129/Sv-WT and 129/Sv-cGKI-KO mice (Pfeifer et al., 1998) were bred and maintained in the animal facilities of University of Regensburg. Experiments are conducted according to the guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. Protocols were approved by local authorities for animal research (Regierung der Oberpfalz, Bayern, Germany, #54-2532.1-26/13) and conducted according to German law for animal care.

Unilateral ureteral obstruction, an established mouse model for chronic renal interstitial fibrosis (Chevalier et al., 2009), was performed according to Schinner et al. (2013) Kidney tissues of WT and cGKI-KO mice were divided into four groups: mice untreated or treated with RLX. Fibrotic tissue was derived from UUO-obstructed kidney, healthy tissue from contralateral kidney served as control. RLX was diluted in 20 mM sodium acetate (pH = 5) and administered continuously through osmotic minipumps (Alzet; model 1007D) immediately after UUO for 7 days (0.5 mg/kg/day).

Under isoflurane inhalation kidney tissue was removed after perfusion with 0.9% NaCl 7 days after UUO. Proteins from kidney tissue were solubilized in 50 mM Tris/2% SDS/phosphatase inhibitor (PhosSTOP, Roche; 1 tablet/5 ml)/protease inhibitors (leupeptin 0.5 μg/ml, PMSF 300 μM, benzamidine hydrochlorid 1 mM, and EDTA 5 mM) for 45 min at 7°C followed by centrifugation at 12400g, 7°C, 45 min. In supernatants, protein content was determined by modified Lowry method (Lowry et al., 1951) and stored at –80°C until analysis.

Protein expression of GAPDH, ERK1/2, P- ERK1/2, P-Smad2, P-VASP (Ser 239), GAPDH (antibodies from Cell Signaling, Danvers, MA, USA), TGF-β, CTGF, PDE5a (antibodies from Santa Cruz Biotechnology, Heidelberg, Germany) and P-PDE5a (Ser 92) (FabGennix, Frisco, TX, USA) were assayed by western blotting. After SDS-PAGE proteins were transferred to PVDF membranes. Donkey anti-goat IgG HRP (santa cruz Biotechnology, Heidelberg, Germany) and donkey anti-rabbit IgG HRP (Dianova GmbH, Hamburg, Germany) were used as secondary antibodies. Quantification was performed by ImageLab™ densitometry software (BioRad, München, Germany). Values were related to corresponding GAPDH values, except P-ERK1/2 is related to ERK1/2. Change of markers from healthy to fibrotic tissue was compared by values of fibrotic tissue in relation to healthy tissue, both untreated WT mice. The influence of treatment on markers was determined only in fibrotic tissue by analyzing values of markers in relation to mean values of untreated fibrotic WT, which were set as 1.

The activity of MMP2 and MMP9 was determined using gelatin zymography. SDS-PAGE was performed with a gel containing 0.1% gelatin. After washing (100 mM NaCl and 2.5% Triton X-100 in 50 mM Tris-HCl, pH 7.5) the gel was transferred to a reaction buffer (200 mM NaCl, 0.02% NaN₃, 0.5 μM ZnCl₂, 1 mM CaCl₂, 2% Triton-X 100, in 50 mM Tris-HCl, and pH 7.5) for enzymatic reaction at 37°C overnight. Gel was stained with Coomassie blue, destained in 10% acetic acid (v/v), and 30% methanol (v/v) and quantified using Image Lab. MMPs in fibrotic tissue were expressed as relative values of markers in fibrotic kidneys from untreated WT mice.

Serelaxin serum levels were determined using Human Relaxin-2 Quantikine^® ELISA (R&D Systems, Wiesbaden-Nordenstadt, Germany). cGMP levels in kidney tissue using cGMP EIA (IBL-Cayman, Hamburg, Germany). Measurements were performed according to manufacturer’s instructions.

Collagen levels in the kidneys were measured by a modified sirius red/fast green method (Lopez-De Leon and Rojkind, 1985), based on selective binding of sirius red to collagen and fast green dye binding to non-collagen proteins. Sirius red/fast green staining was calculated as increase (%) of collagen [ratio collagen/non-protein collagen] after 7 days UUO related to healthy kidney as described previously (Schinner et al., 2013).

Kidney tissues were cut at 4 μm. Immunohistochemistry and quantification was performed according to Schinner et al. (2013) Primary antibodies are mouse anti-α-SMA (Beckman Coulter, Krefeld, Germany), rabbit anti-Col1a1 and rabbit anti-fibronectin (Abcam, Cambridge, UK). Alexa 647-conjugated donkey anti-rabbit and Cy2-conjugated donkey anti-mouse served as secondary antibodies. For quantification the increase after UUO was related to the healthy kidney.

