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. 2025 Aug 11;44(6):899–915. doi: 10.23876/j.krcp.24.234

Tonsil-derived mesenchymal stem cells protect the kidney from gentamicin-induced acute kidney injury by incorporation into damaged renal tubules and amelioration of oxidative and endoplasmic reticulum stresses

Mina Yu 1,*, Dal-Ah Kim 1,*, Eun-Sun Ryu 1,*, Sung Min Jung 1, Sung-Chul Jung 2, Inho Jo 3, Han Su Kim 4, Duk-Hee Kang 1,✉
PMCID: PMC12611628  PMID: 40905041

Abstract

Background

Stem cell-based therapy is one of the tools for acute kidney injury (AKI) treatment. Tonsil tissue is a promising alternative source for the high-yield isolation of mesenchymal stem cells (MSCs). This study was undertaken to investigate the effects of tonsil-derived MSCs (T-MSCs) in animal model of AKI induced by gentamicin (GM).

Methods

Twenty Sprague-Dawley rats were divided into four groups: Control, GM (70 mg/kg/day, intraperitoneal injection for 10 days), GM + T-MSCs (1 × 107 cells, intravenous injection at 1 day after the last vehicle/GM), and T-MSCs. Renal function, apoptosis, and markers of endoplasmic reticulum stress were measured on day 16 after the first vehicle/GM. Oxidative stress was assessed by measuring urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and the expression of glutathione peroxidase (GPx) and catalase. Effects of T-MSCs on GM-induced apoptosis and oxidative stress in NRK cells were also evaluated using a co-culture technique of NRK cells and T-MSC.

Results

In the GM + T-MSCs group, blood urea nitrogen, creatinine, and tubular damage score were lower compared to the GM group. T-MSCs injection decreased apoptotic cells and the expression of Bax, cytochrome c, and cleaved caspase and increased Bcl-2. T-MSC injection decreased urinary 8-OHdG and increased expression of GPx and catalase in the kidneys. Anti-human nuclei and PKH26 staining demonstrated the localization of T-MSCs in the tubules of renal cortex. In-vitro study revealed that T-MSCs or T-MSC-conditioned media ameliorated GM-induced nicotinamide adenine dinucleotide phosphate oxidase-1 expression, hydrogen peroxide generation, and apoptosis of NRK cells.

Conclusion

Our study demonstrated that T-MSCs ameliorated GM-induced AKI by directly incorporating into the damaged renal tubules and exerting antiapoptotic and antioxidative effects.

Keywords: Acute kidney injury, Endoplasmic reticulum stress, Gentamicins, Oxidative stress; Tonsil-derived mesenchymal stem cells

Graphical abstract

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Introduction

Acute kidney injury (AKI) is a common complication among hospitalized patients and is an important cause of in-hospital mortality. The incidence of AKI continues to increase worldwide with an annual growth rate of 11% [1]. Indeed, a single episode of AKI carries a significant mortality risk with observed in-hospital mortality as high as 62% [2]. Many patients with AKI have a mixed etiology in which sepsis, renal ischemia, and nephrotoxicity often coexist and complicate recognition and treatment [3]. Current therapeutic measures in AKI are mainly supportive, including the avoidance of further damage and renal replacement therapy whenever necessary [4].

Stem cell-based therapy is the one which has emerged as a potential tool for AKI treatment [5,6]. Previous studies have shown the therapeutic effect of stem cells employing mesenchymal stem cells (MSCs), hematopoietic stem cells, and induced pluripotent stem cells (iPSCs) in preventing and repairing damaged renal cells in AKI. Among all cell populations applied for therapeutic purposes in AKI, MSCs have been most extensively evaluated showing the benefits in a renal generation in animal models of AKI induced by gentamicin (GM), cisplatin, and ischemia-reperfusion injury [5,7,8]. The responsible mechanisms provided by MSCs in AKI include direct (cell incorporation into the injured renal tubules) [9] and indirect processes, the latter being mediated by humoral factors [10] and the production of extracellular vesicles [11,12].

One of the major limitations of stem cell therapy is related to the availability of a sufficient number of MSCs at the moment in need. Recent studies demonstrated that tonsil-derived MSCs (T-MSCs) can be harvested from the tonsillar tissues after tonsillectomy [13]. Human palatine tonsils contained a multipotent MSC population with multilineage differentiation abilities and immunosuppressive properties equivalent to those of bone marrow-derived MSCs (BM-MSCs) [14,15]. T-MSCs could be successfully isolated and expanded in vitro by means of the standard procedure with a faster proliferation rate than BM-MSCs [15]. As T-MSCs can be easily obtained from surgically removed tissue that is supposed to be discarded, it can be a valuable means of recycling human tissue for stem cell therapy [16,17]. T-MSCs were reported to ameliorate experimental hepatic fibrosis and colitis via modulation of the inflammatory reaction [18]. However, there are no studies to examine the effect of T-MSCs on kidney diseases.

