin (FN1), and the inhibition of their degradation.2 We prevented fibrosis and proliferation of tubular and interstitial have revealed that connective tissue growth factor (CTGF) is cells. In this model, there is upregulation of connective tissue an important factor that mediates profibrotic action of growth factor (CTGF) mRNA expression and extracellular TGFB1 in renal fibroblasts and in unilateral ureteral obstruc- signal-regulated kinase (ERK) phosphorylation, and tion (UUO), an established model of obstructive nephro- adrenomedullin overexpression suppressed both of these pathy.5,6 Introduction of Ctgf antisense oligonucleotide in activities without altering the blood pressure. In NRK-49F the renal interstitium markedly suppresses renal fibrosis, renal fibroblasts, adrenomedullin reduced transforming but does not inhibit Tgfb1 induction and cell proliferation growth factor-b-induced CTGF and fibronectin mRNA in UUO.6 upregulation through the cyclic AMP/protein kinase A Adrenomedullin (ADM), a potent vasorelaxing and natri- signaling pathway, and suppressed ERK phosphorylation and uretic peptide,7 is expressed in renal glomeruli and tubules.8,9 cell proliferation. In the kidneys with an obstructed ureter, ADM exerts its actions by stimulating cyclic AMP (cAMP) adrenomedullin receptor gene expression was upregulated production and by nitric oxide release in target tissues.10–12 In along with cyclic AMP production in kidney slices. The humans, its receptor is composed of calcitonin receptor-like latter effect was partially blocked by a neutralizing receptor (CRLR or CALCRL) and a family of receptor- antibody to adrenomedullin, indicating that an endogenous activity-modifying proteins (RAMPs 1–3). RAMP2/CALCRL peptide-receptor system was activated. Our results show and RAMP3/CALCRL constitute ADM receptors, whereas that overexpression of exogenous adrenomedullin in the RAMP1/CALCRL generates calcitonin gene-related protein ureteral-obstructed kidney prevents tubulointerstitial fibrosis (CGRP or CALCA) receptor.13 We have reported that Ramp1, and cell proliferation through the cyclic AMP-mediated Ramp2, and Calcrl mRNAs are markedly upregulated after decrease of CTGF induction and ERK phosphorylation. UUO in rats, and proposed that the ADM–RAMP system 14 Kidney International (2008) 74, 70–80; doi:10.1038/ki.2008.98; may modulate the process of tubulointerstitial fibrosis. published online 9 April 2008 We and others have shown that ADM may either stimulate KEYWORDS: TGF-b; cyclic AMP; gene expression; extracellular matrix; or inhibit cell proliferation, depending on the cell types. fibroblast ADM has a proliferative action on 3T3 fibroblasts15 and vascular smooth muscle cells,16 but has an antiproliferative action on endothelial and mesangial cells,10,17 and cardiac fibroblasts.18 Systemic administration of ADM, in many cases, potently reduces blood pressure and exerts protective Correspondence: K Mori, Department of Medicine and Clinical Science, 19–21 Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, effects against cardiac and renal injury. Heterozygotic Sakyo-ku, Kyoto 606-8507, Japan. E-mail: [email protected] Adm-deficient mice are predisposed to suffering from fibrotic Received 21 December 2006; revised 4 December 2007; accepted 3 changes in the cardiovascular system and in the kidney, January 2008; published online 9 April 2008 indicating that endogenous ADM possesses organ protective

70 Kidney International (2008) 74, 70–80 T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis original article

effects.22–25 Whether local and direct action of ADM in the AxAM, an adenovirus encoding human ADM gene, was kidney is sufficient to prevent tubulointerstitial fibrosis and transferred into rat kidney via renal vein using hydrody- whether endogenous ADM–ADM receptor system is activated namics-based gene transfer technique.6 Using control AxLacZ in obstructive nephropathy remains to be elucidated. In adenovirus injection, successful gene transfer was verified the present study, we investigated the effects of ADM over- by positive X-gal staining specifically in interstitial cells of expression in renal interstitium upon UUO. Furthermore, we the obstructed kidney, and by the absence of expression in examined the signaling pathway of ADM in the context of the other side of the kidney, liver, spleen, lung, and heart cAMP/protein kinase A (PKA), extracellular signal-regulated (Figure 1).6,26 On day 6 after ureter ligation, exogenous kinase (ERK or MAPK1/MAPK3), and also CTGF, which we human ADM mRNA expression was detected in AxAM- have been proposing as a crucial profibrotic molecule acting infected kidney (Figures 2 and 3). In situ hybridization downsteam of TGFB1 in obstructive nephropathy.6 Finally, revealed that human mRNA was indeed expressed predomi- by in situ mRNA hybridization, we investigated the spatial nantly in the interstitium, and expression in tubules was regulation of ADM–ADM receptor system in UUO. little, if any (Figure 2). In contrast, endogenous rat Adm was expressed in glomeruli and distal nephrons (presumably RESULTS collecting ducts). UUO caused upregulation of rat Adm gene Renal fibrosis induced by UUO is prevented by exogenous expression in inner medullary collecting ducts, but expres- expression of ADM sion pattern in other parts of the kidney did not seem to be To investigate whether exogenous expression of ADM locally altered. These changes were reflected by slight elevation of rat in the kidney ameliorates renal fibrosis induced by UUO, Adm expression levels in the whole kidney (1.2-fold, but not significantly; Figure 3). Gene expression of Tgfb1, the key y molecule involved in tissue fibrosis, was upregulated after UUO by 3.4-fold, and gene transfer of AxLacZ or AxAM did t. kidne pleen eart R Lt. kidney S Liver H Lung not affect the expression level. Gene expression of Ctgf, the critical factor that mediates profibrotic activity of TGFB1 in UUO,5,6 was elevated in the obstructed kidney by 1.6-fold as LacZ compared with the sham-operated kidney. Introduction of AxAM into the kidney markedly inhibited Ctgf mRNA upregulation (by 75±8%) compared with control AxLacZ adenovirus-treated obstructed kidney. Furthermore, gene Figure 1 | Gene transfer into the rat kidney by hydrodynamics- expression of an extracellular matrix protein fibronectin based retrograde gene transfer via left renal vein. (a) X-gal (FN1) was upregulated in the obstructed kidney (4.2-fold staining (in blue) of the left kidney section at 6 days after of sham operation), and was significantly attenuated (by transfection with AxLacZ reporter adenovirus. Arrows indicate the ± outer surfaces (basement membranes) of tubules. Original 81 10%) with the transfer of AxAM. Immunohistochemical magnification 200. (b) Expression of LacZ mRNA in various analysis revealed that FN1-expressing area was increased tissues analyzed by northern blotting. by 3.5-fold in UUO and was remarkably decreased in

a b c

de f

Figure 2 | Distribution of endogenous Adm and exogenous ADM in the rat kidney undergoing UUO. (a–f) In situ mRNA hybridization of the kidney on day 6 after ureter ligation. Gene expression of endogenous rat Adm (in dark blue staining, rAM) was detected in distal nephrons (a, black arrows) and glomeruli (b, red arrows) in sham-operated kidney, and was upregulated in inner medullary collecting ducts in UUO kidney (c, black arrows). By clear contrast, exogenous human ADM (hAM) was expressed in the interstitium of ADM-encoding adenovirus-treated obstructed kidney (e; f, blue arrows), but not in noninfected UUO kidney (d). Original magnification: 100 (d, e), 200 (a, c), and 400 (b, f).

