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Suppression of osteogenic-like differentiation in human renal interstitial fibroblasts by miRNA-410-3p through MSX2

Background The aim of this stay was to determine the effect of calcium ions in promoting osteogenic-like differentiation in human renal interstitial fibroblasts (hRIFs). The role of miRNA-410-3p in upregulating Msh homeobox 2 (MSX2) level in hRIFs was also investigated. Methods Quantitative polymerase chain reaction (qPCR) analysis was used to assess the expression levels of miRNA-410-3p in Randall’s plaque (RP) and normal renal papillary (nRP) tissues. Furthermore, the expression levels of osteogenesis-related protein in the RP and nRP tissues were assessed with qPCR and immunohistochemistry (IHC). hRIFs were cultured from isolated human kidney papilla before treatment with calcium chloride or osteogenic medium, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed at 1, 5, 9, and 14 days post-treatment. Alizarin red staining was used to estimate the deposits of calcium aggregates. After the overexpression or knockdown of miRNA-410-3p, we evaluated the changes in the osteogenic-like differentiation and osteogenesis-related protein by alizarin red staining and qPCR, respectively. A binding relationship between miRNA-410-3p and MSX2 was established through a dual-luciferase reporter gene assay. Rescue experiments demonstrated that miRNA-410-3p regulated the osteogenic-like differentiation by targeting MSX2. Results miRNA-410-3p levels were lower in RP tissue than in control nRP tissues. qPCR and IHC showed that the level of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN) were higher in RP tissues. The calcium deposition of hRIFs showed a time-dependent trend when treated with osteogenic medium or calcium chloride. The overexpression of miRNA-410-3p downregulated the levels of osteogenesis-related expression and attenuated mineralization. The knockdown of miRNA-410-3p yielded the opposite trend. Dual-luciferase reporter gene assay and rescue experiments indicated that miRNA-410-3p could target MSX2, while the overexpression of MSX2 reversed the effects of miRNA-410-3p on osteogenic-like differentiation. Conclusions The current findings suggest that calcium ions could promote the osteogenic-like differentiation of hRIFs and miRNA-410-3p regulates hRIFs osteogenic-like differentiation by inhibiting MSX2.


Introduction
Kidney stones are the most common disease of the urinary tract, and a reported 10-12% of people in industrialized countries will have a urinary stone in their lifetime (1). Kidney stones affect 1 in 11 people in the USA, with over 600,000 Americans estimated to suffer from urolithiasis every year (2). Approximately 5-10% of the Chinese population is expected to be diagnosed with urolithiasis, 25% of which will require hospitalization (3). The main components of kidney stones are calcium stones (about 80%) with the majority being the calcium oxalate (CaOx) type. The mechanisms of kidney stone development, or lithogenesis, are not completely understood. The major theory for predicting lithogenesis is the presence of Randall's plaque (RP), which appears to be the precursor of kidney stone development (4). One study found that most CaOx stones were found to be attached to RP at the renal papilla (5). However, the pathogenesis of RP remains unclear. Hypercalciuria, and the acidification concentration of urine, have all been implicated in RP formation (6). Among these, the involvement of hypercalciuria deserves special focus.
Recently, investigators have found that ectopic calcification is involved in the formation of RP (7). Many studies have shown that typical bone osteoid protein and hydroxyapatite mineralization are found in RP (8)(9)(10). He et al. discovered calcium ions could promote renal epithelial cell differentiation into cells with osteogenic phenotypes in idiopathic hypercalciuric patients (11). Typical bone osteoid proteins were also found to be significantly increased in hypercalciuric rats (12). These studies suggest that hypercalciuria may promote osteogenic-like differentiation of RPs.
MicroRNAs (miRNAs) are small noncoding RNA molecules of approximately 22 nt in length. They have been shown to elicit post-transcriptional gene regulation by degradation or inhibition of target mRNA translation. In recent years, several studies have been reported that miRNAs are associated with bone formation and osteoporosis (13,14). miRNA-410-3p is involved in a variety of biological processes and plays important roles in mammalian development. For instance, it has been implicated in the promotion of apoptosis in rhabdomyosarcoma cells and the attenuation of gemcitabine resistance in pancreatic ductal adenocarcinoma (15,16). However, there are no reports that clarify the role of miRNA-410-3p in osteoblast differentiation.
Studies have shown that Msh homeobox 2 (MSX2) enhances osteogenic differentiation in synergy with bone morphogenetic-2 protein (BMP2). Furthermore, MSX2 was found to promote the expression of critical osteogenesis proteins [runt-related transcription factor 2 (Runx2) and Osterix] in vascular smooth muscle cells (17). However, little is known about the relationship between MSX2 and miRNA in the development of RP. We speculated that human renal interstitial fibroblasts (hRIFs), as one of the main cell components in renal papillary tissues, may have osteogenic-like differentiation potential, and osteogeniclike differentiation may occur under the influence of pathological factors such as high concentration of calcium ions. Thus, this study aimed to investigate whether calcium ions could promote the osteogenic-like differentiation of hRIFs and whether miRNA-410-3p could regulate MSX2 level in hRIFs. Our findings may provide novel concepts for kidney stone treatment.