In this study, we investigated the effect of T-MSCs in the animal model of GM-induced AKI, one of the prevalent causes of AKI. We also explored the mechanisms for the renoprotective effect of T-MSCs in AKI by examining whether T-MSCs incorporate into damaged renal tubules, and ameliorate both oxidative and endoplasmic reticulum (ER) stresses, two major mechanisms of in GM-induced AKI [19,20].

Methods

Reagents

All chemicals and tissue culture plates were obtained from Sigma-Aldrich and Nunc Labware, unless otherwise stated.

Preparation of tonsil-derived mesenchymal stem cells

Human tonsils were obtained after Institutional Review Board approval from Ewha Womans University Mokdong Hospital, Seoul, Republic of Korea (No. ECT 11-53-02) and informed consent from patients undergoing tonsillectomy. T-MSC isolation from tonsil tissue was performed as described previously [17,21]. Briefly, extracted tonsils were minced and digested in a medium containing 210-U/mL collagenase type I (Invitrogen) and 10-μg/mL DNase (Sigma-Aldrich). After filtration through a cell strainer (BD Labware), T-MSCs were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37 °C with 5% CO2. Characterization of T-MSCs is shown in Supplementary method and Supplementary Fig. 1 (available online).

In-vivo experimental model of gentamicin-induced acute kidney injury and cell tracking

Male Sprague-Dawley rats (200–250 g, Orient) were randomly divided into four groups: Control, T-MSC, GM, and GM + T-MSC (n = 5/group) (Supplementary Fig. 2, available online). GM-induced AKI was established by daily intraperitoneal injection of GM (70 mg/kg/day) for 10 days. T-MSCs (1 × 107 cells/mL) were labeled with PKH26 fluorescent dye (Sigma) for in-vivo tracking, following the manufacturer’s protocol. Labeled cells suspended in 500-μL saline solution were injected via the tail vein on day 11. On day 16, all the rats were sacrificed for an assessment of renal function and histologic evaluation of the kidney. Blood urea nitrogen (BUN; QuantiChrom urea nitrogen assay kit, Bioassay Systems), creatinine (QuantiChrom creatinine assay kit), and urinary protein excretion (BCA protein assay kit, Pierce Biotechnology) were measured according to the manufacturer’s protocol.

All animal procedures were approved by the Institutional Animal Care and Use Committees of Ewha Womans University School of Medicine (No. ESM 12-0207).

Histological analysis

Tissue was fixed in Methyl Carnoy’s solution and embedded in paraffin. Then, 3-μm sections were stained with the periodic acid-Schiff reagent and counterstained with hematoxylin. To determine kidney tubular injury, a semiquantitative scoring method was used. Injury was graded from 0 to 4 according to the distribution of lesions: 0, none; 1, <5%; 2, 5%–25%; 3, 25%–75%; 4, >75%.

TdT-mediated dUTP nick-end labeling (TUNEL) staining

Apoptosis of renal cells was identified by TUNEL in-situ cell death detection kit (Roche Diagnostics). After the deparaffinization and dehydration of tissue section, nuclear proteins were stripped from the DNA by incubating in proteinase K for 20 minutes and endogenous peroxidase was blocked with hydrogen peroxide (H2O2). Sections were incubated in a buffer containing TdT and digoxigenin-labeled dUTP, followed by digoxigenin-conjugated peroxidase treatment. TUNEL signal was observed under a fluorescence microscope (×200). Apoptotic cells were stained by green fluorescence, and all cells were marked with blue fluorescence using 4’,6-diamidino-2-phenylindole (DAPI). The apoptotic ratio was calculated as tunnel-positive cells divided by total cell number.

Measurement of urinary 8-hydroxy-2’-deoxyguanosine

To evaluate the oxidative status in rats, urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) was measured using oxidative DNA damage enzyme-linked immunosorbent assay (ELISA) kit (Cell Biolabs). Urine samples were centrifuged at 10,000 ×g for 10 minutes after thawing, and the supernatants were added to an 8-OHdG/bovine serum albumin (BSA) conjugate preabsorbed plate. After a brief incubation at 37 °C, 8-OHdG content was determined by a competitive ELISA.

Western blot analysis

Protein samples from cell lysates or renal cortex tissue were mixed with reducing buffer, boiled, resolved on 10% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and transferred to a polyvinylidene difluoride membrane (MilliporeSigma) via electroblotting. Membranes were blocked with 3% BSA in phosphate-buffered saline (PBS) with Tween 20 (PBST) for 30 minutes at room temperature, then incubated overnight with primary antibodies (Supplementary Table 1, available online). After PBST washes, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, followed by chemiluminescence detection (Santa Cruz Biotechnology). Immunoreactive bands were quantified by densitometry and normalized to β-actin.