Kidney International (2008) 74, 70–80 71 original article T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis

SULAM

TGFB1

CTGF

FN rAM

hAM

28S

5 2 ∗∗ 6 bcd∗ NS NS NS ∗∗NS 4 5 4 3 1 3 2 FN/28S CTGF/28S

TGFB1/28S 2 1 1

0 0 0 SULAM SULAM SULAM

ef i j

g h k l

∗∗ ∗∗ m 100 ∗∗NS ∗∗ n 4 NS

80 3 60 2 40 FN (% area)

Fibrosis score 1 20

0 0 SULAM S U LAM Figure 3 | Effects of ADM gene transfer in the rat kidney undergoing UUO. (a–d) Northern blot analysis of the kidney on day 6 after ureter ligation. S, sham-operated kidney; U, vehicle-treated ureter-ligated kidney; L, AxLacZ-treated obstructed kidney; AM, AxAM (ADM-encoding adenovirus)-treated obstructed kidney. The relative mRNA levels of Tgfb1 (TGF-b), Ctgf (CTGF), and Fn1 (FN) were normalized with 28S ribosomal RNA. rAM, rat Adm; hAM, human ADM. Mean levels of the sham-operated kidney from control rats were determined as 1.0 arbitrary unit. (e–h, m) Immunofluorescence staining of FN1 (FN) using fluorescein-conjugated antibody and quantified relative FN1 þ areas. (i–l, n) Fibrotic areas determined by Masson’s trichrome staining (shown in blue) and semiquantitative scores of tubulointerstitial fibrosis. Original magnification 200. *Po0.05; **Po0.01; NS, not significant; n ¼ 5 per each group.

AxAM-treated kidney (by 67± 9%). Furthermore, fibrosis These observations show that exogenous ADM expressed in the score determined by Masson’s trichrome staining was also renal interstitium inhibited renal fibrosis in UUO. elevated in AxLacZ-treated obstructed kidney compared with sham, and was significantly reduced in AxAM-treated kidney Cell proliferation after UUO is suppressed by exogenous (the scores were 3.2±0.4 for AxLacZ, 1.1±0.1 for AxAM, and expression of ADM 0.1±0.1 for sham). Urinary volume and systolic blood pressure Because cellular proliferation of tubular and interstitial measured by the tail-cuff method were not changed signifi- cells is one of the hallmarks of obstructive nephropathy, we cantly after 6 days of UUO and by AxAM treatment (Figure 4). carried out Western blot analyses for proliferating cell nuclear

72 Kidney International (2008) 74, 70–80 T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis original article

160 12 SU LAM 140 120 10

) PCNA

100 –1 80 8 β-Actin 60 S S 40 U (ml day U

Systolic blood 6

L Urinary volume L p-ERK

pressure (mm Hg) 20 AM AM 0 4 06 06 t-ERK Days Days

b 14 ∗∗NS ∗∗ c 10 ∗∗NS ∗∗ 8 ∗∗ 12 8 6 ∗∗ † 10 4 8 6

body weight) S –1 2 U 6 L 4 AM p-ERK/t-ERK

PCNA/ β -actin 4

(mm g 0 Left kidney weight ratio weight Left kidney 06 2 Days 2 0 Figure 4 | Effects of ADM gene transfection on blood pressure, 0 SULAM S U LAM urinary volume, and kidney weight following renal obstruction in rats. (a) Systolic blood pressure. (b) Urinary volume. (c) Left kidney/body weight ratio. S, sham; U, vehicle de treated UUO; L, AxLacZ-treated UUO; AM, AxAM-treated UUO. Mean±s.e.m. n ¼ 5; **Po0.01 vs each basal level; wPo0.05 vs U and L at day 6. antigen (PCNA) and phosphorylated MAPK1/MAPK3, and immunostaining for PCNA. As shown in Figure 5, PCNA expression in the whole kidney was significantly increased after 6 days of ureter ligation, but upregulation of PCNA f g expression was significantly decreased in AxAM-treated kidney as compared with control AxLacZ-treated obstructed kidney (4.5- vs 9.6-fold of sham operation, respectively). Immunohistochemical study also indicated that the number of PCNA þ -proliferating cells in the obstructed kidney was increased both in the tubules and in the interstitium, and was decreased with ADM treatment. Cellular proliferation (Figure 20 5h and i) and extent of interstitial fibrosis (Figure 3m and n) h 20 i ∗∗NS ∗∗ ∗∗ ∗∗ were proportional to kidney weight/body weight ratio of the NS 15 15 obstructed kidney (Figure 4c). Activation of MAPK1/MAPK3 plays an important role in tubular and interstitial cell 10 10 27 cells (%) proliferation. The level of phosphorylated MAPK1/MAPK3 cells (%) in whole kidney of AxLacZ-treated UUO rats was signifi- 5 5 Interstitium, positive cantly increased (by 7.0-fold), compared with that in sham- positive Renal tubule, 0 0 operated kidney (Figure 5). AxAM treatment significantly SULAM SULAM suppressed MAPK1/MAPK3 phosphorylation in UUO (by Figure 5 | PCNA expression and ERK (MAPK1/MAPK3) 69±13%). These findings indicate that renal cell prolifera- phosphorylation in the kidney of obstructive nephropathy. tion during UUO was inhibited by ADM treatment, which (a) Western blots of PCNA, b-actin, and phosphorylated and total may be due to reduced MAPK1/MAPK3 phosphorylation. MAPK1/MAPK3 (p-ERK and t-ERK) at day 6 after ureteral obstruction. Equal amounts of protein (40 mg per lane) from the whole kidney were separated by electrophoresis. S, ADM inhibits TGFB1-induced Ctgf and Fn1 upregulation Sham-operated kidney; U, vehicle-treated obstructed kidney; through the cAMP/PKA signaling pathway L and lacZ, AxLacZ-treated obstructed kidney; AM, AxAM-treated To address the molecular mechanism of ADM-mediated obstructed kidney. (b, c) The relative levels of PCNA/b-actin and p-ERK/t-ERK measured with densitometer. (d–g) Immunohisto- kidney protection in UUO, we examined direct actions chemistry for PCNA (stained in brown) in the obstructed kidney. of ADM on cultured NRK-49F normal rat renal fibroblasts. Original magnification 400. All the nuclei were further We investigated the effects of ADM on TGFB1-induced visualized with hematoxylin counterstaining for quantification þ expression of CTGF and its downstream molecule FN1, both (not shown). (h, i) The ratio of PCNA cells in renal tubular and interstitial cells. **P 0.01, NS, not significant; n 5. of which are critical mediators of tissue fibrosis.5,6 Ctgf o ¼ expression was strongly increased by TGFB1 in fibroblasts,

