miR-103a-3p Silencing Ameliorates Calcium Oxalate Deposition in Rat Kidney by Activating the UMOD/TRPV5 Axis

Maintaining the balance of calcium (Ca2+) metabolism in the kidney is crucial in preventing the formation of kidney stones. Functionally, the microRNA (miRNA) participating in this process needs to be unveiled. We induced NRK-52E cell injury by oxalate treatment. The role of transient receptor potential cation channel subfamily V member 5 (TRPV5) in oxalate-induced cells was studied by TRPV5 overexpression transfection, qRT-PCR, Western blot, MTT, and crystal adhesion detection. After identifying uromodulin (UMOD) expression in injured cells, we confirmed the interaction between TRPV5 and UMOD by coimmunoprecipitation (CoIP) and cell-surface biotinylation assays. The validation of UMOD-regulating TRPV5 in viability, crystal adhesion, and Ca2+ concentration of oxalate-induced cells was performed. Bioinformatics analysis and luciferase assay were used to identify the miRNA-targeting UMOD. The role of the miR-103a-3p-regulating UMOD/TRPV5 axis was detected by rescue experiments. We constructed a rat model with treatment of ethylene glycol (EG) to investigate the miR-103a-3p/UMOD/TRPV5 axis in vivo by hematoxylin-eosin (H&E) staining, Western blot, and immunohistochemistry (IHC). Upregulation of TRPV5 protected NRK-52E cells from oxalate-induced injury by enhancing cell viability and inhibiting CaOx adhesion. UMOD was depleted in oxalate-induced cells and positively interacted with TRPV5. UMOD silencing reversed the effect of TRPV overexpression on oxalate-induced cells. miR-103a-3p targeted UMOD and was mediated in the regulation of the UMOD/TRPV5 axis in oxalate-induced cells. Downregulating miR-103a-3p mitigated EG-induced CaOx deposition in kidney tissues in vivo by activating the UMOD/TRPV5 axis. miR-103a-3p silencing ameliorated CaOx deposition in the rat kidney by activating the UMOD/TRPV5 axis.


Introduction
Urinary calculi, one of the most common diseases in urology, are caused by the precipitation of oversaturated crystals from the urine in the kidney [1]. Although not as aggressive as malignant tumors, urinary calculi have long had a serious impact on human health due to their high incidence and recurrence rate [2]. Studies have confirmed that the onset of urinary calculi is multifactorial and involves genetics, metabolism, environmental climate, and lifestyle habits [3,4]. Calcium oxalate (CaOx) stones account for the majority of cases of this disease, and most patients with Ca 2+ -containing stones have a combination of hypercalciuria [5]. Physicochemical studies have verified that the saturation of CaOx in urine is related to the concentration of Ca 2+ and that increased urinary calcium predisposes to or promotes the formation of Ca 2+ -containing nephrolithiasis [6]. Kidney stones can lead to hypertension, chronic kidney disease, and end-stage renal disease. Studies have reported that preventive measures, including specific pharmacological interventions and recommendations for lifestyle and nutritional changes, can be pursued after a comprehensive metabolic assessment [7,8]. Various human studies have suggested that diets with a higher intake of vegetables and fruits play a role in the prevention of kidney stones [9][10][11]. At present, an in-depth study should be conducted on the molecular mechanism of hypercalciuria induced by abnormal calcium metabolism in the formation of kidney stones.
TRPV5 is an important protein that regulates the transport of Ca 2+ across cell membranes [12]. In renal tissues, TRPV5 is mainly found in the distal convoluted tubule (DCT) and collecting tubule (CNT) that is responsible for regulating urinary Ca 2+ reabsorption and maintaining Ca 2+ homeostasis in the body [13]. Available evidence suggests that downregulated TRPV5 expression may be a vital pathogenic factor in the formation of hypercalciuria and Ca 2+containing nephrolithiasis [14].
Uromodulin (UMOD), also known as Tamm-Horsfall protein, is the most abundant glycoprotein in urine [15], which is primarily expressed in renal tubular epithelial cells with a variety of physiological functions such as balancing the water-electrolyte metabolism, regulating immunity, preventing kidney stones, and protecting against urinary tract infections [16][17][18]. At present, there are few reports on the specific molecular mechanism of UMOD in kidney stone formation.
MicroRNAs (miRNAs) are a class of small, noncoding, single-stranded RNA molecules with a length of about 20-22 nucleotides, widely existing in mammals [19]. Mature miRNAs can regulate the post-transcriptional expression of target genes by binding fully or partially complementarily to the 3′-UTR of the target mRNA, resulting in the degradation or inhibition of protein translational synthesis [20]. Since miRNAs dramatically affect human physiological activities such as growth and development, and cell apoptosis and metabolism, they are considered to hold great promise for the prevention and treatment of diseases [21]. In recent years, research concerning the role of miRNAs in the onset and development of kidney stones has become a hot topic [22,23].
Therefore, this study probed into the relationship between UMOD and TRPV5 in the prevention of kidney stone formation and elucidated the role of miR-103a-3p in targeting and regulating UMOD, contributing to a deeper understanding of the pathogenesis of kidney stones and providing new ideas for the prevention and management of the disease.
2.3. RNA Extraction and qRT-PCR. Total RNA and miRNA from NRK-52E cells were extracted using Cell RNA Kit (19231ES50, Yeasen, China) and MolPure® Cell/Tissue miRNA Kit (19331ES50, Yeasen, China). Then, HiScript II One Step RT-PCR Kit (P612-01, Vazyme, China) was employed to react with the extract following the operating instructions. The amplification condition was set as follows: 50°C for 30 min, 94°C for 3 min, followed by 30 cycles (94°C for 30 s, 60°C for 30 s, and 72°C for 40 s), and 72°C for 5 min. The qRT-PCR was conducted in the ABI 7500 system (Applied Biosystems, USA). Relative mRNA expressions were analyzed by the 2 −ΔΔCt method [25], with β-actin or U6 using for normalization. All primers sequences were shown in Table 1.
2.5. Cell Viability Assay. Cell suspensions were prepared as described in this study [24]. NRK-52E cells were seeded in 96-well culture plates at a density of 5000 cells/well, and 10 μL methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (40201ES72, Yeasen, China) was used to treat the cells in each well at 37°C for 4 h, followed by SDS-HCL treatment. For cell viability evaluation, the optical density (OD) value at a wavelength of 570 nm was measured by a microplate reader (VL0L00D0, Thermo Fisher, USA).

