Therapeutic potential of BMSC-conditioned medium in an in vitro model of renal fibrosis using the RPTEC/TERT1 cell line

Abstract

We investigated the therapeutic potential of bone marrow-derived mesenchymal stem cell-conditioned medium (BMSC-CM) on immortalized renal proximal tubule epithelial cells (RPTEC/TERT1) in a fibrotic environment. To replicate the increased stiffness characteristic of kidneys in chronic kidney disease, we utilized polyacrylamide gel platforms. A stiff matrix was shown to increase α-smooth muscle actin (α-SMA) levels, indicating fibrogenic activation in RPTEC/TERT1 cells. Interestingly, treatment with BMSC-CM resulted in significant reductions in the levels of fibrotic markers (α-SMA and vimentin) and increases in the levels of the epithelial marker E-cadherin and aquaporin 7, particularly under stiff conditions. Furthermore, BMSC-CM modified microRNA (miRNA) expression and reduced oxidative stress levels in these cells. Our findings suggest that BMSC-CM can modulate cellular morphology, miRNA expression, and oxidative stress in RPTEC/TERT1 cells, highlighting its therapeutic potential in fibrotic kidney disease.

INTRODUCTION

Chronic kidney disease (CKD) affects over 10% of the global population and is characterized by progressive alterations in kidney structure and function (1, 2). Renal fibrosis, a common pathological outcome of CKD, involves the accumulation of extracellular matrix (ECM), leading to disruption of renal function (3, 4). Long-term kidney fibrosis is marked by glomerulosclerosis, vascular sclerosis, and tubulointerstitial fibrosis due to imbalances in kidney repair processes, including cell proliferation, inflammation, differentiation, and ECM synthesis (5). Dysregulation of these processes leads to ECM deposition, conversion to a myofibroblast phenotype, and potentially to kidney failure, scarring, and CKD progression (6). Notably, the significant presence of senescent cells, primarily observed among renal tubular epithelial cells, has been documented in various animal models and the kidneys of patients with CKD (7). This accumulation of senescent cells in renal tissues contributes to the progression of CKD. Progression to end-stage disease necessitates renal replacement therapy. However, renal transplantation has a number of challenges, prompting focus on regeneration of injured nephrons (1).

Mesenchymal stem cells (MSCs), renowned for their multipotent differentiation, homing effect, and immunomodulatory functions, have demonstrated significant potential in tissue regeneration and organ transplantation (8, 9). Recent animal studies have highlighted that both MSCs and their conditioned medium (CM) can enhance kidney function (10), an effect attributed to their paracrine and immunomodulatory activities (11-13). In vitro models, which are more affordable and convenient than animal models, have been used to understand the molecular mechanisms of MSC action in CKD. For example, albumin-induced renal proximal tubule epithelial cells (RPTECs) co-cultured with bone marrow-derived MSCs (BM-MSCs) significantly decreased the levels of epithelial-mesenchymal transition (EMT) and fibrotic markers (14). Cisplatin-induced kidney epithelial cells treated with umbilical cord-derived MSC-conditioned medium inhibited EMT and improved renal fibrosis (11), while umbilical cord-derived MSC exosomes reduced apoptosis and oxidative stress (15). One of the critical challenges in modeling CKD in vitro has been replicating the mechanical environment of the kidney, particularly the stiffness characteristic of healthy versus CKD-affected tissues. While traditional models often utilize tissue culture polystyrene (TCP), this substrate differs significantly from the in vivo conditions, primarily in terms of stiffness, which is typically much higher in TCP than physiological levels. This discrepancy is notable as kidney stiffness is a key diagnostic marker of CKD, with the kidneys of healthy individuals exhibiting stiffness in the range of approximately 3.68-8 kPa in contrast to 5.55-33.86 kPa in patients with CKD (16-18). To address this gap, recent studies have employed mechanically tunable substrates, such as collagen (19, 20), polyacrylamide (PAA) (21), polydimethylsiloxane (22), and synthetic hydrogels (23, 24). The stiffness of these substrates more accurately mimics in vivo stiffness, thereby providing a more representative environment for studying CKD.