Kidney International (2008) 74, 70–80 73 original article T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis

3 3 3 ∗ 2 2 ∗ ∗ * 2 ∗ * 1 1 ** 1 CTGF/28S 0 CTGF/28S 0 0 3 3 3 ∗ ∗ ∗ 2 ∗ 2 ∗ ∗ 2 ∗ 1 FN/28S CTGF/28S 1 FN/28S 1

0 FN/28S Veh 9876AMP GMP 0 –Log[AM] (M) Veh RpAMPKI RpGM LY 0 Veh RpAM PKI TGFB1 AM cAMP TGFB1 TGFB1 Figure 6 | Antifibrotic effects of ADM upon NRK-49F normal rat kidney fibroblasts. (a) Northern blot analysis of TGFB1 (3 ng mlÀ1)- À9 À6 induced upregulation of Ctgf and Fn1 gene expression, and suppression by ADM (AM, final 10 –10 M) and by 8-bromo-cAMP and 8-bromo-cGMP (AMP and GMP, 1 mM each). Veh, vehicle. (b) Effects of protein kinase inhibitors upon the antifibrotic actions of ADM (AM, 1 mM). Rp-cAMP (RpAM, 10 mM) and myristoylated protein kinase A inhibitor amide 14–22 (PKI, 0.5 mM) are inhibitors for PKA; RpGM, protein kinase G inhibitor Rp-cGMP (10 mM); LY, phosphatidylinositol 3-kinase inhibitor LY29400 (10 mM). (c) cAMP/PKA-dependent inhibition of TGFB1-induced fibrotic changes. *Po0.05, **Po0.01 vs TGFB1 alone; wPo0.05 vs TGFB1 þ ADM/cAMP, n ¼ 6.

5 ∗ 4 ∗ 30 min) 4 † 3 ∗ ∗ 3 2 2 cells per 4 c.p.m. per well) c.p.m.

1 4 1

cAMP production 0 0 ( × 10 Veh 987 H-Thymidine incorporation H-Thymidine

Veh 10 9 8 7 3 –Log[AM] (M) (pmol per 10 –Log[AM] (M) PDGF

5 PDGF 4 Veh AMP AM 3 ∗ ∗ p-ERK 2

p-ERK/t-ERK 1 t-ERK 0 Veh AMP AM PDGF À10 À7 Figure 7 | Antiproliferative effects of ADM upon NRK-49F cells. (a) ADM (AM, final 10 –10 M)-stimulated increase of cAMP production. Veh, vehicle. *Po0.05 vs vehicle. (b) Inhibitory effects of ADM upon PDGF-BB (PDGF, 3 ng mlÀ1)-stimulated 3H-thymidine incorporation. *Po0.05 vs vehicle, wPo0.05 vs PDGF alone, n ¼ 6. (c, d) Inhibitory effects of ADM upon MAPK1/MAPK3 phosphorylation. À1 ADM (AM, 1 mM) and cAMP analog (AMP, 1 mM) significantly inhibited PDGF (3 ng ml )-induced phosphorylation of MAPK1/MAPK3. p, phosphorylated; t, total. Equal amounts of protein (40 mg per lane) were subjected to Western blot analysis. *Po0.05 vs PDGF alone, n ¼ 4. and pretreatment with ADM dose dependently inhibited Pretreatment with PKA inhibitors, Rp-cAMP (adenosine 30,50- TGFB1-induced Ctgf mRNA upregulation (Figure 6). Because cyclic monophosphorothioate) and myristoylated PKA inhi- cAMP and cyclic GMP (cGMP) are major intracellular signal- bitor amide 14–22, canceled ADM-induced inhibition of Ctgf ing molecules for ADM action, we examined whether they and Fn1 upregulation (Figure 6). By contrast, protein kinase G can substitute for ADM as to suppression of Ctgf induc- inhibitor Rp-cGMP (guanosine 30,50-cyclic monophosphoro- tion. Membrane-permeable analogs of cAMP and cGMP thioate) and phosphatidylinositol 3-kinase inhibitor LY294002 (8-bromo-cAMP and 8-bromo-cGMP) both effectively had no significant effects. In control experiments, both PKA abolished the upregulation of Ctgf gene expression. Essen- inhibitors reversed inhibitory effects of cAMP itself on TGFB1- tially, similar results were obtained on Fn1 gene expression. induced Ctgf and Fn1 mRNA upregulation. Finally, ADM These findings raised the possibility that ADM inhibits TGFB1- stimulated intracellular cAMP production in NRK-49F cells induced Ctgf and Fn1 upregulation through cAMP, cGMP, (Figure 7a). These findings show that the antifibrotic effects of or both. Therefore, we examined which of cAMP and cGMP is ADM in cultured renal fibroblasts were mediated mainly the predominant mediator of the antifibrotic action of ADM. through the cAMP/PKA cascade.