Immunohistochemistry of apoptosis-related protein, endoplasmic reticulum stress markers, tubular injury markers, and human nuclear antigen

Methyl Carnoy-fixed, paraffin-embedded kidney sections were deparaffinized, rehydrated, and washed with PBS. After blocking with 3% H2O2 and 5% normal serum, sections were incubated overnight at 4 °C with primary antibodies: B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax, Cell Signaling), Lys-Asp-Glu-Leu motif (KDEL, Enzo Life Sciences), kidney injury molecule-1 (KIM-1, Cell Signaling), and neutrophil gelatinase-associated lipocalin (NGAL, Abcam). Controls included omission of the primary antibody and substitution with preimmune mouse serum. The reaction product was visualized with diaminobenzidine (Dako). Images were viewed with a light microscope (BX51, Olympus). For immunofluorescence, sections were incubated with an anti-human nuclei antibody (1:200, MilliporeSigma) overnight, followed by an anti-mouse immunoglobulin G-fluorescein isothiocyanate (FITC) secondary antibody (1:200, Vector Laboratories), and mounted with DAPI (Vector Laboratories). Images were captured using a fluorescence microscope (Nikon Ti2-U, Nikon).

Co-culture with tonsil-derived mesenchymal stem cells and NRK-52E cells

To determine whether T-MSCs imposed any effect on the changes in renal tubular cells induced by GM, we used transwell culture with 0.4-μm inserts (Corning Inc.). NRK-52E cells were seeded in the bottom part of 6-well plates at a density of 1 × 105 cells/mL for transwell culture setup, whereas T-MSCs were seeded at the same density in the corresponding transwell cell culture inserts. NRK-52E cells in the lower well were first treated with 3-mM GM for 24 hours, and T-MSCs grown in the transwell were inserted. In 5 minutes to 24 hours, reactive oxygen species (ROS) production, messenger RNA (mRNA), and protein expression of NRK-52E cells were evaluated by the methods described.

Cell apoptosis assay: flow cytometric analysis for propidium iodide staining and Annexin V-fluorescein isothiocyanate binding

After exposure of co-culture with T-MSCs and NRK-52E cells to GM for 48 hours, cell suspensions were prepared by treating the cells with a trypsin/ethylenediaminetetraacetic acid mixture in DMEM. Cells were washed twice in cold PBS and then resuspended in 1× Annexin V-binding buffer at a concentration of 1 × 106 cells/mL. After incubation of 100-μL cell suspension with 5-μL FITC Annexin V and 5-μL propidium iodide (PI) in the dark for 15 minutes, 400 μL of 1× binding buffer was added to each tube. Samples were then analyzed on a FACS Calibur flow cytometer (Beckton Dickinson) and CellQuest software (Beckton Dickinson). Early and late apoptosis were assessed by measuring the fraction of Annexin V+/PI– and Annexin V+/PI+ cells, respectively.

Measurement of hydrogen peroxide production

H2O2 production was measured using Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen). NRK-52E and T-MSCs were mixed at a ratio of 6:1. Cells were incubated with 100-μM Amplex Red and 0.2-U/mL HRP mixture after exposure to GM. A serial fluorescence was measured using fluorescent ELISA reader at excitation 530 nm and emission 560 nm (Molecular Devices).

Measurement of NADPH oxidase activity

NRK-52E and T-MSCs were mixed at a ratio of 6:1 and exposed to GM (3 mM) for 5 to 120 minutes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activity was measured by a luminescence assay of superoxide production in a 50-mM phosphate buffer containing 1-mM EGTA (ethylene glycol-bis[β-aminoethyl ether]-N,N,N′,N′-tetraacetic acid), 150-mM sucrose, 5-μM lucigenin as the electron acceptor, and 100-μM NADPH as the substrate with an addition of 100 μL of cell homogenate. Nox activity was expressed as the rate of relative chemiluminescence.

Real-time polymerase chain reaction

Total RNA was extracted using RNeasy mini kit (Qiagen) and reverse-transcribed into complementary DNA (cDNA) with the Superscript First Strand Synthesis System (Life Technologies BRL). Real-time polymerase chain reaction (PCR) was performed on an ABI PRISM 7000 using SYBR Green PCR Master Mix (Applied Biosystems) following the manufacturer’s instructions. Reactions included 5-μL cDNA, 10-μL SYBR Green PCR Master Mix, and validated primers (Supplementary Table 2, available online). The relative mRNA expression levels of the target genes in each sample were calculated using the comparative cycle threshold (Ct) method, normalized to β-actin. The Ct value is the cycle number at which the fluorescence signal is greater than a defined threshold. In the statistical analysis, all PCR procedures were replicated at least three times.