Tissue samples
The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of The Xiangya Hospital, Central South University (No. 201703258) and informed consent was taken from all the patients. RP tissues were obtained from patients with CaOx stones who underwent percutaneous nephrolithotomy. Donors of normal renal papillary (nRP) tissues were patients with renal tumors who underwent nephrectomy. Samples were obtained from papillary tissues with no tumor invasion. All samples were surgically collected and immediately stored at -80 ℃.

hRIFs isolation and culture
hRIF isolation and culture proceeded as described by Rodemann et al. (18). hRIFs were obtained from normal human kidney papillary tissues (10 g). Cell isolation was performed with enzyme mix [15 mL, 0.2% Collagenase I (Sigma-Aldrich, USA) and 0.25% trypsin (Sigma-Aldrich, USA) at 37 ℃ for 60 min. The cell suspension was then filtered through a 200-mesh nylon net and centrifuged at 800 rpm for 5 min. The culture medium (DMEM, 10% fetal bovine serum (FBS; BI, Israel), 100 U/mL penicillin (BI, Israel) and 100 μg/mL streptomycin (BI, Israel)] was added to the precipitate, and the cells were maintained at 37 ℃ in 5% CO 2 for 1 hour, and then the culture medium was replaced with new media to filter the adherent fibroblasts. The culture medium was replaced every 3 days.

RNA extraction and quantitative real-time polymerase chain reaction (qPCR) analyses
RNA extracted from tissue samples were isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. The primer sequences for qPCR are displayed in Table S1. qPCR analyses were performed on MX3000p real-time PCR (Agilent, USA). The relative miRNA and mRNA expression levels were analyzed using the 2 -ΔΔCt method.

Immunohistochemistry (IHC)
IHC was performed to evaluate the osteogenesis-related proteins expression in RP tissue. Paraffin-embedded sections were dewaxed and rehydrated before treatment with 3% hydrogen peroxide for 30 minutes. Antigen retrieval and incubation with anti-Runx2 antibody (RRID: AB-10888180), anti-osteocalcin (anti-OCN) antibody, and anti-osteopontin (anti-OPN) antibody (RRID: AB-2194998) were performed. After 24 h, the tissue sections were incubated with horseradish-conjugated secondary antibodies. Subsequently, all sections were stained with hematoxylin. Positive protein expression was indicated by brown granules in the cytoplasm or matrix. The staining intensity was graded into four levels: 0 was negative staining, 1 was low intensity, 2 was moderate intensity, and 3 was high intensity. The percentages of positively stained cells were also divided into four levels: 0 was 0%, 1 was ≤25%, 2 was ≤50%, 3 was ≤75%, and 4 was ≤100%. Histological scores were determined by the sum of intensity scores and the percentage of positively stained cells. Protein expression levels were considered low if histological scores were ≤3, while scores ≥4 and ≤ 7 were considered high.

Alizarin red staining
Alizarin red staining was performed to estimate the deposits of calcium aggregates. After treatment with osteogenic induction medium, the hRIFs were washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and subsequently stained with 1% alizarin red staining for 5 min. Calcified nodules were observed under an inverted microscope and the absorbance at 570 nm was read in triplicate.

Western blot
Western blot was conducted to examine the protein expression of target genes. After hRIFs were induced and transfected, cells were harvested for Western blot analysis. The cells were homogenized in ice-cold suspension and lysate (Beyotime, China) was added for total protein extraction. An equivalent amount of protein was fractionated on SDS polyacrylamide gels before immunoblotting with the following main antibodies: anti-Runx2 (Abcam, UK), anti-OCN (Abcam, UK) or anti-OPN (ZSGB-BIO, China). The membrane was incubated with a peroxidase-conjugated secondary antibody. The blotted bands were quantified using Image J (Bethesda, USA).

