Minichromosome maintenance 4 plays a key role in protecting against acute kidney injury by regulating tubular epithelial cells survival and regeneration

Graphical abstract

Highlights

• •The upregulation of MCM4 during AKI development is an adaptive response.
• •MCM4 exerts an essential role in AKI by regulating 53BP1/p53/p21 signaling axis.
• •MAD2B regulates the transcription level of MCM4 by effecting the level of E2F1.
• •Identification of MCM4 as the potential therapeutic target during AKI development.

Abstract

Introduction

Minichromosome maintenance 4 (MCM4), a constituent of the MCM family, playing a pivotal role in DNA replication. Although MCM4 expression has been widely linked to various malignant tumors, its role in kidney diseases is not well-studied. This study primarily investigates the role and underlying mechanism of MCM4 in acute kidney injury (AKI).

Objectives

Characterizing a novel target of MCM4 in patients with AKI.

Methods

We used CRISPR/Cas9 gene editing to delete MCM4 gene in tubular cells from C57BL/6J mice. Adeno-associated virus 9 harboring MCM4 was administered via intraparenchymal injection into the kidney to enhance MCM4 expression in vivo. These mice were used to established cisplatin- and ischemic reperfusion injury (IRI)-induced AKI mouse models, for detecting the functional role of MCM4 in the pathological process of AKI.

Results

MCM4 level was increased in the tubules of cisplatin- and IRI-induced AKI mouse models. Compare to wide-type mice, MCM4 knockout mice demonstrated greater degree of histological damage and a higher ratio of apoptotic tubular cells, as well as kidney dysfunction upon cisplatin- and IRI-induced AKI models. Conversely, MCM4 overexpression ameliorated the severity of kidney injury and promoted regenerative capacity of tubular cells during AKI development. Mechanically, loss of MCM4 induced the expression of p53-binding protein 1, activating the p53/p21 pathway and exacerbating AKI progression. Additional, MAD2B, as an upstream molecule of MCM4, regulates the transcription level of MCM4 by affecting the level of E2F1.

Conclusions

These findings demonstrate that MCM4 upregulation during AKI development is an adaptive response that preserves tubular cell regenerative capacity and limits the severity of renal injury, thus highlighting the potential value of MCM4 as a biomarker or therapeutic target in patients with AKI.

Introduction

Acute kidney injury (AKI) is a global public health problem characterized by the sudden deterioration of kidney function, posing a high risk for chronic kidney disease [1], [2]. Timely identification of AKI is essential for optimizing treatment strategies and reducing kidney injury [3]. Proximal tubular epithelial cells (PTCs) are highly susceptible to various types of injury but possess remarkable regenerative potential [4]. Moderately injured PTCs was mainly replaced by self-proliferation of surviving PTCs, promoting the adaptive repair and restoring kidney function; however, heavily injured PTCs undergo apoptosis and necrosis, resulting in maladaptive repair and fibrosis [4], [5]. Thus, PTCs play a significant role in kidney injury and repair, understanding their biology function might be determinant for AKI prognosis.

The tumor suppressor protein p53 is involved in AKI development and post-AKI kidney repair by regulating multiple cellular processes [6]. Although substantial evidence has demonstrated that DNA damage, hypoxia, and reactive oxygen species are major triggers of p53 activation, the specific interacting proteins and regulatory mechanisms remains to be discovered [7]. The essential function of p53-binding protein 1 (53BP1) in the DNA damage response is well-established [8], with increasing focus on its interaction with p53, which contributes to the regulation of p53 target gene expression and signal transduction pathways [9], [10]. However, the functional role of 53BP1 in AKI pathogenesis and its cooperation with p53 in AKI development and kidney repair require further investigation.

Minichromosome maintenance 4 (MCM4), part of the MCM2-7 complex, is crucial for initiating DNA synthesis, with its expression being cell-cycle-dependent and modulated during cellular aging [11], [12]. MCM4 deficiency impairs cell viability, enhances cisplatin sensitivity, and inhibits the proliferation of lung cancer cells [13], [14]. MCM4 plays a role in checkpoint activation during DNA damage, resulting in G1-phase cell cycle arrest and apoptosis [15], [16]. In various carcinomas, MCM4 shows potential as a proliferation marker, surpassing Ki67 and PCNA in sensitivity [17], [18]. However, the biological function of MCM4 during AKI development is not well understood.