Crystal Cell Adhesion Detection.
After oxalate treatment, NRK-52E cells (2 × 10 5 cells/well) were resuspended in a 6well plate for obtaining over 90% cell confluence. Then, the cells were washed with PBS and intubated in DMEM containing 40 μg/mL with Ponceau S-labelled CaOx monohydrate (COM) crystals at room temperature for 10 minutes (min). After being washed multiple times with PBS to remove unbound COM crystals, images were captured by an inverted microscope (IX-71, Olympus, Japan) and the number of adherent crystals in 10 randomized fields was calculated.

Coimmunoprecipitation (CoIP).
The physiological interaction between TRPV5 and UMOD was detected using CoIP Kit (abs955, Absin, China) in NRK-52E cells transfected   Relative UMOD protein expression levels ⁎⁎     ) were used in in vivo study. All rats were divided into 4 groups of control, EG, EG + antagomir, and EG + antagomir-NC, with ten rats in each group. During housing, in the control group, the rats were only provided with tap water; in the EG group, the rats were treated with 1% EG; in the EG + antagomir group, the rats were treated with 1% EG and intravenously injected with miR-103a-3p antagomir; and in the EG + antagomir-NC group, the rats were treated with 1% EG and intravenously injected with miR-103a-3p antagomir-NC. After 4 weeks, all rats were sacrificed by cervical dislocation under anesthesia (pentobarbital sodium, 40 mg/kg, P-010, Merck, USA) and kidney tissues were collected for histological study. The miR-103a-3p antagomirs used in this experiment were obtained from Ribobio (Guangzhou, China).     2.14. Statistical Analysis. All experiments were repeated three times individually, with measurement data describing as the mean ± standard deviation. Differences compared between multiple groups were analyzed by one-way analysis of variance. GraphPad Prism 8.0 (GraphPad Software, USA) was used for data analysis, and a p value < 0.05 indicated a statistically significant difference.