Statistical analysis

Data are shown in box-and-whisker plot displaying minimum, median, upper and lower quartiles, and maximum. Differences in parameters at each time point and the concentration of GM or T-MSCs were compared by paired t test and one-way analysis of variance (ANOVA). Differences in the various parameters among groups were evaluated by one-way ANOVA followed by Tukey multiple comparisons. Significance was defined as p <0.05.

Results

Effect of tonsil-derived mesenchymal stem cell on kidney weight, proteinuria, and renal function

Kidney weight/body weight was higher in rats in the GM group compared to control rats, which was slightly decreased in the GM + T-MSCs group (Supplementary Figs. 2 and 3, available online). An increase in urinary protein excretion observed in the GM group was alleviated in the GM + T-MSCs group by 42% (Fig. 1A). Higher BUN and creatinine concentrations in the GM group were also significantly ameliorated in the GM + T-MSCs group (Fig. 1B, C). Tubular dilatation with interstitial cell infiltration in the kidneys of the GM group was alleviated by T-MSC injection (Fig. 1D, E).

Figure 1. Effect of T-MSCs on proteinuria, BUN, creatinine, and renal pathology.

Figure 1.

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In 10 days of gentamicin (GM) administration, proteinuria (A), BUN (B), and creatinine (C) were increased in the GM group. In the GM + T-MSCs group, renal function was significantly preserved compared to the GM group. Tubular dilatation and interstitial fibrosis were evident in the GM group at 10 days, which were preserved in the GM + T-MSCs group. Representative periodic acid-Schiff staining of the renal cortex (D: Control [a], GM [b], GM + T-MSCs [c], T-MSCs [d]; magnification, ×200; scale bar = 100 μm) with quantitation of tubular damage (E) is shown. Data are shown in box plots.

BUN, blood urea nitrogen; T-MSC, tonsil-derived mesenchymal stem cell.

*p < 0.05 vs. Control and T-MSCs, #p < 0.05 vs. GM.

Effect of tonsil-derived mesenchymal stem cell in apoptosis and the expression of the apoptosis-related proteins in gentamicin-induced acute kidney injury

GM administration resulted in an increase in apoptosis of renal tubular cells with upregulation of proapoptotic factors such as Bax, cytochrome c, cleaved caspase-9 and -3, and a decrease in antiapoptotic Bcl-2 (Fig. 2). T-MSC injection ameliorated apoptosis of renal tubular cells in GM-induced AKI shown as a decrease in TUNEL (+) cells in renal tubules (Fig. 2A), and reversed the changes in the expression of pro- and antiapoptotic factors in the kidneys (Fig. 2B–D).

Figure 2. Effect of T-MSCs in apoptosis and the expression of pro- and antiapoptotic proteins in an animal model of acute kidney injury.

Figure 2.

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(A) Gentamicin (GM) induced apoptosis of renal tubular cells assessed by TdT-mediated dUTP nick-end labeling (TUNEL) staining, which was alleviated in the GM + T-MSCs group. (A) Control (a–c), GM (d–f), GM + T-MSCs (g–i), T-MSCs (j–l); magnification, ×100; scale bar = 100 μm. Representative TUNEL staining with a quantitation bar is shown. Green fluorescein TUNEL (+) renal tubular cells are co-localized with nuclear 4’,6-diamidino-2-phenylindole (DAPI) staining (blue). (B, C) GM administration upregulates the expression of proapoptotic factors such as B-cell lymphoma 2 (Bcl-2)–associated X (Bax), cytochrome c (Cyt c), cleaved caspase-9 and -3 with a decrease in antiapoptotic Bcl-2. T-MSCs reverse the changes in the expression of pro- and antiapoptotic factors in the kidneys. Representative Western blots (B) with quantitation bar (C) are shown. (D) Immunohistochemistry reveals an increase in Bax in dilated renal tubular epithelial cells with a decrease in Bcl-2 in the GM group is alleviated in the GM + T-MSCs group (Control [a, e], GM [b, f], GM + T-MSCs [c, g], T-MSCs [d, h]; magnification, ×200; scale bar = 100 μm). Data are shown in box plots.

T-MSC, tonsil-derived mesenchymal stem cell.

*p < 0.05 vs. Control and T-MSCs, #p < 0.05 vs. GM.