In this study, we used a PAA gel platform to simulate the stiffness characteristic of kidneys in patients with CKD and cultured RPTEC/TERT1, a human telomerase reverse transcriptase (hTERT)-immortalized renal epithelial cell line. We found that cells cultured on a stiffer matrix exhibited elevated levels of α-smooth muscle actin (α-SMA), indicative of activated fibrogenic cells. Notably, treatment with bone marrow-derived mesenchymal stem cell-conditioned medium (BMSC-CM) significantly reduced the expression of α-SMA and vimentin under both soft and stiff conditions, suggesting reversal of fibrotic tendencies. In contrast, the epithelial marker E-cadherin and aquaporin 7 (AQP7), a water-selective membrane channel, showed increased expression following BMSC-CM treatment, especially on stiff substrates. To elucidate the molecular mechanisms of action of BMSC-CM, we conducted miRNA profiling, and identified 43 differentially expressed microRNAs (miRNAs). Target prediction and pathway analysis highlighted critical biological pathways, including cell senescence. In addition, we observed elevated levels of reactive oxygen species (ROS) under stiff conditions, which were markedly diminished following BMSC-CM treatment. Our results demonstrated that RPTEC/TERT1 can be cultured under physiologically relevant conditions of stiffness using the PAA gel platform, providing a valuable tool to monitor the in vitro therapeutic effects of BMSC-CM.

RESULTS

Matrix stiffness-induced morphological and α-SMA expression changes in RPTEC/TERT1

To explore the influence of substrate stiffness on the cellular morphology and expression of EMT markers in RPTEC/TERT1 cells, we created a fibrotic environment by developing PAA gels with two distinct levels of stiffness: a softer matrix at 3.9 kPa and a stiffer matrix at 32.7 kPa (Fig. 1A). The RPTEC/TERT1 cell line was then cultured on these substrates (Fig. 1B). While cell viability remained consistent between both substrates, cells cultured on the stiffer matrix exhibited increased proliferation (Supplementary Fig. 1). On the first day, cells on the softer substrate were smaller in size and had a larger aspect ratio compared with those on the stiffer substrate (Fig. 1C, D). However, after 5 days of culture, despite a similar cell area on both substrates (Fig. 1C), RPTEC/TERT1 on the stiff substrate developed significantly longer protrusions (Fig. 1D). Our analysis further extended to evaluating the expression of EMT markers. Upon transferring RPTEC/TERT1 from PAA gels to Transwell membrane filters for polarization, as described previously (25, 26), we noted a substantial increase in the level of α-SMA, a marker associated with mesenchymal cells, particularly in cells cultured on stiff substrates. This was in contrast to the expression of the epithelial-associated marker E-cadherin and another mesenchymal-associated marker, vimentin, which showed similar expression levels on both the soft and stiff matrices (Fig. 1E). Taken together, our findings suggest that RPTEC/TERT1 undergo morphological changes and express an elevated level of α-SMA when cultured on a stiff substrate.

Fig. 1.

The impact of matrix rigidity on RPTEC/TERT1 cellular morphology and EMT marker expression. (A) Young’s moduli (kPa) of soft and stiff PAA gels. The lines represent the median values of five independent experiments. (B) Morphology of RPTEC/TERT1 on days 1 and 5 of culture under different stiffness conditions. Scale bars, 100 μm. (C) Cell area and (D) cell aspect ratio of RPTEC/TERT1 on days 1 and 5. Central box, first to third quartile; middle line, median (n = 45 cells per group, three independent experiments, ^#P < 0.0001, Student’s two-tailed t test). (E) Representative immunofluorescence images of mesenchymal markers (α-SMA and vimentin) and an epithelial marker (E-cadherin) of RPTEC/TERT1 cultured on PAA gel plates. Scale bars, 25 μm.

BMSC-CM attenuates fibrosis and upregulates epithelial markers in RPTEC/TERT1

Further investigations were performed to assess the potential therapeutic effects of BMSC-CM. The CM generated from human BMSCs cultured in human platelet lysate was applied to RPTEC/TERT1 cultured under varying substrate stiffness conditions. After treatment with BMSC-CM for 24 h, we observed a notable increase in the cell area under both substrate conditions (Fig. 2A, B). The enhanced cellular protrusions were particularly evident in cells cultured on the soft substrate (Fig. 2C). Moreover, we observed significant decreases in the protein levels of mesenchymal markers, including α-SMA and vimentin, following BMSC-CM treatment, while the level of E-cadherin was increased (Fig. 2D, E). In addition, the AQP7 level, following the addition of water in the Transwell system, showed a substantial increase with BMSC-CM treatment, especially under stiff matrix conditions (Fig. 2D, E). These findings suggested that BMSC-CM not only modulates the morphological characteristics of RPTEC/TERT1 but also influences the expression of key markers, highlighting its potential therapeutic role.

Fig. 2.