Upregulation of TRPV5 Protected NRK-52E Cells from
Oxalate-Induced Injury. Based on the pivotal role of TRPV5 in regulating Ca 2+ transport in renal tubular cells, we first performed TRPV5 overexpression in NRK-52E cells. As demonstrated in Figure 1(a), a significant increase of the TRPV5 mRNA level was detected by qRT-PCR (p < 0:001). Western blot has also measured the high level of TRPV5 protein in transfected cells (Figure 1(b), p < 0:001). After oxalate treatment in NRK-52E cells, we found that the expression of TRPV5 was downregulated compared to that of the control group, which was reversed by transfecting TRPV5-overexpressing plasmid (Figure 1(c), p < 0:001). In addition, the detection of Western blot presented similar outcomes (Figure 1(d), p < 0:001 ). The following MTT assay indicated that oxalate attenuated cell viability, which was partially overturned after TRPV5 overexpression (Figure 1(e), p < 0:05). The detection of crystal adhesion showed that TRPV5 overexpression strikingly inhibited the adhesion of CaOx induced by oxalate in NRK-52E cells (Figure 1(f)).

Oxalate Induction Downregulated UMOD Expression
which Was Positively Associated with the Enrichment of TRPV5 on the Cell Surface. The mRNA expression of UMOD was found to be downregulated in oxalate-induced NRK-52E cells (Figure 2(a), p < 0:001), so as its protein level (Figure 2(b), p < 0:01). The results of CoIP verified that UMOD was considerably coimmunoprecipitated by antiflag-tagged TRPV5, and conversely, TRPV5 was coimmunoprecipitated by anti-UMOD (Figure 2(c)). Next, we upregulated UMOD expression in NRK-52E cells which was verified by qRT-PCR and Western blot. The remarkable elevation of UMOD expression determined in mRNA (Figure 2(d), p < 0:001) and protein levels ( Figure 2(e), p < 0:001) signified the successful transfection. Furthermore, the results of cell-surface biotinylation assay revealed that TRPV5 was much in abundance on the surface of NRK-52E cells cotransfected with UMOD and TRPV5 overexpression, compared with its negative control (Figure 3(a)).

UMOD Regulated the Protecting Role of TRPV5 in
Oxalate-Induced Cells. (Figure 3(a)). In order to unravel the interplay of UMOD and TRPV5 in oxalate-induced NRK-52E cells, we conducted siUMOD transfection. QRT-PCR determined that UMOD mRNA is expressed at a low level in siUMOD-transfected cells (Figure 3(b), p < 0:001), and the similar result in its protein level was measured by Western blot (Figure 3(c), p < 0:001). Subsequently, MTT and crystal adhesion assays demonstrated that siUMOD notably offset the promoting effect of TRPV5 on cell viability ( Figure 3(d), p < 0:01) and the inhibiting effect of TRPV5 on the CaOx adhesion (Figure 3(e)) in NRK-52E cells induced by oxalate. Besides, we measured the Ca 2+ content after divergent transfections. As results described in Figure 3(f), TRPV5 markedly restrained oxalate-induced reduction in intracellular Ca 2+ concentration of NRK-52E cells, which was partially restored with the addition of siUMOD (p < 0:001).

miR-103a-3p Silencing Mitigated EG-Induced CaOx
Deposition in Kidney Tissues In Vivo by Activating the UMOD/TRPV5 Axis. For further bolstering our findings in vitro, we constructed the hyperoxaluria rat model for the in vivo study. As illustrated in Figures 6(a) and 6(b), substantial deposition of CaOx crystals in kidney tissues of the EG group was observed comparing to that in the control group and this trend was reversed by miR-103a-3p antagomir (p < 0:001). Then, the results of Western blot indicated that EG strongly downregulated both UMOD and TRPV5 protein levels, which was markedly restored by miR-103a-3p antagomir (Figure 6(c), p < 0:05). By IHC, we noticed that TRPV5 expression was evidently downregulated in the EG group compared to the control group and it was upregulated in the EG + antagomir group compared to the EG + antagomir-NC group ( Figure 6(d)).

