Essential roles of oncostatin M receptor β signaling in renal crystal formation in mice

Oncostatin M (OSM), a member of the IL-6 family of cytokines, has important roles in renal diseases. The relationship between OSM and kidney stone disease, however, remains unclear. To investigate the roles of OSM in the development of kidney stone disease, we generated a mouse model of renal crystal formation using OSM receptor β (OSMRβ)-deficient mice (OSMRβ−/− mice). There were fewer renal crystal deposits in OSMRβ−/− mice than in wild-type (WT) mice. Crystal-binding molecules (osteopontin, annexin A1, and annexin A2), inflammatory cytokines (TNF-α and IL-1β), and fibrosis markers (TGF-β, collagen 1a2, and α-smooth muscle actin) were also decreased in the kidneys of OSMRβ−/− mice compared with those in WT mice. Immunofluorescence staining showed that OSMRβ was expressed in renal tubular epithelial cells (RTECs) and renal fibroblasts in the model of renal crystal formation. In the cultured RTECs and renal fibroblasts, OSM directly induced the expression of crystal-binding molecules and fibrosis markers. Expressions of inflammatory cytokines were increased by stimulation with OSM in cultured renal fibroblasts. OSM may promote the formation of renal crystal deposits by directly acting on RTECs and renal fibroblasts to produce crystal-binding molecules and inflammatory cytokines.


Formation of GOx-induced renal crystal deposits in OSMRβ −/− mice.
To investigate the roles of OSM signaling in renal crystal formation, WT and OSMRβ −/− mice were given intraperitoneal injection of GOx. On day 0, no renal crystal deposits were observed in either WT or OSMRβ −/− mice (Fig. 2). On day 3, some There were no differences in the amount of renal crystal deposits between WT and OSMRβ −/− mice on day 3 (Fig. 2b). On day 6, renal crystal deposits were markedly increased in WT mice (Fig. 2). However, renal crystal deposits in OSMRβ −/− mice were significantly decreased compared with those in WT mice on day 6 ( Fig. 2).

Expressions of crystal-binding molecules in the kidneys of OSMRβ −/− mice.
To investigate the roles of OSM signaling in the expressions of crystal-binding molecules after GOx injection, we elucidated the expressions of osteopontin (OPN), ANXA1, and ANXA2 in the kidneys of WT and OSMRβ −/− mice. On day 0, there were no differences in the mRNA expressions of these crystal-binding molecules between WT and OSMRβ −/− mice ( Fig. 3a-c). In WT mice, the mRNA expressions of OPN, ANXA1, and ANXA2 were increased on day 3 and day 6 compared with those on day 0 ( Fig. 3a-c). The mRNA expressions of these crystal-binding molecules in OSMRβ −/− mice were significantly suppressed, compared with those in WT mice on day 3 and day 6 ( Fig. 3a-c). In addition, Western blot analysis confirmed the significant suppression of protein expressions of these crystal-binding molecules in OSMRβ −/− mice on day 3 (Fig. 4a,b) and day 6 ( Fig. 4c,d).

Expressions of markers of kidney injury and fibrosis in the kidneys of OSMRβ −/− mice.
To assess the roles of OSM signaling in GOx-induced kidney injury and fibrosis, we investigated the expressions of genes related to kidney injury (kidney injury molecule-1, KIM-1), and fibrosis (transforming growth factor-β, TGF-β; type 1 collagen α2 chain, Col1a2; α-smooth muscle actin, αSMA; and tissue inhibitor of metalloproteinase 2, Timp2). The expressions of KIM-1, TGF-β, and Col1a2 were up-regulated by GOx injection in the kidneys of WT mice on day 3, but were decreased on day 6 ( Fig. 3h-j). In addition, αSMA and Timp2 were increased on day 3 and day 6 compared with those on day 0 in WT mice (Fig. 3k,l). In the kidneys of OSMRβ −/− mice on day 3, the expressions of KIM-1, TGF-β, Col1a2, and αSMA were significantly suppressed compared with WT mice (Fig. 3h-k). Western blot analysis confirmed the significant suppression of protein expressions of TGF-β and Col1a2 in OSMRβ −/− mice on day 3 ( Fig. 4i,j). On day 6, the expression of KIM-1 in OSMRβ −/− mice was lower than that in WT mice (Fig. 3h). There were no significant changes in the expression of Timp2 mRNA in the kidneys between WT and OSMRβ −/− mice on day 3 and day 6 ( Fig. 3l). The ratio of areas with renal crystal deposits. The ratio was quantified by calculating the percentage of the area containing crystal deposits to the total kidney area. Data are expressed as mean ± SEM; n = 6 per group. *P < 0.05 compared with WT mice. www.nature.com/scientificreports/