Effect of tonsil-derived mesenchymal stem cell in endoplasmic reticulum stress and tubular injury in gentamicin-induced acute kidney injury

Since ER stress is one of the mechanisms of GM-induced AKI via inducing the apoptosis of renal tubular cells [22], we investigated the effect of GM and/or T-MSCs in the expression of ER stress markers. Glucose-regulated protein (GRP) 78/94 expression in the kidneys was significantly increased in the GM group, which was alleviated by T-MSCs (Fig. 3A–D). In particular, the enhanced expression of C/EBP homologous protein (CHOP) in GM-induced AKI was almost completely blocked by T-MSCs (Fig. 3B, D). To further evaluate the extent of tubular injury, we analyzed KIM-1 and NGAL expression using immunohistochemical and Western blotting. Both markers were markedly increased in the GM group, indicating severe tubular damage, but significantly reduced with T-MSC treatment (Fig. 3E, F). These findings further support the renoprotective effects of T-MSCs in GM-induced AKI.

Figure 3. Effect of T-MSCs on ER stress and tubular injury in an animal model of acute kidney injury.

Figure 3.

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(A–D) Gentamicin (GM) administration increases the expression of KDEL, a marker of ER stress, in damaged renal tubules in the renal cortex, which is ameliorated in the GM + T-MSCs group (magnification, ×200; scale bar = 100 μm). Representative immunohistochemical staining (A: Control [a], GM [b], GM + T-MSCs [c], T-MSCs [d]), Western blotting of glucose-regulated protein (GRP) 78/94 and C/EBP homologous protein (CHOP) (B) with quantitation bar (C, D) are shown. (E, F) The expression of tubular injury markers kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) is significantly increased in the GM group, indicating tubular damage, but is reduced with T-MSCs treatment. (E) Representative immunohistochemical (IHC) staining (magnification, ×200; scale bar = 100 μm). Representative IHC staining (E: Control [a, e], GM [b, f], GM + T-MSCs [c, g], T-MSCs [d, h]), Western blot analysis and quantification (F) are presented. Data are shown in box plots.

ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; T-MSC, tonsil-derived mesenchymal stem cell.

*p < 0.05 vs. Control and T-MSCs, #p < 0.05 vs. GM.

Effect of tonsil-derived mesenchymal stem cell in local oxidative stress in gentamicin-induced acute kidney injury

GM-induced AKI was associated with an increase in urinary excretion of 8-OHdG, a marker of oxidative stress (Fig. 4A). GM injection also resulted in a decrease in antioxidant enzymes such as glutathione peroxidase (GPx) and catalase (Fig. 4B). T-MSC treatment decreased oxidative stress with an enhanced expression of GPx and catalase, which indicated a favorable oxidant-antioxidant environment provided by T-MSCs. There were no changes in superoxide dismutase (SOD) by GM or T-MSCs (Fig. 4B).

Figure 4. Effect of T-MSCs on oxidative stress in the kidneys of an animal model of acute kidney injury.

Figure 4.

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(A) Urinary excretion of 8-hydroxy-2’-deoxyguanosine (8-OHdG), a marker of oxidative stress, is increased in the gentamicin (GM) group. (B) GM injection also results in a decrease in antioxidant enzymes such as glutathione peroxidase (GPx) and catalase. T-MSC treatment decreases oxidative stress with an enhanced expression of GPx and catalase whereas there is no change in the expression of superoxide dismutase 2 (SOD2) by GM or T-MSCs. Representative Western blotting with a quantitation bar is shown. Data are shown in box plots.

T-MSC, tonsil-derived mesenchymal stem cell.

*p < 0.05 vs. Control and T-MSCs, #p < 0.05 vs. GM.

Localization of injected tonsil-derived mesenchymal stem cells in the kidneys of gentamicin-induced acute kidney injury

To evaluate the homing ability of T-MSCs and their contribution to reno-protection, we tracked injected cells using PKH26 dye and a human-specific nuclear marker. PKH26-labeled T-MSCs were detected in the cytoplasm of renal tubular cells 5 days after injection in GM-induced AKI rats (Fig. A5), confirming their incorporation into the injured kidney. In addition, immunofluorescence staining with an anti-human nuclei antibody revealed T-MSCs localized in the renal tubules and interstitial areas of the cortex (Fig. 5B), further supporting their presence in damaged renal tissue.

Figure 5. Localization of T-MSCs in the kidney of GM-induced AKI.

Figure 5.

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(A, B) PKH26-stained T-MSCs (A: Control [a–c], GM + T-MSCs [d–f, j], T-MSCs [g–i]) and nuclei of T-MSCs (B: Control [a, b], T-MSCs [c, d], GM + T-MSCs [e–k]) are detected in renal tubules and interstitial area in 5 days after intravenous injection of T-MSCs in the animal model of GM-induced AKI. There was no positive staining of anti-human nuclei in control (a, b) and T-MSCs only (c, d) groups. In the GM + T-MSCs group, green fluorescent staining was detected in renal tubular cells (e–i) and tubulointerstitial area (j, k). Representative PKH26 (red) and human nuclei (green; red arrowheads) with nuclear 4’,6-diamidino-2-phenylindole (DAPI) (blue) staining is shown (magnification, ×400; scale bar = 50 μm).