Isolation of total RNAs from kidney tissue, determination using quantitative RT-PCR as well as calculation was described previously (Schinner et al., 2013). mRNA levels of αSMA, fibronectin, Col1a1, MMP2, and MMP9 were detected. 18S rRNA served as housekeeping gene. The ΔΔC_T (cycle threshold) value is calculated from the difference of the corresponding control (C) and fibrosis-induced kidneys (F) [ΔΔC_T = ΔC_T(C) - ΔC_T(F)]. Then, the ratio of expression (r) was determined [r = 2^ΔΔCT].

Serum creatinine was determined by a previously published HPLC method with minor modifications (Schramm et al., 2014). Serum (10 μl) was mixed with 50 μl perchloric acid to precipitate proteins. The tube was mixed, kept at 4°C for 15 min, then centrifuged (5 min, 10,800 g). 5 μl of the supernatant was injected into the HPLC apparatus (Prominence LC20 series equipped with a LC20A photometric detector set at 234 nm; Shimadzu, Duisburg, Germany). Separation was performed using a Zorbax 300-SCX 5 μm, 150 × 4.6 mm, analytical column (Agilent, Waldbronn, Germany) and a mobile phase consisting of 5 mM sodium acetate (pH = 5.1)/acetonitrile (800:200, v:v). Creatinine eluted after 6.3–6.5 min at a flow rate of 1.0 ml/min (column temperature 35°C).

All data are expressed as mean ± SEM. Statistical differences between two means were calculated by unpaired student’s t-test (two-tailed, confidence interval 95%). Statistical significance was marked by asterisks (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001). n indicates number of animals. For data analysis, GraphPad Prism, version 6 (GraphPad software, Inc., La Jolla, CA, USA), was used.

Plasma levels of relaxin were determined through RLX ELISA in WT and cGKI-KO mice. In untreated mice serum levels were at 11.4 ± 1.4 pg/ml for WT (n = 11) and at 0.04 ± 0.04 pg/ml for cGKI-KO (n = 5). In RLX-treated mice serum levels increased to 20361 ± 2290 pg/ml for WT (n = 14) and 13357 ± 3122 pg/ml for cGKI-KO mice (n = 4, not significantly different for WT and cGKI-KO), which was highly statistically significant compared to untreated mice (WT: p < 0.001; cGKI-KO: p = 0.0018).

cGMP levels were investigated in kidney tissue. Figure 1A shows that cGMP was higher in fibrotic tissue 7 days after UUO than in the contralateral kidney (42.8 ± 5.7 vs. 25.9 ± 5.2 pmol/g). After RLX treatment cGMP levels of fibrotic kidneys further increased significantly (95.0 ± 12.5 pmol/g).

The activity of cGKI can be determined by quantification of cGKI-specific VASP phosphorylation at Serine 239. In Figure 1B an increase of VASP phosphorylation was observed after RLX treatment, in healthy and in fibrotic WT tissues. The increase in WT mice, expressed as relative values in kidneys from untreated WT mice, changed from 0.94 ± 0.12 (n = 5) to 1.68 ± 0.32 (n = 5) after treatment. In cGKI-KO this effect was lacking (0.79 ± 0.22; n = 3 vs. 0.86 ± 0.17; n = 2).

mRNA of α-SMA, a marker of myofibroblast differentiation, (Nagamoto et al., 2000) was increased in both WT and cGKI-KO after UUO. A reduction of mRNA was observed after treatment with RLX in WT, whereas no effect was seen in cGKI-KO after treatment (Figure 2A). As expected, in unobstructed renal tissue only vascular smooth muscle cells were immunostained with α-SMA, in UUO-obstructed kidneys enhanced interstitial expression was observed (data not shown). In WT, α-SMA protein was elevated compared to the contralateral kidney, after RLX treatment the increase was significantly reduced. In cGKI-KO no significant reduction of α-SMA protein expression through RLX was demonstrated (Figure 2B).

Fibronectin and Col1A1 are components of ECM, whose gene expressions are upregulated in fibrosis through TGF-β signaling. mRNA of both genes were elevated in fibrotic WT kidney 7 days after UUO compared to the contralateral kidney. Fibronectin and Col1A1 mRNA were reduced through RLX in WT (Figures 3B and 4A). Protein expression was strongly elevated by more than 40% for fibronectin and Col1A1 in both WT and cGKI-KO mice, compared to kidneys without UUO (for fibronectin, Figures 3A,C). Significant reduction of both proteins through RLX treatment was observed only in WT after 7 days of treatments.

Protein expression of total collagen was elevated by 21.4% ± 0.99 in fibrotic WT and significantly decreased through RLX treatment to an elevation of 16.6% ± 1.7 compared to unobstructed kidneys (p = 0.0126). In cGKI-KO, effects of RLX on ECM accumulation were not observed (Figures 3A,C and 4B,C).