This study firstly reveals that the adaptive regulation of that MCM4 in PTCs during AKI, where it plays a kidney-protective role, promoting the regenerative capacity of PTCs and decreasing cell apoptosis, thereby limiting the severity of renal injury.

Materials and methods

Animal models

Eight-week-old male C57BL/6J mice, weighing around 20 g, were obtained from Charles River (Beijing, China). In AKI mouse models, mice received an intraperitoneal injection of 25 mg/kg cisplatin (Sigma-Aldrich, St. Louis, MO, USA) and were euthanized after 3 days. The IRI-induced AKI model was created by clamping the bilateral renal pedicles of mice with an atraumatic vascular clip for 35 min. During the operation, mice were maintained at a body temperature of 36.6–37.2℃, and mice were euthanized two days after procedure.

Generation of tubule-specific MCM4 knockout mice

MCM4^fl/fl mice (C57BL/6J) and tamoxifen-inducible KSP-Cre mice were obtained from Cyagen Bioscience Inc. (Guangzhou, China). KSP-Cre mice were generating by introducing the tamoxifen-inducible Cre recombinase protein expression element downstream of the stop codon of the mouse Cdh16 gene using CRISPR/Cas9 technology. MCM4^fl/fl mice and tamoxifen-inducible KSP-Cre mice were cross mated to generate conditional MCM4 knockout mice. The mice received a daily intraperitoneal injection of tamoxifen (100 mg/kg, dissolved in corn oil) (Sigma-Aldrich, St. Louis, MO, USA) for 5 consecutive days to induce MCM4 deletion in renal tubular cells (Cre⁺/MCM4^fl/fl). Ten days after MCM4 deletion, the Cre⁺/MCM4^fl/fl mice were used to establish the AKI mouse models.

Generation of tubule-specific MAD2B knockout mice

Tubule-specific MAD2B knockout mice (Cre⁺/MAD2B^fl/fl) were generated by crossing MAD2B^fl/fl mice and PEPCK-Cre mice to deplete MAD2B in renal tubular epithelial cells, as previously described [19].

Adeno-associated virus delivery within the kidney

Adeno-associated virus 9 (AAV9) vectors harboring MCM4 (AAV-OE-MCM4) and a negative control AAV9 (AAV-OE-Ctrl) were obtained from Vigenebio (Shandong, China). Intraparenchymal injection of AAV-OE-MCM4 was performed to achieve MCM4 to overexpress MCM4 expression in tubular cells in vivo. 100 μL (4 × 10¹¹ pfu/mL) of AAV-OE-MCM4 (or AAV-OE-Ctrl) virus was injected into the left renal cortex using a 31G needle. The same procedure was repeated in the right kidney 7 days later. A total of 200 μL of AAV9 particles were used to inhibit MCM4 expression in renal tubules, as previously described [20]. One month following the second injection, AKI mouse models were established.

Histopathological assessment

Hematoxylin and eosin (HE) staining was performed to assess histopathological changes in tubules following manufacturer's instructions (Beyotime, Shanghai, China). Tubular injury was assessed by the percentage of damaged tubule lesions and graded as follows: 0 for no damage, 1 for less than 25 %, 2 for 25–50 %, 3 for 50–75 %, and 4 for more than 75 %, following the method outlined in [19].

Immunohistochemistry (IHC) staining

The 3 μm tissue sections were incubated with primary antibodies overnight at 4℃, followed by 1 h incubation with secondary antibodies at 37℃. Finally, sections were stained using DAB, with nuclei counterstained by Hematoxylin. Stained samples were observed and imaged using a Nikon Ni-E bright field microscope (Tokyo, Japan). Antibodies involved are summarized in Table S1.

Immunofluorescence (IF) staining

Renal sections (2 μm) or cell coverslips were fixed with 4 % paraformaldehyde for 15 min. 0.2 % triton X-100 were utilized to increase the permeability of the cell membrane for 15mins. Sections were blocked with 10 % donkey serum for 1 h, then incubated with primary antibodies overnight at 4℃ and with secondary antibodies for 1 h at 37℃. DAPI was used to stained with nuclei. Representative figures were captured by inverted fluorescence microscope. Antibodies involved are summarized in Table S1.