74 Kidney International (2008) 74, 70–80 T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis original article

14 ADM suppresses proliferation and MAPK1/MAPK3 Cont. ** ‡ 14 phosphorylation of renal fibroblasts Cont.+AM Adrenomedullin has a proliferative action on 3T3 ﬁbroblasts15 12 UUO 12 16 UUO+AM * ‡ ** ‡ and vascular smooth muscle cells, but has an antiprolifera- 10 10 10 tive action on endothelial and mesangial cells. We examined ‡ the effects of ADM on renal ﬁbroblast proliferation. ADM 8 8 inhibited platelet-derived growth factor (PDGF)-BB-induced † † † † 6 † 6 DNA synthesis in a dose-dependent fashion (Figure 7). Next, * cAMP production we examined the effects of ADM on MAPK1/MAPK3 cAMP production 4 4 phosphorylation, as MAPK1/MAPK3 phosphorylation plays 27 2 2

a key role in the induction of interstitial cell proliferation. tissues per 5 min) (pmol per mg wet (fmol per isolated glomeruli per 5 min) Preincubation with ADM or a membrane-permeable cAMP 0 0 analog significantly inhibited PDGF-induced MAPK1/ Cont. UUO Cont. UUO Cont. UUO Cont. UUO MAPK3 phosphorylation (Figure 7). These results indicate Whole kidney Medulla Cortex Glomeruli that ADM inhibited MAPK1/MAPK3 phosphorylation and 14 renal fibroblast proliferation. Cont. 12 Cont.+ascites Cont.+AM Ab cAMP production is augmented in UUO kidney, partially by 10 UUO UUO+ascites endogenous ADM ‡‡ 8 UUO+AM Ab In this study, we have demonstrated that exogenous ADM 8 † † inhibits Ctgf and Fn1 upregulation and cell proliferation in 6 ††† * † 6 UUO kidney and in cultured renal fibroblasts, and that * * cAMP production cAMP production 4 4 cAMP plays a crucial role in these ADM actions. Using cAMP * as a reporter, we investigated whether the endogenous 2 2 (pmol per mg wet tissues per 5 min) (pmol per mg wet ADM–ADM receptor system is activated in UUO. Basal 0 0 (fmol per isolated glomeruli per 5 min) cAMP production of renal tissue slices from whole kidney Cont. UUO Cont. UUO Cont. UUO Cont. UUO was increased by 2.4-fold in UUO compared with control Whole kidney Medulla Cortex Glomeruli (Figure 8). This was also true when the medulla, cortex, or Figure 8 | Effects of ADM upon cAMP production rate in glomeruli was examined separately. Addition of ADM kidney tissue slices and in isolated glomeruli from UUO rats. (a) Basal and ADM (AM)-induced cAMP production. (b) Blockade increased cAMP production in the medulla and glomeruli of cAMP production by neutralizing ADM (AM) antibody. from control and UUO rats, indicating the presence of wPo0.05, zPo0.01 vs basal (ADM-untreated conditions) of control active ADM receptors. To evaluate the contribution of (non-UUO kidney); *Po0.05, **Po0.01, significant changes by endogenous ADM in enhanced rate of cAMP production in ADM (a) or by neutralizing ADM antibody treatment (b); n ¼ 6. UUO kidney, ADM-neutralizing antibody was added to renal tissue slices. Basal cAMP production in control kidney counterparts, transient coexpression of rat Calcrl and Ramp2 was not affected significantly by ADM-neutralizing antibody or Ramp3 generated the receptors specific to ADM, and that (Figure 8). In contrast, the antibody partially inhibited cAMP Calcrl and Ramp1 generated the CALCA receptor. elevation in whole kidney, medulla, cortex, and glomeruli from UUO rats. As a control, control ascites gave no effects. Ramp2 and Ramp1 are upregulated in renal medulla, cortex, These findings indicate that the endogenous ADM–ADM and glomeruli after UUO receptor system was activated in UUO kidney. To further characterize the role of RAMPs 1–3 in UUO, we analyzed their renal distribution and the change of expres- Transient coexpression of Calcrl (CRLR) and Ramp2 or Ramp3 sion. Northern blot analyses revealed that Ramp1 was mainly generate ADM receptor, and Calcrl and Ramp1 generate upregulated in medulla and cortex, Ramp2 in medulla, CALCA (CGRP) receptor cortex, and glomeruli at 6 days after UUO, whereas Ramp3 In humans and mice, functional reconstitution of RAMPs 1–3 was unchanged (Figure 10). Through all these in vivo and and CALCRL as ADM or CALCA receptor has been in vitro experiments, it is suggested that upregulation of reported.13,28–30 To clarify the rat ADM receptor selectivity, Ramp2 expression activated the ADM–ADM receptor system we made expression vectors for rat Ramp’s and Calcrl and in UUO kidney. transfected them into COS-7 cells. As shown in Figure 9, when Ramp2 or Ramp3 was co-transfected with Calcrl, ADM Ramp1 is expressed in all nephron segments, upregulated markedly increased cAMP production, but when Ramp1 and evenly, and newly induced in interstitium after UUO Calcrl was co-transfected, ADM induced little cAMP Finally, we investigated renal distribution of Ramp2 gene production. In contrast, only when Ramp1 was co-transfected expression before and after UUO (Figure 11). In situ with Calcrl, CALCA markedly increased cAMP production hybridization revealed that Ramp2 was expressed in virtually (Figure 9). These results indicate that similarly to human all nephron segments including glomeruli in sham-operated

Kidney International (2008) 74, 70–80 75 original article T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis

kidney. Especially, distal nephrons (such as thick ascending expression was induced in interstitium adjacent to dilated limbs of Henle and collecting ducts) expressed Ramp2 abun- distal nephrons. These ﬁndings suggest that renal interstitium dantly. After UUO, Ramp2 gene expression was enhanced was an important target of endogenous and exogenous ADM evenly in nephron segments. At the same time, de novo in the present study.

10 pCAGGS/Neo 10 pCAGGS/Neo R1/CRLR R1/CRLR DISCUSSION 8 R2/CRLR 8 R2/CRLR cells) R3/CRLR cells) R3/CRLR 4 CRLR 4 CRLR In the present study, we locally overexpressed exogenous 6 R1 6 R1 R2 R2 ADM predominantly in the renal interstitial cells in obstruc- R3 R3 4 4 ted nephropathy by injecting ADM-encoding adenovirus via renal vein, and revealed the protective action of ADM, which 2 2 was characterized with suppression of interstitial ﬁbrosis and cAMP (pmol per 10 0 cAMP (pmol per 10 0 of cellular proliferation. Administration of ADM adenovirus 012108 6 0121086 also caused downregulation of CTGF, FN, PCNA, and –Log[AM] (M) –Log[CGRP] (M) phosphorylation of MAPK1/MAPK3 in the kidney. Unlike Figure 9 | Cyclic AMP production in COS-7 cells overexpressing systemic delivery of the Adm gene19,20 or administration of Ramps 1–3 and/or Calcrl (CRLR). ADM (a, AM) or CALCA 21 (b, CGRP)-induced cAMP production. pCAGGS/Neo, mock; R1–R3, ADM in previous reports, we did not observe blood Ramps 1–3. Average of n ¼ 4. pressure reduction in our model, highlighting the importance