Statistical analysis
Data are expressed as the mean ± standard deviation (SD). The statistical significance between two groups was analyzed by a Student's t-test. P values <0.05 were considered statistically significant. Data analyses were performed using Statistical Package for the Social Sciences (SPSS version 17.0, Chicago, IL, USA).

Low expression of miRNA-410-3p in RP tissues
qPCR analysis of miRNA-410-3p expression in RP and nRP tissues displayed low levels of miRNA-410-3p in RP compared with control nRP tissues ( Figure 1A). This suggests that miRNA-410-3p may participate in the progression of RP formation.

Osteogenesis-related protein expression in RP tissues
qPCR and IHC were employed to assess the expression levels of osteogenesis-related protein in RP and nRP tissues. qPCR revealed higher protein levels of Runx2, OCN, and OPN in RP tissues ( Figure 1B), with the same trend noted via IHC staining ( Figure 1C). The results indicate that osteogenic-like differentiation may participate in the  (C) MTT assay showed the cell proliferation ability of hRIFs was inhibited at 5, 9, 14 days compared to control group; (D) the hRIFs were treated with osteogenic medium, the calcium deposition showed a time-dependent trend and was most significantly at 14 days by alizarin red staining (×100); (E) the hRIFs were treated with calcium chloride, the calcium deposition showed a time-dependent trend and was most significantly at 14 days by alizarin red staining (×100); (F) the absorbance was detected at 570 nm, the results showed the deposits of calcium aggregates were significantly at 9 and 14 days by treated with osteogenic medium or calcium chloride. *, P<0.05. hRIFs, human renal interstitial fibroblasts; MTT assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

Establishment and identification of hRIFs
hRIFs were isolated from human kidney papilla, and identified by morphological examination and immunofluorescence staining after extraction and culture.
The results revealed hRIFs to be shuttle-shaped cells (Figure 2A) with positive vimentin expression and negative epithelial membrane antigen expression ( Figure 2B).

The hRIFs showed osteogenic-like differentiation potential
MTT assay was performed at 1, 5, 9, and 14 days after the hRIFs were treated with osteogenic medium or calcium chloride. The results showed that cell proliferation was inhibited at 5, 9, and 14 days compared to the control group ( Figure 2C). Alizarin red staining was performed to estimate the deposits of calcium aggregates. Calcium deposition increased over time, and was most abundant at day 14 ( Figure 2D,E,F). hRIFs treated with calcium chloride during osteogenic-like differentiation displayed decreasing miRNA-410-3p levels with the prolongation of induction ( Figure 3A). In contrast, osteogenesis proteins Runx2 ( Figure 3B), OCN ( Figure 3C), and OPN ( Figure 3D) levels increased under the same conditions. The results suggest that hRIFs possess osteogenic-like differentiation potential.

miRNA-410-3p regulates the osteogenic-like differentiation of hRIFs by inhibiting MSX2
To further confirm the inhibitory mechanism of miRNA-410-3p in hRIF osteogenic-like differentiation, bioinformatics analysis was performed. A potential binding sequence between miRNA-410-3p and MSX2 was identified by TargetScan ( Figure 6A), with binding confirmed by the dual-luciferase reporter. Luciferase activity was reduced in cells co-transfected with miRNA-410-3p mimics and WT-MSX2 ( Figure 6B). Furthermore, real-time qPCR and Western blot analysis revealed the ability of miRNA-410-3p to effectively reduce the expression levels of MSX2 ( Figure 6C,D), while the miRNA-410-3p inhibitor elevated MSX2 expression ( Figure 6C,E). The above results suggested that miR-410-3p participated in the osteogeniclike differentiation of hRIFs by directly targeting MSX2.

MSX2 promoted osteogenic-like differentiation of hRIFs
To determine the effect of MSX2 on osteogenic-like differentiation in hRIFs, these cells were transfected with NC (non-specific control), miRNA-410-3p mimics, or miRNA-410-3p mimics plus MSX2 plasmids. MSX2 protein decreased when miRNA-410-3p was overexpressed. This phenomenon was reversed by MSX2 overexpression (Figure 7A,B). A negative correlation was observed between the volume and content of calcified nodules with miRNA-410-3p overexpression. However, co-transfection of pcDNA-MSX2 ( Figure 7C,D) led to a reversal of this trend. MSX2 was also shown to overcome the downregulation of    Figure 7E) and Osterix ( Figure 7F) during miRNA-410-3p overexpression. The above findings demonstrate that low expression levels of miRNA-410-3p enhanced the upregulation effects of MSX2 on the osteogenic-like differentiation of hRIFs.