Cell culture and treatment

NRK-52E cells and HK-2 cells were obtained from ATCC. Cells were cultured in DMEM medium (Gibco, NY, USA) supplemented with 10 % fetal bovine serum (FBS). Cisplatin treatment involved administering 20 μM cisplatin for 12 h, while hypoxia and reperfusion experiments consisted of 24 h of hypoxia followed by 2 h of reperfusion. PFT-α and T-5225, both at 50 μM concentrations from Selleck Chemical, were administered for 2 h prior to cisplatin treatment.

Lentivirus-mediated gene expression

NRK-52E cells were grown in 6-well culture plates at a density of 20–30 %. Cells were treated with a combination of co-transfection reagents A and P, along with lentivirus. NRK-52E cells were infected at a multiplicity of infection (MOI) of 30. The medium was replaced 12 h post-transfection. GFP fluorescence was observed 24 h post-transfection. 2 μg/ml puromycin was introduced to the medium to eliminate non-transfected cells, and western blot analysis was employed to assess knockdown efficiency after 72 h post-transfection.

siRNA-mediated knockdown

Specific MCM4 siRNA (si-MCM4) and 53BP1 siRNA (si-53BP1) were synthesized by RiboBio (Guangzhou, China). Each well was transfected with 5 μl of siRNA using 5 μl of Lipofectamine 2000 (Invitrogen, CA, USA). After 48 h in culture, the knockdown efficiency of siRNA transfection was detected by western blot.

The sequence of si-MCM4 was as follows: 5′-TGATGATGATTTCCTAACA-3′.

The sequence of si-53BP1 was as follows: 5′-CCTCTACTTGGGAATGAAA-3′.

Western blot

Western blot was performed as previously described [21]. Total protein from kidney cortex or cells were extracted in RIPA buffer for at least 30mins on ice. The centrifugation supernatant was subjected to SDS-PAGE and transferred to 0.45 μm PVDF membranes. Finally, after incubating with primary antibodies overnight at 4℃ and followed with secondary antibodies for 1 h, ECL luminescent solution is used for strip development. Antibodies involved are summarized in Table S1.

Cell viability assay

The methylthiazolyldiphenyl-tetrazolium (MTT) bromide assay kit (MTT; CK04, Dojindo, Japan) was used to determine cell viability. NRK-52E cells were transfected with si-MCM4 and then seeded on 96 well plate. Different doses of cisplatin was incubated for 12 or 24 h after cell adherence. Cultured medium was replaced, and cells were incubated with MTT solution (0.5 mg/ml) for 4 h, followed by optical density measurement at 450 nm.

Colony formation assay

NRK-52E cells (500 cells per well) were seeded in a six-well plate, transfected with si-MCM4 and incubated with varying doses of cisplatin for 7 days. After discarding the cultured medium, cells were fully cover with 4 % paraformaldehyde for 20 min, followed by staining with crystal violet for another 20 min. Colonies were observed under inverted microscope (Olympus, Tokyo, Japan).

EdU incorporation assay

NRK-52E cells were cultured and seeded on 96-well plate. For the EdU incorporation assay, NRK-52E cells were incubated with 200 μ L EdU mixture (10 μM) for 2 h in darkness. After discarding the cultured medium, cells were fully fixed with 4 % paraformaldehyde for 20 min, followed by staining with the EdU reaction mixture for 30 min. The nuclei were stained by DAPI for 5 min. Representative figures were captured by confocal microscope. Representative figures were captured by confocal microscope.

Flow cytometric analysis

For apoptosis analysis, poured off the medium, properly digested the NRK-52E cells with pancreatic enzymes, then collected and centrifuged the cells, and discarded the supernatant. Adding 100 μL Annexin V binding buffer (640930, BioLegend, CA, USA) into cells to prepare cells suspension. The suspension was mixed with 2 μL APC-Annexin V and 2 μL 7-AAD, then incubated in the dark for 30 min. The mixtures were transferred to a 5 mL flow tube, supplemented with 100 μL of Annexin V binding buffer, and cell apoptosis was assessed using flow cytometry.

For cell cycle analysis, poured off the medium, properly digested the NRK-52E cells with pancreatic enzymes, then collected and centrifuged the cells, and discarded the supernatant. The suspension was added with 75 % ethanol to fix cells overnight at 4 ℃, centrifuge and collect cell precipitates. Adding 200 μL PI/RNase dyeing solution (KGA511, KeyGEN, Nanjing, China) to the cell mixture and incubate for 30 min, and cell cycle was measured by flow cytometry.