Control UUO WKMed Cx GIWK Med Cx GI RAMP1 RAMP2

RAMP3

28S

12 8 ∗∗ 4 ∗∗ ∗∗ 10 ∗∗ 6 8 6 4 ∗ 2 ∗∗ ∗ ∗ 4 RAMP3/28S RAMP2/28S

RAMP1/28S 2 2 0 0 0 WMCG WMCG WMC G WMCG WMC G WMC G Control UUO Control UUO Control UUO Figure 10 | Distribution of the Ramp family mRNA expression in UUO kidneys.(a) Distribution and changes of mRNA expressions of the Ramp family in the kidney after UUO. (b–d) The relative mRNA levels of Ramp’s normalized with 28S ribosomal RNA in the control and obstructed kidney at day 6. The expression levels in the whole kidney from control (sham operated) rats were defined as 1.0 arbitrary unit. WK or W, whole kidney; Med or M, medulla; Cx or C, cortex; Gl or G, glomerulus. *Po0.05, **Po0.01 vs control. n ¼ 5.

abc

def

Figure 11 | Localization and changes of Ramp2 mRNA expression in the kidney after UUO. (a, b) In sham-operated kidney, Ramp2 was expressed in all nephron segments, such as glomeruli, proximal tubules (P), and distal nephrons (black arrows). (c) Control sense probe gave no signals in UUO kidney. (d–f) In UUO kidney, Ramp2 was upregulated evenly in nephrons, and was newly induced in interstitium (red arrows) adjacent to dilated distal nephrons (D). Original magnification: 40 (a, c, d) and 200 (b, e, f).

76 Kidney International (2008) 74, 70–80 T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis original article

of direct renal actions of ADM in modulating the patho- the obstructed kidney, and that overexpression of Ramp2 and physiology of UUO. Calcrl reconstitutes a functional rat ADM receptor. There- In the process of renal interstitial fibrosis, renal fibroblasts fore, we propose that upregulation of Ramp2 expression plays play a key role by their involvement in the proliferation, an important role in the activation of the endogenous migration, and synthesis of growth factors and matrix ADM–ADM receptor system in UUO, and this may augment proteins.31–33 ADM stimulates not only cAMP production the protective effects of exogenous ADM. but also calcium mobilization in cultured endothelial cells. Furthermore, we investigated renal localization of rat The latter effects result in an increased nitric oxide produc- Adm, Ramp2, and human ADM by in situ hybridization. tion to enhance cGMP accumulation in the vasculature, Exogenous human ADM, introduced by retrograde adeno- thereby contributing to vasodilatory effects of ADM in virus infection via renal vein, was expressed predominantly concert with increased cAMP production.11,12 In this study, in the interstitium. In a previous report, it was demonstrated we showed that ADM significantly suppressed TGFB1- that the gene delivery method used in this study specifically induced upregulation of Ctgf and Fn1 expression in NRK- targets interstitial fibroblasts, and spares endothelial cells and 49F renal fibroblasts. ADM as well as membrane-permeable proximal tubules.26 Despite highly localized expression, analogs of cAMP and cGMP, both of which are intracellular exogenous ADM exerted potent protective effects upon both signaling molecules of ADM, effectively abolished the tubular and interstitial cells. This may be explained by upregulation of Ctgf and Fn1 mRNA. Pretreatment with possibilities that the gene delivery enhanced secretion of PKA inhibitor reversed this suppression, but neither protein protective factors (including ADM) and, at the same time, kinase G inhibitor nor phosphatidylinositol 3-kinase inhi- suppressed release of deteriorating factors (such as CTGF) bitor affected the response, demonstrating the critical role of from renal interstitium, and that these soluble factors acted the cAMP/PKA pathway in fibrogenesis of these cells. in paracrine manners. We also found that, in UUO kidneys, Sustained activation of MAPK1/MAPK3 in renal tubular Ramp2 expression was upregulated along all nephron and interstitial cells in UUO provides a possible mechanism segments and was also induced in the interstitium. Over- of the proliferative response against interstitial injury.27 We expression of exogenous ADM in interstitium did not reflect and others have reported potent inhibitory effects of ADM a physiologic response to UUO (because we did not find on mesangial cell proliferation,10,34–37 but little is known upregulation of endogenous rat Adm in the interstitium), but about the antiproliferative action of ADM on tubulointer- turned out to be an excellent strategy to carry out gene stitial cells in vivo and in vitro. The present study revealed therapy. that the tubulointerstitial proliferation was attenuated in In summary, we demonstrate here that adenovirus- the kidney overexpressing ADM gene with the inhibition mediated ADM gene delivery into the kidney ameliorates of MAPK1/MAPK3 phosphorylation.24 Furthermore, ADM the histologic alterations caused by UUO. The results also decreased PDGF-induced thymidine incorporation and suggest that the renoprotective effects of ADM in tubuloin- MAPK1/MAPK3 phosphorylation in NRK-49F cells, consis- terstitial proliferation and fibrosis are not due to systemic tently with a recent work reporting that ADM inhibited basal blood pressure reduction, but due to cAMP-dependent CTGF and EGF (epidermal growth factor)-stimulated proliferation suppression and inhibition of MAPK1/MAPK3 phosphory- in these cells.38 Because interstitial volume expansion in lation. These findings indicate that ADM may offer a new obstructed kidney has been attributed to fibroblast differ- therapeutic strategy to prevent renal fibrosis in obstructive entiation/proliferation and extracellular matrix deposition,39 nephropathy and perhaps in other chronic nephropathies the suppressive effects of ADM on the increasing renal mass leading to tubulointerstitial fibrosis. of the ligated kidney are likely the results of antiproliferative and antifibrotic effects of ADM on renal fibroblasts. Interes- MATERIALS AND METHODS tingly, suppression of CTGF activity in UUO by introduction Materials of antisense oligonucleotide through renal vein only inhi- Human ADM and CALCA (CGRP) were obtained from Peptide bited fibrotic changes but did not affect renal cell prolifera- Institute (Osaka, Japan). 8-Bromo-cAMP and 8-bromo-cGMP were 6 tion, although overexpression of ADM via a similar route in from Sigma (St Louis, MO, USA), and Rp-cGMP (final 10 mM) was this study reduced both tubulointerstitial fibrosis and from Biolog (La Jolla, CA, USA). Rp-cAMP (10 mM), myristoylated proliferation. These findings highlight the potentially distinct PKA inhibitor amide 14–22, and LY294002 were purchased from effects of the future strategy to treat fibrosis-associated renal Calbiochem (San Diego, CA, USA). diseases either by CTGF blockade or by ADM administration. Construction of recombinant adenovirus We and others have shown that ADM possesses a potent Recombinant adenovirus encoding human ADM cDNA was inhibitory activity against oxidative stress, which may also 42 synthesized by COS-TPC method using Adenovirus Expression play a role in its antifibrotic effects described in the present 22,23,40,41 Vector Kit (Takara Shuzo, Otsu, Japan). Briefly, human ADM cDNA study. (nucleotides (nt) 124–737) containing the complete coding region of In the UUO kidney, cAMP production rate was enhanced, ADM7 was ligated into cosmid cassette vector pAxCAwt, which which was partially blocked with ADM-neutralizing anti- contains the CAG promoter consisting of cytomegalovirus imme- body. We show here that Ramp2 expression is upregulated in diate early enhancer and chicken b-actin/rabbit b-globin hybrid