AKI, acute kidney injury; GM, gentamicin; T-MSC, tonsil-derived mesenchymal stem cell.

*p < 0.05 vs. Control and T-MSCs, #p < 0.05 vs. GM.

Effect of tonsil-derived mesenchymal stem cells on apoptosis and the expression of the apoptosis-related protein in cultured renal tubular cells

In cultured renal tubular cells (NRK-52E cells), GM (3 mM) induced apoptosis (Fig. 6). Co-culture of T-MSCs (upper chamber) with NRK-52E cells (lower chamber) using transwell resulted in a significant decrease in GM (3 mM)-induced apoptosis at 48 hours assessed by flow cytometry and TUNEL staining (Fig. 6A–E). Co-culture of NRK-52E cells with T-MSCs resulted in a decrease in the percentage of early and late apoptotic cells, which was associated with altered expressions of Bax and Bcl-2 (Fig. 6F).

Figure 6. Effect of co-culture of renal tubular cells with T-MSCs in GM-induced apoptosis.

Figure 6.

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(A–D) GM (3 mM) induces apoptosis in cultured renal tubular cells (NRK-52E cells) assessed by FACScan analysis (A–C) and TdT-mediated dUTP nick-end labeling (TUNEL) staining (D: Control [a–c], GM [d–f], T-MSCs [g–i], GM + T-MSCs [j–l]). (F) Co-culture of T-MSCs (upper chamber) with NRK-52E (lower chamber) cells using transwell results in a significant decrease in apoptosis at 48 hours. GM-induced alteration of B-cell lymphoma 2 (Bcl-2)–associated X (Bax) and Bcl-2 is ameliorated by T-MSC co-culture. Representative early apoptotic (Annexin V [+]/PI [–]), late apoptotic (Annexin V [+]/PI [+]), and total apoptotic with quantitation bar (B, C). Representative TUNEL staining (D: green fluorescein with nuclear 4’,6-diamidino-2-phenylindole [DAPI] staining; magnification, ×200; scale bar = 100 μm) with quantitation bar (E) is shown. Representative Western blotting of Bax and Bcl-2 with quantitation bar is shown (F). Data are shown in box plots.

FITC, fluorescein isothiocyanate; GM, gentamicin; PI, propidium iodide; T-MSC, tonsil-derived mesenchymal stem cell.

*p < 0.05 vs. Control and T-MSCs, #p < 0.05 vs. GM without T-MSC.

Effect of tonsil-derived mesenchymal stem cell in endoplasmic reticulum stress in cultured renal tubular cells

GM increased GRP78/94 and CHOP expression in cultured NRK-52E cells from 12 hours, which was alleviated by co-culture with T-MSCs (Fig. 7).

Figure 7. Effect of T-MSCs in GM-induced endoplasmic reticulum stress in renal tubular cells.

Figure 7.

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(A, B) GM (3 mM) induces the expression of glucose-regulated protein (GRP) 78/94 and C/EBP-homologous protein (CHOP) in NRK-52E cells, which are blocked by T-MSC co-culture. Representative Western blotting of GRP78/94 and CHOP with quantitation bar is shown (n = 5). Data are shown in box plots.

GM, gentamicin; T-MSC, tonsil-derived mesenchymal stem cell.

*p < 0.05 vs. others.

Effect of tonsil-derived mesenchymal stem cells on oxidative stress in cultured renal tubular cells

GM increased ROS generation from 15 minutes shown as H2O2 generation and dichlorofluorescin diacetate staining (Fig. 8A, B). An early increase in ROS production was accompanied by enhanced activity of Nox (Fig. 8C). Not only an increase in Nox activity, but GM also increased the transcription of Nox (Fig. 8D). NOX1 was the major isoform of Nox expressed in NRK-52E cells, which was upregulated by GM from 1 hour. T-MSCs partially inhibited an upregulated NOX1 mRNA expression induced by GM (Fig. 8D, E). The decrease in antioxidant enzymes, GPx, and catalase, by GM in NRK-52E cells was also reversed by T-MSCs (Fig. 8F).

Figure 8. Effect of T-MSCs in GM-induced oxidative stress of renal tubular cells.

Figure 8.