Discussion
In the context of increasingly diverse surgical treatments against kidney stones, what should be underscored lies in prominent methods to prevent its occurrence and recurrence [27]. The most common component of kidney stones is CaOx crystals. There are many factors that influence the development of Ca 2+ -contained kidney stones, with hypercalciuria being by far the main recognized risk factor [28]. Tsuruoka et al. demonstrated that through the renal tubules, reduced Ca 2+ reabsorption is instrumental in the induction of hypercalciuria and that active calcium reabsorption could be regulated by various factors [29]. Later, Worcester et al.
proposed that postprandial urinary Ca 2+ levels were starkly higher in patients with hypercalciuria compared to the controls, even when blood parathyroid hormone secretion and renal load were at the same level [30]. Collectively, it is connoted that impaired renal tubular reabsorption may be the foremost reason for hypercalciuria. This study therefore explored the underlying molecular mechanism that regulates urinary Ca 2+ levels from the perspective of Ca 2+ reabsorption, so as to put forward momentous channels to prevent and treat kidney stones. TRPV5 acts as a renal tubular epithelial Ca 2+ channel that mediates Ca 2+ transport and reabsorption in the kidney [31]. Jang et al. pointed out that TRPV5 protein expression was overtly downregulated in the renal tissues of hypercalciuric rats but was restored by hydrochlorothiazide administration, indicating that the mechanism of hydrochlorothiazide treatment for hypercalciuria may be achieved through upregulation of TRPV5 protein expression in the renal tissues [32]. Studies have validated that high oxaluria induces reactive oxygen species (ROS) production, damages renal tubular epithelial cells, and alters the structure of phosphatidylserine in the cell membrane, leading to adhesion of CaOx to the surface of renal tubular epithelial cells [33]. In the previous research, we uncovered that 13 Disease Markers remarkably downregulated TRPV5 appeared after inducing NRK-52E cell injury by oxalate, the trend of which was reversed after transfection of TRPV5 overexpression. Additionally, upregulation of TRPV5 enhanced cell viability and inhibited CaOx adhesion in NRK-52E cells induced by oxalate, indicating that TRPV5 generates a critical preventive effect on oxalate-induced CaOx stone formation in the kidney.
As a primary channel for Ca 2+ reabsorption, the activity and expression of TRPV5 could be regulated by multiple factors [34]. For instance, a recent study confirmed that UMOD prevents the formation of kidney stones by stimulating Ca 2+ reabsorption via TRPV5 [35]. Moreover, it was noted that 87.5% of mice with UMOD knockout formed calcium oxalate and calcium phosphate stones in the CNT, renal medulla, and papillae at 15 months of age. All these findings suggested that UMOD could interact with TRPV5 in the formation of kidney stones, which has not been borne out yet. However, our study identified for the first time the existence the interaction that UMOD positively regulated TRPV5 expression functioning in injured NRK-52E cells by strengthening cell viability, reducing CaOx adhesion, and enhancing the reabsorption of Ca 2+ . miRNAs are involved in the development and progression of kidney diseases through their targeted regulation of mRNAs. miR-21 was found to contribute oxalate-induced renal tubular cell injury by upregulating PPARA [36]. In addition, Jiang et al. reported that inhibiting miR-155-5p to upregulate MGP expression could observably attenuate oxalate-induced oxidative stress injury in the kidney [37]. However, few studies have devoted to ascertaining the mechanism of miRNA regulation on UMOD affecting kidney stone formation. In order to gain a further insight into the pathogenesis of this disease, this study employed bioinformatics analysis and luciferase assay to find the miRNA, miR-103a-3p, potentially regulating UMOD. In renal disease research, the miR-103a-3p cycle has been confirmed to make profound impacts upon renal inflammation and fibrosis [38]. Peters et al. also suggested that miR-103a-3p could be regarded as a potential biomarker in chronic kidney disease [39]. In our study, interestingly, we verified the molecular mechanism of miR-103a-3p in kidney stones that miR-103a-3p silencing could alleviate oxalate-induced injury in NRK-52E cells by activating the UMOD/TRPV5 axis. Furthermore, we proved this regulatory mechanism even more strongly through in vivo experiments in EG-induced rat models. However, there is a lack of consideration in this study as it involves a single type of cell and a relatively small number of study variables in the in vitro experiments.

Conclusions
Overall, our current study unraveled that miR-103a-3p silencing ameliorates CaOx deposition in the rat kidney by activating the UMOD/TRPV5 axis, which lays a novel theoretical foundation for prevention, diagnosis, and treatment strategies towards kidney stones from the perspective of Ca 2+ reabsorption.

Data Availability
The analyzed datasets generated during the study are available from the corresponding author upon reasonable request.