Kidney International (2008) 74, 70–80 77 original article T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis

promoter,43,44 and was co-transfected into 293 cells together with Northern blot analysis EcoT221-digested DNA-TPC (from Ad5dlx) to generate replication- Total RNA from the kidney or cultured cells was extracted by deficient adenovirus AxAM.42 the acid guanidinium thiocyanate–phenol–chloroform method. Northern blot analysis was performed as described previously.14 32 Unilateral ureteral obstruction and in vivo gene transfer P-labeled cDNA probes for rat Ctgf (nt 1221–1803), Tgfb1 (nt 1142–1546), Fn1 (nt 619–1082),6 Adm (nt 19–591),48 Calcrl using hydrodynamics-based technique 46 14 All animal experiments were conducted in accordance with our (nt 2001–2668), and rat Ramp family were prepared by standard Institutional Guidelines for Animal Research. Viral vectors were reverse transcription-PCR. The amount of RNA loaded in each lane introduced into rat kidney by means of a recently developed was normalized with 28S ribosomal RNA. hydrodynamics-based gene transfer technique.6,26 In brief, male cAMP measurement Wistar rats weighing B200 g were anesthetized with pentobarbital, Basal cAMP production and ADM-stimulated cAMP production in the the left kidney was exposed by midline incision, and the left ureter cells and tissues were measured by radioimmunoassay.10 In brief, was ligated with 4-0 silk at two points.6 The recombinant adenovirus, cultured COS-7 and NRK-49F cells grown to confluence in 24-well AxAM (5 109 PFU dissolved in 750 ml of Ringer solution; Sigma) or plates were washed twice with serum-free DMEM and preincubated for AxLacZ (control adenovirus AxCALacZ, a gift from Dr Y Kanegae, 20 min at 371Cin400ml DMEM containing 0.1% bovine serum Tokyo University) was then injected into the left renal vein using a albumin and 0.5 mM isobutylmethylxanthine (IBMX; Sigma). Human 24-gauge SURFLO intravenous catheter (Terumo, Tokyo, Japan) after ADM or CALCA was added to the culture and cells were further clamping the left renal vein. Blood flow was reestablished 10 min incubated for 30 min at 371C. After incubation, 1 ml of ice-cold ethanol after injection. Rats were killed 6 days after UUO or sham operation. was added to the culture to disrupt the cells and the mixture was centrifuged at 15 000 r.p.m. for 10 min at 41C. The supernatant was Histology and immunohistochemistry vacuum dried and the pellet dissolved in assay buffer was subjected to For histological analysis, sagittal kidney sections fixed with 4% RIA (Yamasa, Tokyo, Japan). For rat tissue slice cAMP measurement, buffered paraformaldehyde were embedded in paraffin and 2-mm- 1.5-mm-thick renal tissue slices prepared by a tissue slicer (Medical 5 thick sections were stained with Masson’s trichrome. The fibrotic Agent, Tokyo, Japan) or isolated glomeruli were incubated in 0.1% 45 area was measured semiquantitatively using a computer-aided bovine serum albumin/Hank’s balanced salt solution (Sigma) and manipulator (KS400; Carl Zeiss Vision, Munich, Germany). Thirty 0.5 mM IBMX with or without 10 mM human ADM for 5 min at 371C randomly selected fields were examined by two investigators without and were homogenized, and the cAMP content was measured. For knowledge of the origin of the slides, and graded semiquantitatively neutralization experiment, anti-ADM monoclonal antibody10 or (0–4) according to the area of positive staining (score 0, o5%; 1, control mouse ascites was added 10 min before ADM addition. 5–25%; 2, 25–50%; 3, 50–75%; and 4, 475%). Then, the mean score per section was calculated. Immunohistochemistry for PCNA was Western blot analysis performed using mouse anti-PCNA antibody and LSAB þ kit Western blot analysis was performed as described6 using anti-PCNA, 6 (DAKO Japan, Kyoto, Japan). PCNA-positive cells in renal tubules anti-phospho-MAPK1/MAPK3 (ERK1/2), anti-total MAPK1/ and in the interstitium were counted separately in 20 high-power MAPK3 (New England Biolabs, Boston, MA, USA), and anti-b- fields. Immunofluorescence study of FN1 was carried out with actin antibodies (Sigma; as an internal control). mouse anti-FN ED-A antibody (Abcam, Cambridge, UK). Ratio of positively stained areas was computer analyzed using NIH Image In situ mRNA hybridization 6 and expressed as percentages. To prepare cRNA probes, cDNA fragments encoding rat Adm (nt 3–573),48 rat Ramp2 (nt 103–691),49 and human ADM (nt 124–741)7 Cell culture, DNA transfection, and proliferation study were amplified by standard reverse transcription-PCR and ligated COS-7 cells and NRK-49F cells were maintained in DMEM into pGEM-T Easy plasmid vector (Promega, Madison, WI, USA). (Dulbecco’s modified Eagle’s medium; Sigma) containing 10% fetal Digoxigenin-labeled sense and antisense probes were synthesized calf serum, 100 U mlÀ1 penicillin, and 100 mg mlÀ1 streptomycin. with SP6 and T7 RNA polymerases (Roche Diagnostics, Penzberg, The cDNA encoding rat Calcrl,46 modified to provide a consensus Germany) after linearizing the plasmids. In situ hybridization was Kozak sequence,47 and Ramp cDNAs14 were cloned into pCAGGS/ carried out by an automated application (Ventana Medical Systems, Neo (a gift from Dr J Miyazaki, Osaka University). Transient Tucson, AZ, USA).50 The rat kidneys were fixed in 4% paraformal- transfection into COS-7 cells was carried out using Effectene dehyde and embedded in paraffin. Sections of 8-mm thickness were Transfection Reagent (QIAGEN, Santa Clarita, CA, USA). For deparaffinized, fixed, hydrogen chloride treated, and were subjected TGFB1 stimulation, cells at B90% confluence were made quiescent to cell conditioning and protease digestion. Hybridization was in serum-free DMEM supplemented with 10 mgmlÀ1 insulin, performed with antisense or sense probe (10 ng per side) at 601C 10 mg mlÀ1 transferrin, and 10 ng mlÀ1 selenium (Sigma). After for 5 h in RiboHybe hybridization solution (Ventana Medical 24 h of serum starvation, cells were treated with protein kinase Systems). Hybrids were detected with alkaline phosphatase- inhibitors 30 min before the addition of 1 mM ADM, 1 mM 8-bromo- conjugated anti-digoxigenin antibody (Roche Diagnostics) and nitro cAMP, or 8-bromo-cGMP. One hour later, fibroblasts were blue tetrazolium chloride 5-bromo-4-cloro-3-indolyl phosphate stimulated with 3 ng mlÀ1 of human TGFB1 (R&D Systems, 4-toluidinium salt. The sections were counterstained with Fast Red. Minneapolis, MN, USA) and incubated for 48 h, or incubated with 3ngmlÀ1 of PDGF-BB (Becton Dickinson Labware, Bedford, MA, Statistical analysis USA) for 18 h. For proliferation study, cells were pulsed with 3H- Data are expressed as the mean±s.e.m. Statistical analysis was thymidine for the final 6 h, and the tracer incorporated into the cells performed using analysis of variance followed by Scheffe’s test. was measured in a liquid scintillation counter.10 P-value o0.05 was considered statistically significant.