Inhibitory effects of BMSC-CM on fibrosis and promotion of epithelial marker and aquaporin 7 (AQP7) expression in RPTEC/TERT1. (A) Morphology of BMSC-CM-treated RPTEC/TERT1 cultured under different stiffness conditions. Scale bars, 100 μm. (B) Cell area and (C) cell aspect ratio of RPTEC/TERT1 in BMSC-CM-treated and untreated groups. Central box, first to third quartile; middle line, median (n = 45 cells per group, three independent experiments, **P < 0.01, ^#P < 0.0001, Student’s two-tailed t test). (D) Representative immunofluorescence images for mesenchymal markers (α-SMA and vimentin), an epithelial marker (E-cadherin), and a tubular marker (AQP7) in RPTEC/TERT1. Scale bars, 25 μm. (E) Quantitative measurement of the fluorescence intensities of α-SMA (soft, n = 23; stiff, n = 48; soft + CM, n = 32; stiff + CM, n = 31, three independent experiments, **P < 0.01, ^#P < 0.0001, Student’s two-tailed t test), vimentin (n = 48 cells per group, two independent experiments, ^#P < 0.0001, Student’s two-tailed t test), E-cadherin (soft, n = 43; stiff, n = 35; soft + CM, n = 28; stiff + CM, n = 43, three independent experiments, ^#P < 0.0001, Student’s two-tailed t test), and AQP7 (soft, n = 48; stiff, n = 48; soft + CM, n = 47; stiff + CM, n = 47, two independent experiments, **P < 0.01, ^#P < 0.0001, Student’s two-tailed t test) staining.

Impact of BMSC-CM on miRNA expression profiles and associated pathways in RPTEC/TERT1

To understand the molecular mechanisms underpinning the effects of BMSC-CM on RPTEC/TERT1, particularly under stiff conditions, we conducted comprehensive global miRNA profiling using the NanoString nCounter human v3 miRNA expression panel. This analysis identified 43 differentially expressed miRNAs (25 upregulated and 18 downregulated) in RPTEC/TERT1 treated with BMSC-CM compared with untreated controls (P < 0.05) (Fig. 3A, B).

Fig. 3.

Modulation by BMSC-CM of the miRNA profiles and associated pathways in RPTEC/TERT1. (A) Volcano plot of the differentially expressed miRNAs obtained from miRNA expression profiling in the stiff and stiff + CM groups. (B) Heatmap of 43 miRNAs differentially expressed between the stiff + CM and stiff samples. (C) Schematics of target prediction and pathway analysis of the differentially expressed miRNAs. (D) Predicted targets of the miRNAs were analyzed using IPA software, and the top 10 canonical pathways are shown.

To understand the potential mechanisms underlying regulation of the miRNAs differentially expressed in BMSC-CM-treated RPTEC/TERT1, we attempted to determine their mRNA targets. A total of 6,126 targets of the 43 miRNAs were extracted from the miRNA target filter with high confidence levels using Ingenuity Pathway Analysis (IPA) software. We then analyzed the cellular functions and networks (Fig. 3C). The top 10 canonical pathways enriched among the target genes were associated with various biological processes and signaling pathways, including cell senescence, hepatic fibrosis signaling, regulation of the EMT by growth factor pathways, and wound healing signaling pathway (Fig. 3D). Taken together, distinct subsets of miRNAs exhibited differential expression in response to BMSC-CM treatment. Moreover, the putative target mRNAs of these miRNAs showed significant enrichment in diverse biological processes, notably cellular senescence.

Reduced oxidative stress in RPTEC/TERT1 by BMSC-CM treatment

ROS production and oxidative stress are critical contributors to cellular senescence, a state of permanent cell cycle arrest. Given the identification of senescence pathways as the top canonical pathways influenced by BMSC-CM treatment, we used the oxidation-sensitive fluorescent probe 2’7’-dichlorofluorescin diacetate (H₂DCF-DA) to assess intracellular ROS levels. This analysis demonstrated significant decreases in ROS production upon BMSC-CM treatment in RPTEC/TERT1 cultured under both soft and stiff conditions (Fig. 4A). Furthermore, we confirmed decreased mitochondrial superoxide anion levels by MitoSOX staining, with significantly decreased levels of MitoSOX observed in the BMSC-CM-treated groups, including those cultured under both soft and stiff PAA gel conditions (Fig. 4B). In addition, we monitored the phosphorylation status of p53 at serine 15, a key factor in cellular senescence pathways. Consistent with the decreased levels of ROS following BMSC-CM treatment, we noted a significant decrease in phosphorylated (p)-p53-positive cells after 24 h of exposure to BMSC-CM in RPTEC/TERT1 cultured on both soft and stiff PAA gels (Fig. 4C). Quantitative analysis revealed that the p-p53 level was reduced by 32.2% and 34.5% under soft and stiff conditions, respectively (Fig. 4D). Taken together, these findings suggested that BMSC-CM has a multifaceted effect on RPTEC/TERT1 cultured on PAA gel, particularly by regulating oxidative stress levels, thereby reinforcing its therapeutic potential under fibrotic conditions.