Discussion
In 1937, RP were identified and defined as interstitial deposits of calcium phosphate (CaP) and calcium carbonate, appearing as "type I" papillary calcification on the renal papillary surface. These findings were based on the dissection of over 1,000 kidney pairs (19). In general, stone formation results from precipitation of urinary salts after exposure of primary plaques to urine (20,21). RP, while observed in both non-stone formers and stone formers, is more commonly observed in the latter group (22)(23)(24). The presences of calcified lesions have been shown to be precursors of stone formation. Analysis by Evan et al. clarified the microscopic structure of RP; they found that plaque originated in the basement membrane of the thin loops of Henle and extended through the interstitium to the region inferior to the urothelium (25). Khan et al., under scanning and transmission electron microscopic analysis (24), found plaque to appear as a small protuberance. Detailed scanning analyses showed plaques to be aggregates of spherical CaP crystals mixed with fibers of varying thickness and other cellular products. Urine  (6). Collectively, these studies suggest that hypercalciuria is closely related to the formation of RP and kidney stones.
Ectopic calcification is an established process in various pathological processes, including kidney stone formation and atherosclerosis (26). There is a body of evidence which supports vascular calcification as an actively regulated process which is similar to bone development. Lineage tracing studies in mouse models have shown that vascular smooth muscle cells can transdifferentiate into osteochondrogenic cells (27). Bone osteoid proteins such as BMPs, Runx2, and MSX2 have been shown to be involved in these processes. Recent studies have found that renal epithelial cells also have the capacity to become osteogenic. Jia et al. have shown that bone-related factors (BMP2, Runx2, Osterix, and OPN) were significantly increased in genetic hypercalciuric rats (28). Using genome-wide analysis, Okada et al. found that the expression of OPN in renal tubular cells and the migration of macrophages in the interstitial space around crystals were necessary for stone formation (29).
Kageyama et al. found Madin-Darby canine kidney cells could produce CaP crystal on the basal side when grown in monolayers (30). When these cells were exposed to a highoxalate environment, and to CaOx and CaP crystals, the resulting NADPH oxidase activation and reactive oxygen production led to osteogenic phenotypes. Umekawa et al. found oxalate and CaOx crystals upregulated OPN in renal fibroblasts (31). Evan et al. found that Runx2 was expressed in the RP of hypercalciuria patients (positive in the interstitial cells but not tubular epithelial cells) (32). However, idiopathic calcium stone formers showed no expression of Runx2 at sites of RP. In the study of Mezzabotta et al., mesenchymal cells which did not produce calcified nodules stained positive for Runx2 and OPN (33). These studies indicated that the RP may be a perivascular niche in which epithelial and interstitial cells undergo osteogenic differentiation under specific conditions. hRIFs are one of the main cells in kidney interstitial tissue. Studies have shown that the fibroblasts in multiorganization have osteogenic differentiation potential (34,35). However, there are no reports on the osteogeniclike differentiation of hRIFs. Based on the above evidence, we speculated that hypercalciuria may induce the formation of RP by stimulating osteogenic-like differentiation of kidney interstitial tissue. In this study, we found osteogenesis-related proteins to be highly expressed in RP tissues. The hRIFs showed osteogeniclike differentiation potential after treatment with calcium. Additional investigations confirmed miR-410-3p regulated the osteogenic-like differentiation by directly targeting MSX2 in hRIFs. The low-expression of miRNA-410-3p led to a stimulatory effect of MSX2 on the osteogenic- In summary, the results of our study revealed that hRIFs have osteogenic-like differentiation potential, and that miRNA-410-3p regulates hRIFs osteogenic differentiation by inhibiting MSX2. These results are relevant to enhancing our understanding of the pathogenesis of kidney stones and indicate a novel potential therapeutic target.

Acknowledgments
Funding: This research was supported by grants from the National Natural Science Foundation of China (81770705) and the Youth Science Foundation of Xiangya Hospital (2019Q02). Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of the Xiangya Hospital, Central South University (No. 201703258) and informed consent was taken from all the patients.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the noncommercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/ licenses/by-nc-nd/4.0/.