MMPs are relevant for the degradation of ECM. mRNA of MMP2 was 6.8-fold (± 0.58) increased in fibrotic tissue, whereas MMP9 was not increased in that pathological condition (0.88-fold ± 0.53). Through RLX treatment, only the elevated levels of MMP2 mRNA were significantly decreased (4.3-fold ± 0.61), no changes were found for MMP9 mRNA after treatment (0.76-fold ± 0.067; Figures 5C,E). Zymography distinguishes between latent and active MMP proteins (Figures 5A,B). In WT, latent and active forms of MMP2 were elevated in fibrosis (5.9-fold ± 1.3; 4.2-fold ± 0.84), protein expression of latent MMP9 was significantly enhanced in fibrotic tissue (3.0-fold ± 0.42), active form significantly decreased (0.69-fold ± 0.098) compared to contralateral kidneys (n = 12, respectively).

Figures 5D,F show regulation of MMPs only in fibrotic tissue expressed as relative values of markers in kidneys from untreated WT mice. Consistent with data from mRNA, latent and active forms of MMP2 were both significantly reduced through RLX treatment in fibrotic kidneys. Through reduction of latent MMP9 and increase of active MMP9 after treatment physiological conditions were nearly restored.

The above described effects on MMP2 and MMP9 were not observed in cGKI-KO mice after treatment (Figures 5D,F).

As mentioned above, cGMP levels were elevated through RLX and, so far, cGKI-KO mice showed no antifibrotic effects, which suggested the involvement of the NO/cGMP/cGKI pathway in the antifibrotic effect of RLX. Subsequently, several signaling molecules were analyzed by western blotting, which are involved in fibrosis. Representative western blots demonstrated modulation of the selected markers in fibrotic conditions compared to healthy (Figure 6A).

Downstream to RLX, protein expression of phosphorylated ERK 1/2 (P-ERK1, P-ERK2) was analyzed. P-ERK1 was significantly elevated in kidneys of WT mice compared to the contralateral after undergoing UUO for 7 days (P-ERK1:1.8-fold ± 0.24; n = 7). cGMP is degraded by PDE5a, which is strongly upregulated in fibrosis (6.0-fold ± 1.3; n = 10), whereas phosphorylation of PDE5a at the cGMP-dependent phosphorylation site serine 92 (Thomas et al., 1990; 0.68-fold ± 0.12; n = 7) was decreased. TGF-β is a profibrotic cytokine, that was 5.3-fold (± 1.1; n = 12) elevated in the fibrotic kidneys compared to the contralateral renal WT tissue. Its downstream profibrotic signaling is dependent on Smad or -independent via ERK1/2 phosphorylation. Both P-Smad2 (1.926 ± 0.2384; n = 12) and ERK-1 (see above) were significantly elevated in fibrotic renal WT tissue. Additionally, further TGF-β transcription genes, e.g., CTGF were analyzed in this experiment. It was confirmed, that CTGF levels were elevated 3.3-fold (± 0.77; n = 12) in obstructed WT kidneys.

Protein levels of all signaling markers were significantly different in healthy and fibrotic kidney tissue from WT mice – P-PDE5a was reduced, remaining markers increased in fibrosis.

Figure 6B illustrates the influence of RLX on markers only in fibrotic tissue of WT and cGKI-KO. Values are related to untreated fibrotic WT kidneys, which were set as 1.

The phosphorylation of ERK1 normalized to total ERK1 was significantly reduced in WT through RLX treatment, but not in cGKI-KO.

eNOS and nNOS were increased in fibrosis, but were not further enhanced after treatment with RLX (data not shown).

The cGMP degrading PDE 5a is slightly enhanced through RLX in WT, and furthermore RLX significantly enhanced phosphorylation of PDE5a, indicating enhanced activity of PDE5a in fibrosis. The treatment-dependent increase in PDE5a phosphorylation is lacking in cGKI-KO. TGF-β itself remained unchanged in fibrotic tissue despite RLX treatment, but downstream signaling of TGF-β was affected through treatment. RLX significantly reduced P-Smad2 in WT, but not in cGKI-KO. Signaling via Smad-independent pathway was regulated through ERK1/2 phosphorylation (Leask and Abraham, 2004). As mentioned above, ERK1 phosphorylation was significantly reduced after treatment only in WT kidneys.

As previously stated, collagen, fibronectin and myofibroblast differentiation (α-SMA) was decreased (see above). 7 days after UUO continuous infusion of RLX decreased CTGF significantly in WT, but not in cGKI-KO.

Kidney function was measured by serum creatinine levels analyzed 7 days after UUO. Figure 7 shows that serum creatinine increased significantly after UUO, but renal performance improved significantly through RLX treatment by reducing serum creatinine levels from 1.0 mg/l ± 0.049 to 0.80 mg/l ± 0.069 in WT. cGKI-KO did not improve kidney function through RLX.