Localization of OSM and OSMRβ protein in the kidneys after GOx injection. To identify OSM-
and OSMRβ-expressing cells in the kidney of the mouse model of renal crystal formation, we performed immunofluorescence staining in the kidney of mice on day 3, when the renal expression of OSMRβ had reached a peak by the injection of GOx (Fig. 1). The expression of OSM was strongly observed in the EpCAM-negative renal tubular epithelial cells (RTECs) (Fig. 6a-c). In contrast, intense expression of OSMRβ was observed in the EpCAM-positive RTECs (Fig. 6d-f). These OSMRβ-positive RTECs expressed OPN (Fig. 6g-i). In addition,  www.nature.com/scientificreports/ and ANXA2 were increased by stimulation with OSM in both RTECs (Fig. 7b) and renal fibroblasts (Fig. 7c).
In addition, OSM stimulation induced the expression of TNF-α mRNA in the renal fibroblasts ( Fig. 7c), but not in RTECs (Fig. 7b). Stimulation with OSM did not affect the mRNA expression of MCP-1 in RTECs and renal fibroblasts (Fig. 7b,c). Furthermore, mRNA expressions of TGF-β ( Fig. 7b) and Col1a2 (Fig. 7c) were induced by OSM in the RTECs and renal fibroblasts, respectively. The expression of αSMA, a marker of myofibroblasts, was not changed by the stimulation with OSM in renal fibroblasts (Fig. 7c). The effects of OSM on the expression of these molecules were abolished in RTECs and fibroblasts obtained from the kidneys of OSMRβ −/− mice (Fig. S2).
In addition, Western blot analysis confirmed the significant increases in protein expressions of these molecules in RTECs and renal fibroblasts stimulated with OSM ( Fig. 8).