Fig. 4.

Decreased oxidative stress in RPTEC/TERT1 following treatment with BMSC-CM. Representative fluorescence images of RPTEC/TERT1 stained with (A) H₂DCF-DA and (B) MitoSOX. Scale bars, 100 μm. (C) Representative fluorescence images of p-p53 (green), p53 (red), and F-actin (pink). Scale bars, 50 μm. (D) Quantification of p-p53 intensity in RPTEC/TERT1 (soft, n = 26; stiff, n = 26; soft + CM, n = 30; stiff + CM, n = 27, two independent experiments, ^#P < 0.0001, Student’s two-tailed t test).

DISCUSSION

Our research utilizing the PAA gel platform and cultured RPTEC/TERT1 has contributed to understanding the mechanisms of renal fibrosis and the therapeutic potential of MSCs. We demonstrated that BMSC-CM significantly reduced fibrotic marker levels in these cells, suggesting reversal of the fibrotic process, consistent with previous research underscoring the potential of MSCs in treating fibrosis (13).

Renal fibrosis, leading to end-stage kidney disease, is typically marked by excessive ECM deposition and loss of renal function. Traditional treatments have focused on inhibiting ECM production or enhancing its degradation. However, recent research identified kidney fibroblast activation and tubular epithelial cell injury as central factors involved in fibrosis (27-29). In this context, the RPTEC/TERT1 cell line serves as a critical model for understanding the fibrotic process and evaluating the therapeutic efficacy of MSC-CM.

The use of PAA gel platforms in the present study allowed us to emulate the mechanical environment of CKD in vivo. PAA gels offer biocompatibility and transparency, and their tunable stiffness enabled us to replicate the mechanical properties of renal tissue in CKD (24). This approach is crucial for understanding the often-overlooked biomechanical aspects of fibrosis in conventional in vitro models.

In the present study, we identified 43 miRNAs with differential expression in RPTEC/TERT1 treated with BMSC-CM under fibrotic conditions. This is significant as miRNAs play crucial roles in regulating gene expression and cellular processes involved in kidney diseases, including renal fibrosis (30-32). Notably, miR-21 acts as a profibrotic factor, and its upregulation is correlated with increased severity of kidney fibrosis and renal function decline, particularly in diabetic nephropathy (33, 34). Conversely, miR-27b-3p shows antifibrotic properties, effectively suppressing TGF-β1-induced EMT and reducing fibrotic marker levels, thereby attenuating renal fibrosis in vitro and in unilateral ureteral obstruction mouse models (30, 35). Intriguingly, our miRNA profiling analysis further revealed that BMSC-CM treatment led to upregulation of miR-27a-3p and miR-205, consistent with previous findings indicating that miR-205 and the miR-200 family are significantly downregulated during EMT (36). Given the regulatory role of miRNAs in fibrosis, the ability of BMSC-CM to alter miRNA expression suggests that it may have therapeutic potential. In addition, our study underscored the need for further research to elucidate the precise mechanisms by which BMSC-CM influences miRNA expression and its subsequent impact on renal fibrosis. Such investigations could involve in-depth functional analyses of the identified miRNAs, their target genes, and the associated cellular pathways. This will not only enhance our understanding of the pathophysiology of renal fibrosis but also aid in the development of miRNA-based therapeutic strategies for CKD.

In conclusion, this study highlighted the promising role of MSCs in regenerative medicine, particularly in the treatment of fibrotic diseases. BMSC-CM exhibited significant antifibrotic effects on renal cells, not only reversing changes in fibrotic markers in RPTEC/TERT1 but also altering the oxidative microenvironment. These findings elucidated the cellular and molecular mechanisms underlying renal fibrosis and support the use of MSC-based therapies in CKD and other fibrotic conditions. The insights gained from this study contribute substantially to the field, paving the way for future research in MSC-based therapeutic development for CKD and related diseases, thereby marking significant advances in the understanding and treatment of renal fibrosis.

MATERIALS AND METHODS

Materials and methods are available in the Supplemental information.

Funding Statement

ACKNOWLEDGEMENTS This work was supported by the 2022 Sabbatical Year of Soonchunhyang University, the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1I1A3048341), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI18C0283).