78 Kidney International (2008) 74, 70–80 T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis original article

ACKNOWLEDGMENTS 21. Nishikimi T, Mori Y, Kobayashi N et al. Renoprotective effect of chronic We gratefully acknowledge Dr Y Kanegae (Tokyo University) and adrenomedullin infusion in Dahl salt-sensitive rats. Hypertension 2002; 39: Dr J Miyazaki (Osaka University) for providing plasmids, Dr K Kangawa 1077–1082. (National Cardiovascular Center Research Institute, Osaka) for 22. Shimosawa T, Shibagaki Y, Ishibashi K et al. Adrenomedullin, an valuable discussion, Ms J Nakamura and Ms S Saito for technical endogenous peptide, counteracts cardiovascular damage. Circulation 2002; 105: 106–111. assistance, and Ms S Doi and Ms A Sonoda for secretarial assistance. 23. Kawai J, Ando K, Tojo A et al. Endogenous adrenomedullin protects This work was supported in part by research grants from against vascular response to injury in mice. Circulation 2004; 109: the Japanese Ministry of Education, Culture, Sports, Science 1147–1153. and Technology; the Japanese Ministry of Health, Labour and 24. Niu P, Shindo T, Iwata H et al. Protective effects of endogenous Welfare; Research for the Future Program of the Japan Society adrenomedullin on cardiac hypertrophy, fibrosis, and renal damage. for the Promotion of Science; Smoking Research Foundation; Circulation 2004; 109: 1789–1794. and the Salt Science Research Foundation. 25. Luodonpaa M, Leskinen H, Ilves M et al. Adrenomedullin modulates hemodynamic and cardiac effects of angiotensin II in conscious rats. Am J Physiol Regul Integr Comp Physiol 2004; 286: R1085–R1092. 26. Maruyama H, Higuchi N, Nishikawa Y et al. Kidney-targeted naked DNA REFERENCES transfer by retrograde renal vein injection in rats. Hum Gene Ther 2002; 1. Nath KA. Tubulointerstitial changes as a major determinant in the 13: 455–468. progression of renal damage. Am J Kidney Dis 1992; 20:1–17. 27. Masaki T, Foti R, Hill PA et al. Activation of the ERK pathway precedes 2. Border WA, Noble NA. Transforming growth factor b in tissue fibrosis. tubular proliferation in the obstructed rat kidney. Kidney Int 2003; 63: N Engl J Med 1994; 331: 1286–1292. 1256–1264. 3. Eddy AA. Molecular insights into renal interstitial fibrosis. J Am Soc 28. Buhlmann N, Leuthauser K, Muff R et al. A receptor activity modifying Nephrol 1996; 7: 2495–2508. protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin 4. Basile DP. The transforming growth factor b system in kidney disease and gene-related peptide receptor when coexpressed with human RAMP1. repair: recent progress and future directions. Curr Opin Nephrol Hypertens Endocrinology 1999; 140: 2883–2890. 1999; 8: 21–30. 29. Fraser NJ, Wise A, Brown J et al. The amino terminus of receptor activity 5. Yokoi H, Mukoyama M, Sugawara A et al. Role of connective tissue modifying proteins is a critical determinant of glycosylation state and growth factor in fibronectin expression and tubulointerstitial fibrosis. Am ligand binding of calcitonin receptor-like receptor. Mol Pharmacol 1999; J Physiol Renal Physiol 2002; 282: F933–F942. 55: 1054–1059. 6. Yokoi H, Mukoyama M, Nagae T et al. Reduction in connective tissue 30. Kuwasako K, Shimekake Y, Masuda M et al. Visualization of the growth factor by antisense treatment ameliorates renal tubulointerstitial calcitonin receptor-like receptor and its receptor activity-modifying fibrosis. J Am Soc Nephrol 2004; 15: 1430–1440. 7. Kitamura K, Kangawa K, Kawamoto M et al. Adrenomedullin: a novel proteins during internalization and recycling. J Biol Chem 2000; 275: hypotensive peptide isolated from human pheochromocytoma. Biochem 29602–29609. Biophys Res Commun 1993; 192: 553–560. 31. Wiggins R, Goyal M, Merritt S et al. Vascular adventitial cell expression of 8. Eto T, Kitamura K. Adrenomedullin and its role in renal diseases. Nephron collagen I messenger ribonucleic acid in anti-glomerular basement 2001; 89: 121–134. membrane antibody-induced crescentic nephritis in the rabbit. A cellular 9. Mukoyama M, Sugawara A, Nagae T et al. Role of adrenomedullin and source for interstitial collagen synthesis in inflammatory renal disease. its receptor system in renal pathophysiology. Peptides 2001; 22: Lab Invest 1993; 68: 557–565. 1925–1931. 32. Strutz F. Novel aspects of renal fibrogenesis. Nephrol Dial Transplant 1995; 10. Michibata H, Mukoyama M, Tanaka I et al. Autocrine/paracrine role of 10: 1526–1532. adrenomedullin in cultured endothelial and mesangial cells. Kidney Int 33. Gonzalez-Cuadrado S, Bustos C, Ruiz-Ortega M et al. Expression of 1998; 53: 979–985. leucocyte chemoattractants by interstitial renal fibroblasts: up-regulation 11. Shimekake Y, Nagata K, Ohta S et al. Adrenomedullin stimulates by drugs associated with interstitial fibrosis. Clin Exp Immunol 1996; 106: two signal transduction pathways, cAMP accumulation and Ca2+ 518–522. mobilization, in bovine aortic endothelial cells. J Biol Chem 1995; 270: 34. Chini EN, Choi E, Grande JP et al. Adrenomedullin suppresses mitogenesis 4412–4417. in rat mesangial cells via cAMP pathway. Biochem Biophys Res Commun 12. Hirata Y, Hayakawa H, Suzuki Y et al. Mechanisms of adrenomedullin- 1995; 215: 868–873. induced vasodilation in the rat kidney. Hypertension 1995; 25: 790–795. 