Discussion
A member of the IL-6 family of cytokines, OSM, is associated with the development of a variety of human diseases, including rheumatoid arthritis 25 , metabolic syndrome 26 , and inflammatory bowel disease 22 . Although the expression of OSM increases in the kidneys of patients with obstructive nephropathy due to urolithiasis 27 , the functional roles of OSM in the development of kidney stone disease have been unclear. In the present study, www.nature.com/scientificreports/ we used a mouse model of renal crystal formation in OSMRβ −/− mice to address this question. In the kidneys of OSMRβ −/− mice, there was considerably less formation of GOx-induced crystal deposits than in WT mice on day 6, suggesting that OSM signaling may promote the formation of renal crystal deposits in the process of kidney stone formation. Renal macrophages were recently reported to play an important role in the formation of crystal deposits. Generally, activated macrophages are divided into two types: classically activated macrophages (M1 macrophages) www.nature.com/scientificreports/ and alternatively activated macrophages (M2 macrophages) 28,29 . In the process of kidney stone formation, M1 macrophages promote crystal deposit formation by increases in the expressions of inflammatory cytokines and crystal-binding molecules 18 . We previously reported that OSM switches the phenotype of macrophage from M1 to M2 type in adipose tissue macrophages, peritoneal exudate macrophages, and a macrophage cell line, RAW264.7 cells 23 . Our initial hypothesis was therefore that OSM might inhibit kidney stone formation through decreases in M1 type of renal macrophages. In the present study, however, there were no significant differences in the number of M1 macrophages and in the expression of MCP-1 between WT and OSMRβ −/− mice on day 3. MCP-1 is a chemokine that recruits monocytes to the site of injury. Moreover, the expression of OSMRβ was hardly detected in the renal macrophages. From these findings, OSM is suggested to have no association with the recruitment and phenotypic changes of renal macrophages in the process of kidney stone formation. Crystal-binding molecules, such as OPN, ANXA1, and ANXA2, have been reported to be important for the crystal aggregation, crystal adhesion to RTECs, and crystal retention in renal tubules [30][31][32] . Experiments using cell lines of RTEC have shown that high calcium state and calcium oxalate crystals directly induce the expressions of these crystal-binding molecules 30,33,34 . Meanwhile, the mechanism of the production of crystal-binding www.nature.com/scientificreports/ molecules in the process of kidney stone formation is not fully understood. In the present study, OSM induced the expressions of OPN, ANXA1, and ANXA2, in RTECs isolated from the mouse model of renal crystal formation. In addition, our in vivo study showed that strong expression of OSMRβ was observed in the RTECs during renal crystal formation. The expressions of OPN, ANXA1, and ANXA2 induced by GOx injection were significantly lower in the kidneys of OSMRβ −/− mice than in those of WT mice. OSM may therefore promote the formation of renal crystal deposits through the expression of crystal-binding molecules in the process of kidney stone formation. In addition to the RTECs, OSMRβ was expressed in the renal fibroblasts. Fibroblasts are mainly responsible for the synthesis of extracellular matrix (ECM) proteins, such as collagen and elastin, in both physiological and pathological conditions 35 . In addition to these functions, fibroblasts have been reported to be associated with a variety of biological processes, including inflammatory and immune responses, angiogenesis, and carcinogenesis of adjacent epithelial cells [36][37][38] . Previously, Umekawa et al. reported that the expressions of OPN and MCP-1 are induced in NRK-49F cells, a normal rat kidney fibroblast-derived cell line, stimulated