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(A–C) GM (3 mM) induces reactive oxygen species (ROS) generation from 15 minutes shown as H2O2 generation (A) and dichlorofluorescin diacetate (DCF-DA) staining (B: Control [a], GM [b], T-MSCs [c], GM + T-MSCs [d]) with an increase in NADPH oxidase (Nox) activity (C) in NRK-52E cells. (D) GM also increases the transcription of NOX1 messenger RNA (mRNA), but not NOX2 and NOX4. (E, F) T-MSCs partially inhibited ROS generation and upregulated expression of NOX1 mRNA induced by GM. In addition, GM (3 mM) decreases the expression of glutathione peroxidase (GPx) and catalase in NRK-52E cells, which is also partially reversed by T-MSC. Representative DCF-DA staining (B: magnification, ×100; scale bar = 100 μm) and Western blotting with quantitation bar (F) are shown (n = 5). Data are shown in box plots.

GM, gentamicin; T-MSC, tonsil-derived mesenchymal stem cell.

*p < 0.05 vs. Control and T-MSCs, #p < 0.05 vs. GM without T-MSC.

Discussion

In this study, we demonstrated that T-MSCs alleviated GM-induced AKI via incorporation into damaged renal tubular cells. T-MSCs injection after GM administration led to a decrease in oxidative stress with amelioration of enhanced Nox activity and NOX1 transcription, which was accompanied by increased production of antioxidant enzymes. An enhanced ER stress and apoptosis in the kidneys of GM-induced AKI and cultured renal tubular cells were also attenuated by T-MSCs. Injected T-MSCs were identified at the renal tubules of GM-injected rats in 5 days of intravenous T-MSC administration.

Stem cell-based therapy has recently emerged as a potential and powerful therapeutic tool for AKI treatment. Different types of stem cells, including iPSCs, spermatogonial stem cells, and MSCs have been studied as therapeutic strategies for AKI [23]. Among available stem cells from different sources, MSC-based therapies have shown multiple beneficial outcomes in a vast amount of research without any serious side effects. Previous studies demonstrated the renoprotective effect of BM-MSCs in AKI [5,24]. However, BM-MSCs collection has some limitations. The collection is relatively invasive and bone marrow does not always provide a sufficient amount of MSCs [23,24]. To overcome these limitations, other potential sources for stem cells with the possibility of therapeutic applications have been extensively investigated [25,26].

The aim of our study is to investigate the therapeutic potential of T-MSCs from a novel source of MSCs, human palatine tonsil, in the treatment of GM-induced AKI. Human palatine tonsil is an attractive alternative source of adult stem cells for several reasons. First, it is readily available as surgically removed “waste tissue.” None of the invasive procedures are needed to cultivate MSCs. Second, the doubling time of T-MSCs in proliferation capacity is significantly lower than that of BM-MSCs, suggesting a higher proliferation rate [14]. The low population doubling time is an advantage of T-MSCs, as the required cell number for therapeutic purposes can be obtained in a shorter period of time. The increased proliferation capacity can be explained by donor age. Several studies have reported that the overall expansion potential, proliferation rate, and frequency of MSCs decrease with donor age [27,28]. As tonsillectomy is usually performed at a young age, T-MSCs can be a more suitable therapeutic tool compared with MSCs from other sources. Therapeutic effect of T-MSCs has been examined in acute liver injury [16], diabetes mellitus [29], and senile osteoporosis [30,31]; however, there is no investigation in kidney disease.

In this study, injection of T-MSCs in AKI-induced rats ameliorated renal dysfunction, as shown by decreases in urinary protein excretion, BUN, and creatinine levels. The histological indices of injury in the renal cortex and outer medulla were also improved by T-MSCs. Moreover, we demonstrated the antiapoptotic and antioxidative effects of T-MSCs in GM-induced AKI. T-MSCs treatment resulted in decreased numbers of apoptotic cells, by down-regulating apoptotic genes and up-regulating antiapoptotic genes with an alleviation of ER stress. Also, T-MSCs suppressed oxidative stress as reflected by the decrease in the level of urinary 8-OHdG. Concomitantly, T-MSCs increased the expression of antioxidant enzymes (GPx, catalase, and SOD) in the renal tissue.

Our study demonstrated both the direct and paracrine effects of T-MSCs. We clearly observed that the injected T-MSCs were preferentially located inside the damaged renal tubules in vivo, in contrast to the absence of such cells in the kidneys of the T-MSCs group (without GM), suggesting a direct effect of T-MSCs. This was confirmed by immunostaining with anti-human nuclei, which revealed the localization of the injected T-MSCs in the renal tubules and interstitial areas. In addition, the co-culture of renal tubular cells and T-MSCs using transwell resulted in a decrease in the percentage of apoptotic cells, suggesting the paracrine effect of T-MSCs, in vitro. Currently, the proposed mechanisms responsible for the beneficial effect of stem cell therapy include direct incorporation of injected cells or indirect acting through paracrine/endocrine effects on renal progenitor cells. In the previous studies that demonstrated the effects of BM-MSCs [32], adipose-derived MSCs [33], and cord blood-derived MSCs [34] in AKI, the number of MSCs in the kidney was quantitatively very low and they were almost exclusively localized in the peritubular areas. Therefore, it was suggested that MSCs are likely to promote regeneration through paracrine action, rather than a direct repopulation. On the other hand, there were other studies showing MSCs to integrate into damaged tubules and differentiate into renal epithelial cells, supporting the findings that stem cells improve the kidney function and structure directly, by migrating to the kidney and populating the renal cortex [7,35]. We did not investigate the paracrine effects of T-MSCs in animal model of AKI using conditioned media or T-MSC–derived extracellular vesicles. To further understand the renoprotective mechanism of T-MSCs, additional in-vivo experiments to explore the paracrine effect of T-MSCs with an analysis of secretosome will be necessary.