35. Kohno M, Yasunari K, Minami M et al. Regulation of rat mesangial cell 13. McLatchie LM, Fraser NJ, Main MJ et al. RAMPs regulate the transport and migration by platelet-derived growth factor, angiotensin II, and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998; adrenomedullin. J Am Soc Nephrol 1999; 10: 2495–2502. 393: 333–339. 36. Parameswaran N, Nambi P, Brooks DP et al. Regulation of glomerular 14. Nagae T, Mukoyama M, Sugawara A et al. Rat receptor-activity-modifying mesangial cell proliferation in culture by adrenomedullin. Eur J Pharmacol proteins (RAMPs) for adrenomedullin/CGRP receptor: cloning and 1999; 372: 85–95. upregulation in obstructive nephropathy. Biochem Biophys Res Commun 37. Haneda M, Araki S, Sugimoto T et al. Differential inhibition of 2000; 270: 89–93. mesangial MAP kinase cascade by cyclic nucleotides. Kidney Int 1996; 50: 15. Withers DJ, Coppock HA, Seufferlein T et al. Adrenomedullin stimulates 384–391. DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 38. Parameswaran N, Hall CC, Bomberger JM et al. Regulation of cells. FEBS Lett 1996; 378: 83–87. adrenomedullin signaling in kidney interstitial fibroblasts. Cell Physiol 16. Iwasaki H, Eguchi S, Shichiri M et al. Adrenomedullin as a novel growth- Biochem 2003; 13: 391–400. promoting factor for cultured vascular smooth muscle cells: role of 39. Klahr S, Morrissey J. Obstructive nephropathy and renal fibrosis. Am J tyrosine kinase-mediated mitogen-activated protein kinase activation. Physiol Renal Physiol 2002; 283: F861–F875. Endocrinology 1998; 139: 3432–3441. 40. Moriyama T, Kawada N, Nagatoya K et al. Fluvastatin suppresses oxidative 17. Plank C, Hartner A, Klanke B et al. Adrenomedullin reduces mesangial cell stress and fibrosis in the interstitium of mouse kidneys with unilateral number and glomerular inflammation in experimental ureteral obstruction. Kidney Int 2001; 59: 2095–2103. mesangioproliferative glomerulonephritis. Kidney Int 2005; 68: 41. Miyashita K, Itoh H, Arai H et al. The neuroprotective and 1086–1095. vasculo-neuro-regenerative roles of adrenomedullin in ischemic brain 18. Tsuruda T, Kato J, Kitamura K et al. An autocrine or a paracrine role of and its therapeutic potential. Endocrinology 2006; 147: 1642–1653. adrenomedullin in modulating cardiac fibroblast growth. Cardiovasc Res 42. Miyake S, Makimura M, Kanegae Y et al. Efficient generation of 1999; 43: 958–967. recombinant adenoviruses using adenovirus DNA-terminal protein 19. Dobrzynski E, Wang C, Chao J et al. Adrenomedullin gene delivery complex and a cosmid bearing the full-length virus genome. Proc Natl attenuates hypertension, cardiac remodeling, and renal injury in Acad Sci USA 1996; 93: 1320–1324. deoxycorticosterone acetate-salt hypertensive rats. Hypertension 2000; 43. Kanegae Y, Lee G, Sato Y et al. Efficient gene activation in mammalian 36: 995–1001. cells by using recombinant adenovirus expressing site-specific Cre 20. Zhang JJ, Yoshida H, Chao L et al. Human adrenomedullin gene delivery recombinase. Nucleic Acids Res 1995; 23: 3816–3821. protects against cardiac hypertrophy, fibrosis and renal damage in 44. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression hypertensive Dahl salt-sensitive rats. Hum Gene Ther 2000; 11: 1817–1827. transfectants with a novel eukaryotic vector. Gene 1991; 108: 193–199.

Kidney International (2008) 74, 70–80 79 original article T Nagae et al.: Direct actions of adrenomedullin in renal fibrosis

45. Kasahara M, Mukoyama M, Sugawara A et al. Ameliorated glomerular 48. Sakata J, Shimokubo T, Kitamura K et al. Molecular cloning and biological injury in mice overexpressing brain natriuretic peptide with renal activities of rat adrenomedullin, a hypotensive peptide. Biochem Biophys ablation. J Am Soc Nephrol 2000; 11: 1691–1701. Res Commun 1993; 195: 921–927. 46. Chang CP, Pearse II RV, O’Connell S et al. Identification of a seven 49. Tomoda Y, Kikumoto K, Isumi Y et al. Cardiac fibroblasts are major transmembrane helix receptor for corticotropin-releasing factor and production and target cells of adrenomedullin in the heart in vitro. sauvagine in mammalian brain. Neuron 1993; 11: 1187–1195. Cardiovasc Res 2001; 49: 721–730. 47. Aiyar N, Rand K, Elshourbagy NA et al. AcDNAencodingthecalcitonin 50. Alchi B, Nishi S, Kondo D et al. Osteopontin expression in acute renal gene-related peptide type 1 receptor. JBiolChem1996; 271: 11325–11329. allograft rejection. Kidney Int 2005; 67: 886–896.

80 Kidney International (2008) 74, 70–80