with calcium oxalate crystals 39 . In the present study, OSM induced the expressions of crystal-binding molecules, including OPN, ANXA1, and ANXA2, in the cultured renal fibroblasts. This suggests that OSM is a novel potent inducer of crystal-binding molecules in the fibroblasts as well as the RTECs. TNF-α was also induced by stimulation with www.nature.com/scientificreports/ OSM in the renal fibroblasts, while the expression of MCP-1 was not affected by OSM. From these findings, OSM seems to regulate inflammation through the production of inflammatory cytokines rather than by recruitment of M1 macrophages in the process of kidney stone formation. Kidney stone disease is a risk factor for renal fibrosis and subsequent CKD 1-3 . In the process of renal fibrosis, TGF-β is important for the differentiation of renal fibroblasts into myofibroblasts, which are a main source of extracellular matrix (ECM) proteins, including collagens and fibronectin 40,41 . In the present study, on day 3, the expressions of TGF-β and αSMA, a marker of myofibroblasts, were lower in the kidneys of OSMRβ −/− mice than those in WT mice. Suppression of Col1a2 expression was also observed in the kidneys of OSMRβ −/− mice. In addition, in vitro study revealed that OSM directly induced the expression of TGF-β in the RTECs, while αSMA was not affected by OSM in the renal fibroblasts. These findings suggest that OSM produces TGF-β in RTECs and that the differentiation into myofibroblasts is indirectly induced. The expression of Col1a2, one of the ECM proteins related to fibrosis, was enhanced by OSM in the renal fibroblasts. OSM signaling thus promotes renal fibrosis in the process of kidney stone formation.
In humans, CaOx kidney stones mainly form as overgrowths on sub-epithelial plaques of calcium phosphate in the renal papillae, known as Randall's plaque 1,3 . Several lines of evidence have suggested that CaOx crystals in the tubules and interstitium cause the formation of Randall's plaque 1 . Several mouse models have been developed to study the mechanisms underlying the formation of CaOx crystals and subsequent Randall's plaque. To date, there are no mouse models for Randall's plaque, except for a mutant mouse lacking ABCC6 (ATP-binding cassette sub-family C member 6) 42 . However, mutations in ABCC6 are rarely responsible for kidney stone disease in humans 43 . Generally, single-gene mutations only account for 10-20% of kidney stone disease 44,45 , and the common pathogenesis of kidney stone disease is believed to involve the environmental and lifestyle factors. A mouse model of CaOx kidney stone disease using non-genetically engineered (normal) mice has therefore been considered to be suitable for investigation of the pathogenesis of human CaOx kidney stones. Okada et al. 46 have established a mouse model forming CaOx crystals in normal mice (C57BL/6 mice) by intraabdominal injection of GOx 46 , and we used this model in the present study. Unfortunately, it is likely that the crystals will not develop into Randall's plaque in this mouse model, as they disappeared by day 15 46 . However, intratubular crystals are grown within a very short time period, 3-6 days 46 , compared with the other mouse model using C57BL/6 mice, the high-oxalate/calcium-free diet-fed model, which needs 14 days to grow crystals 47 . The mouse model used in the present study is easy to prepare and may therefore be a good model for investigating the common pathogenesis of CaOx crystal formation in humans.
In the present study, we demonstrated that the deficiency of OSM signaling suppressed the formation of renal crystal deposits and OSM directly induced the production of crystal-binding molecules in the RTECs and renal fibroblasts. Our results strongly suggest that OSM contributes to the formation of renal crystal deposits through the induction of crystal-binding molecules in the process of kidney stone formation.