We assessed the oxidative stress in kidney tissue to evaluate the protective effect of T-MSCs. The antiapoptotic and anti-inflammatory effect of MSCs is demonstrated in various studies [5,36]. However, the antioxidative effect of MSCs on AKI has not been thoroughly investigated. ROS are important mediators exerting toxic effects on various organs including the kidney, and oxidative stress plays a crucial role in the pathogenesis of GM-induced AKI [37,38]. This study confirmed that administered T-MSCs via intravenous route could reduce oxidative stress, which was associated with a differential increase in the expression of antioxidant enzymes. In addition to their antioxidative effects, T-MSCs may also protect against AKI by inhibiting apoptosis and promoting extracellular matrix remodeling. Our unpublished single-cell RNA-sequencing analysis suggests that key regulators such as forkhead box O3, glutathione peroxidase 4, Wnt signaling components, and meteorin contribute to these effects by enhancing antioxidant defense, regulating apoptosis, and supporting tubular regeneration. These findings highlight the therapeutic potential of T-MSCs in AKI recovery. Another novel finding of this study was a differential regulation of Nox isoforms by GM in cultured renal tubular cells, our result showed that NOX1 was the most abundant isoform in NRK-52E cells and the only isoform that was upregulated by GM.

In addition, we also demonstrated the role of ER stress in GM-induced apoptosis in the kidneys. Upregulation of ER chaperone protein and CHOP was noted in GM-induced AKI and cultured renal tubular cells. An amelioration of proapoptotic ER stress by T-MSCs was verified both in in-vivo and in-vitro models. In previous studies, T-MSCs alleviated high-fat diet–induced ER stress in pancreatic cells [25]. Adipose-derived MSC injection suppressed the expression of apoptotic ER stress, CHOP, and CASPASE12 in dog kidneys [39,40]. The limitation of this study is not to examine the migration of T-MSCs in other organs, which needs to be further investigated in future studies.

This is the first study investigating T-MSCs as a new potential therapeutic tool for GM-induced AKI. Our study demonstrated the feasibility of T-MSCs application and the proof of concept for the treatment of GM-induced AKI. Our study demonstrated the direct and paracrine effects of T-MSCs on GM-induced AKI to improve the oxidative/antioxidant environment in the kidney.

These results suggest that T-MSCs ameliorated GM-induced AKI by directly incorporating into the damaged renal tubules, exerting antiapoptotic and antioxidative effects. T-MSCs also showed a beneficial effect on the functional and morphological recovery of a damaged kidney. We suggest that T-MSCs as a potential tool of efficient and effective cell therapy for AKI based on easily obtainable characteristics, and superior proliferation and differentiation capacities compared to other MSCs.

Footnotes

Conflicts of interest

All authors have no conflicts of interest to declare.

Funding

This research was supported by the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Health & Welfare) (23A0201L1) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (2020R1A2C3007759).

Data sharing statement

The data presented in this study are available from the corresponding author upon reasonable request.

Authors’ contributions

Conceptualization: MY, DHK

Data curation: SMJ

Methodology: IJ, SCJ

Formal analysis: DAK, ESR

Funding acquisition, Supervision: DHK

Writing–original draft: MY, DAK, DHK

Writing–review & editing: SCJ, IJ, HSK, DHK

All authors read and approved the final manuscript.

Supplementary Materials

Supplementary data are available at Kidney Research and Clinical Practice online (https://doi.org/10.23876/j.krcp.24.234).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

j-krcp-24-234-Supplementary-Method.pdf (399.7KB, pdf)
j-krcp-24-234-Supplementary-Table-1.pdf (407.3KB, pdf)
j-krcp-24-234-Supplementary-Table-2.pdf (397.9KB, pdf)
j-krcp-24-234-Supplementary-Fig-1.pdf (596KB, pdf)
j-krcp-24-234-Supplementary-Fig-2.pdf (497KB, pdf)
j-krcp-24-234-Supplementary-Fig-3.pdf (100KB, pdf)

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