TUNEL assay

Apoptotic cells in frozen tissue sections were identified using an in situ Apoptosis Detection kit, following the manufacturer's instructions (Roche, Mannheim, Germany). The sections were fully covered with 4 % paraformaldehyde for incubating 20 min, then permeabilized with 0.3 % Triton X-100 for 20 min. After discarding the permeating solution and cleaning with PBS, the TUNEL reaction mixture was added into the sections to fully cover the tissues, incubating at 37 ℃for 1 h in the dark, as previously described [21]. The apoptotic cells were examined using a fluorescence microscope (Olympus, Tokyo, Japan).

Dual-luciferase reporter assay

The wild-type promoter sequence of 53BP1 was cloned into pGL3 reporter plasmids, and c-Jun overexpression plasmids were synthesized by GeneChem (Shanghai, China). Luciferase activity was assessed via chemiluminescence using a dual-luciferase reporter assay system (Promega, USA), according to the manufacturer's protocol.

Statistical analysis

All data in this study are expressed as means ± SEM. A two-tailed unpaired Student’s t-test was utilized for comparisons between two groups, and one-way ANOVA with Tukey’s post-test was applied for comparisons involving more than three group. Statistical data were analyzed and visualized by GraphPad Prism 8.0 software (La Jolla, USA), and P < 0.05 was considered to have significant difference.

Results

MCM4 is highly unregulated in renal tubules during AKI development

We investigated the specific role of MCM4 in kidney disease, particularly its involvement in AKI. Immunoblotting analysis confirmed the abundant expression of MCM4 in TECs (Fig. 1A), and immunofluorescence (IF) assays further showing the concentrated nuclear expression of MCM4 in TECs (Fig. 1B). In the cisplatin-induced AKI mouse model, Scr and BUN levels were significantly elevated (Fig. 1C). Immunochemistry (IHC) staining (Fig. 1D and E) revealed that MCM4 was upregulated in cisplatin-induced AKI mice, and the co-staining of MCM4 and proximal tubular markers (LTL) indicated the renal localization of MCM4 in tubules (Fig. 1F). Immunoblotting analyses further confirmed the increased level of MCM4 in AKI mouse, along with induction of NGAL, a known marker of tubular injury (Fig. 1G and H). MCM4 upregulation was similarly noted in the kidneys of mice with IRI-induced AKI (Fig. 1I-N). In vitro experiments corroborated our in vivo results, demonstrating increased MCM4 expression in cultured TECs following cisplatin and hypoxia reoxygenation (H/R) treatments (Fig. 1O-R). Overall, our results demonstrate the substantial elevation of MCM4 in tubular cells after AKI stimulations, indicating its potential role during AKI development. However, as AKI developed, MCM4 expression was decreased in late stage (at day 4) of AKI, which might be attributed to the speed of TECs proliferation in different stages during AKI development (Additional file1: Fig. S1).

Fig. 1.

The upregulation of MCM4 in AKI mouse models and cultured TECs. (A) Representative western blotting of MCM4 in TECs. HK-2: human proximal tubular epithelial cells. NRK-52E: rat proximal tubular epithelial cells. (B) Representative confocal microscopic images illustrating the distribution of MCM4 in TECs. Scale bar: 6.25 μm. (C) Graphical representation of Scr and BUN levels in serum from sham and cisplatin treatment group. (D and E) Representative immunohistochemical staining images of MCM4 in sham and cisplatin treatment group. Scale bar: 50 μm. （F）Representative immunofluorescence staining showing the co-localization of MCM4 and proximal tubular markers (LTL) in sham and cisplatin treatment group. Scale bar: 25 μm. (G and H) Representative Western blotting and quantification of MCM4 in sham and cisplatin treatment group. (I) Graphical presentation of the levels of Scr and BUN in serum from sham and IRI group. (J and K) Representative immunohistochemical staining images of MCM4 in sham and IRI group. Scale bar: 50 μm. （L）Representative immunofluorescence staining showing the co-localization of MCM4 and LTL in sham and IRI group. Scale bar: 25 μm. (M and N) Representative Western blotting and quantification of MCM4 in sham and IRI group. (O and P) Representative western blotting and quantification of MCM4 in cisplatin-treated NRK-52E cells. (Q and R) Representative western blotting and quantification of MCM4 in HR-treated NRK-52E cells. N = 6 for each group. All data were represented as mean ± SEM. ***P < 0.001.