Methods
Animals. Eight-week-old male C57BL/6 J mice were purchased from Nihon SLC (Hamamatsu, Japan). Generation of OSMRβ −/− mice followed the protocol as previously described 48 . OSMRβ +/+ (WT) and OSMRβ −/− littermates were obtained from our breeding colony using heterozygous breeding pairs. All mice were housed in specific pathogen-free facilities under light [12-h (h) light/dark cycle]-, temperature (22-25 °C)-, and humidity (50-60% relative humidity)-controlled conditions. The mice had free access to food (MF; Oriental Yeast, Tokyo, Japan) and water. All experimental procedures were approved by the Animal Research Committees of Wakayama Medical University, and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, revised 1978) and the in-house guidelines for the care and use of laboratory animals of Wakayama Medical University.
Injection of GOx in mice. As described elsewhere 46 , we prepared a mouse model of renal crystal formation by intraperitoneal injection of GOx (Sigma, St. Louis, MO). Briefly, mice were intraperitoneally injected with GOx dissolved in PBS once daily at a dose of 80 mg/kg. Day 0 was the first day of GOx injection and the "day 0" samples were taken from mice before the injection. Mice were injected with GOx once daily for 3, 6, or 9 days, and then sacrificed 24 h (h) after the final injection of GOx (the samples on day 3, 6, or 9, respectively). For example, the samples of "day 3" were taken from mice injected with GOx on day 0, day 1, and day 2, and sacrificed on day 3.
Pizzolato staining and quantification of the amount of crystal deposits. The mice were deeply anaesthetized with isoflurane and transcardially perfused with ice-cold 0.9% NaCl followed by 4% paraformaldehyde. The kidney was then quickly removed, post-fixed in the same fixative at 4 °C for 4 h, and cryoprotected in 30% sucrose in 0.1 M PBS for 16 h. The specimens were embedded in an optimal cutting temperature (OCT) medium (Sakura Finetek, Torrance, CA), frozen rapidly in cold n-hexane on dry ice, and stored at -80 °C. Frozen sections were cut on a cryostat at 6-μm thickness. Pizzolato staining was performed to detect oxalate-containing crystals as described elsewhere 49 . Briefly, the solution for Pizzolato staining was prepared by a mixture of equal volumes of 5% silver nitrate and 30% hydrogen peroxidase. Frozen sections were incubated with the solution for 30 min (min). During the incubation, the sections were exposed to light from a 60-W incandescent lamp at a distance of 15 cm. The sections were then washed with distilled water and counterstained with nuclear fast red (Vector Laboratories, Burlingame, CA). Whole images of the kidney sections were acquired on a light microscope (BZ-7000, Keyence, Tokyo, Japan) using the image stitching system. The total area and positive area by Pizzolato staining in each kidney section were measured using Image J (US National Institutes of Health, Bethesda, MD). Positive area by Pizzolato staining was normalized by the total area of the kidney section. Coronal sections Scientific RepoRtS | (2020) 10:17150 | https://doi.org/10.1038/s41598-020-74198-3 www.nature.com/scientificreports/ of the kidney containing the cortex, medulla, and papilla, were used for quantification of crystal deposits. Six sections were selected at 120 μm intervals from the midcoronal plane of each kidney.
Immunofluorescence staining. Immunofluorescence staining was performed as described previously 23 .
Briefly, the mice were deeply anaesthetized with isoflurane and transcardially perfused with ice-cold 0.9% NaCl followed by ice-cold modified Zamboni's fixative (2% paraformaldehyde and 0.2% picric acid in 0.1 M PBS). The kidney was quickly removed and postfixed in the same fixative at 4 °C for 3 h. All specimens were then immersed in 20% sucrose in 0.1 M PBS for 16 h. The specimens were embedded in an OCT medium, frozen rapidly in cold n-hexane in dry ice, and stored at -80 °C. Frozen sections were cut on a cryostat at 6-μm thickness. The sections were preincubated with 5% normal donkey serum at room temperature for 1 h, followed by incubation with primary antibodies at 4 °C for 16 h. The primary antibodies were used at the following dilution: . The plot of a forward-versus side-scatter was used as the first gate to gate out aggregates and debris (Fig. S3). To identify individual live cells, the events were then gated based on side scatter versus 7-AAD (Fig. S3). Next, the CD45, F4/80, and CD11b-triple-positive cells were selected as macrophages in the kidney (Fig. S3). M1 and M2 macrophages were identified as Ly6C-and CD206-positive cells, respectively, in the macrophage fraction as previously described 17 . Single color controls were used to set the compensation and gates.
Isolation of RTECs and renal fibroblasts from kidney. First, the cells obtained from kidney were incubated with anti-CD16/CD32 antibodies to block Fc binding. For the isolation of RTECs, the cells were incubated with APC-conjugated anti-EpCAM antibody (clone G8.8, eBioscience). The stained cells were sorted using the anti-APC Multisort kit (Miltenyi Biotec) and the autoMACS Pro Separator (Miltenyi Biotec). The sorted cells were cultured in DMEM supplemented with 10% FCS, 100 U/ml of penicillin (Invitrogen), and 100 μg/ml of streptomycin (Invitrogen) for three days, and used as RTECs.
According to the previously reported method for the isolation of renal fibroblasts 50 , the cells isolated from the kidney were plated on the collagen-coated six-well plates (Corning, Corning, NY) in DMEM supplemented with 10% FCS, 100 U/ml of penicillin (Invitrogen), and 100 μg/ml of streptomycin (Invitrogen). The cells were then passaged at confluence and the fourth passage cells were used as renal fibroblasts.
These cells were then treated with vehicle or 50 ng/ml of recombinant mouse OSM (R&D Systems) and maintained for 1 and 2 h. All cells were cultured at 37 °C in a humidified atmosphere of 5% CO 2 .
Quantitative real-time PCR. Quantitative real-time PCR was performed with some modifications as described previously 23 . Briefly, total RNAs from the kidney, cultured RTECs, and cultured renal fibroblasts were prepared using TRI reagent (Molecular Research Center, Cincinnati, OH). Using the total RNA, the cDNA was synthesized with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The following TaqMan

Statistical analysis.
Results are shown as mean ± SEM. Comparison between the two groups was analyzed by Student's t-test. For multiple group comparisons, ANOVA followed by the post hoc Bonferroni test was used. For all statistical tests, the significance threshold was p < 0.05.

Data availability
Scientific RepoRtS | (2020) 10:17150 | https://doi.org/10.1038/s41598-020-74198-3 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.