Proximal tubule-specific MCM4 knockdown aggravates cisplatin-induced kidney dysfunction and tubular injury

MCM4^fl/fl mice were created using a Cre-LoxP recombination system to study the role of MCM4 in AKI pathology. Inducible Ksp-Cre mice were bred with MCM4^fl/fl mice to create tubule-specific MCM4 knockout mice (Cre⁺/MCM4^fl/fl), with genotypes verified via tail genotyping (Fig. 2A and B). MCM4 expression was suppressed by tamoxifen injection in Cre⁺/MCM4^fl/fl mice, followed by cisplatin administration to establish AKI mouse models (Fig. 2C). As depicted in Fig. 2D and E, Cre⁺/MCM4^fl/fl mice exhibited a more pronounced increase in Scr and BUN levels compared to Cre^-/MCM4^fl/fl mice after cisplatin injection. HE staining and IHC staining revealed that genetic deletion of MCM4 aggravated cisplatin-induced kidney damage, as evidenced by the marked severity of histological injury and the upregulation of NGAL expression (Fig. 2F-H), which was further confirmed through immunoblotting analysis of NGAL and Bax expression (Fig. 2I and J). In kidney tissues from cisplatin-treated Cre⁺/MCM4^fl/fl mice, there was an increase in TUNEL-positive cells and a decrease in Ki67-positive cells (Fig. 2K and L). These findings indicate that loss of MCM4 promotes tubular cell apoptosis and inhibits regenerative capacity of tubular cells after AKI injury, aggravating cisplatin-induced renal injury and impeding kidney repair.

Fig. 2.

Proximal tubule-specific MCM4 knockdown aggravates cisplatin-induced kidney dysfunction and tubular injury. (A) Generation of conditional knockout mice with specific ablation of MCM4 in proximal tubular epithelial cells, using the Cre-LoxP recombination system. (B) Genotyping confirmation of conditional knockout through tail preparation and PCR at 2 weeks of age. (C) Experimental design: Mice was intraperitoneal injected with tamoxifen for 5 consecutive days to induce deletion of MCM4 expression in tubular cells, followed with intraperitoneal injection of cisplatin to establish the cisplatin-induced AKI mouse model after 10 days. (D and E) Graphical representation of Scr and BUN levels in serum from different experimental groups. (F to H) Representative images of immunohistochemical staining and quantification of MCM4 and NGAL expression in different groups. Graphical representation of the tubular damage score in different groups. Scale bar: 50 μm. (I and J) Representative western blotting and quantification of MCM4, NGAL, and Bax in different groups. (K and L) Representative images showing TUNEL-positive cells and Ki67-positive cells in different treatment groups. Scale bar: 50 μm. N = 6 for each group. All data were represented as mean ± SEM. ***P < 0.001, ^###P < 0.001.

Furthermore, intraparenchymal injection of AAV9 carrying MCM4 (AAV-sh-MCM4) into mouse kidneys further confirmed the exacerbation of cisplatin-induced kidney dysfunction and renal injury upon MCM4 knockdown in tubular cells in vivo (Additional file1: Fig. S2).

MCM4 deletion exacerbates IRI-induced kidney dysfunction and tubular injury

To confirm the function of MCM4 in other AKI models, we examined its involvement in IRI-induced AKI (Fig. 3A). IRI-induced Cre⁺/MCM4^fl/fl mice showed exacerbated kidney dysfunction and injury compared to IRI-induced Cre^-/MCM4^fl/fl mice, indicated by elevated Scr and BUN levels, increased tubular damage scores, and higher NGAL and Bax expression (Fig. 3B-H). Moreover, MCM4 knockdown weakened tubule cells' resistance to cell death and hindered their proliferation under IRI conditions (Fig. 3I and J). These findings collectively indicate that MCM4 knockdown aggravates tubular cells injury during AKI development, underscoring the significant role of MCM4 in different types of AKI.

Fig. 3.

Proximal tubule-specific MCM4 knockdown exacerbates IRI-induced kidney dysfunction and tubular injury. (A) Experimental design: Intraperitoneal injection of tamoxifen for 5 days to induce the deletion of MCM4 expression in renal tubules. To establish the IRI-induced acute kidney injury model, bilateral renal pedicles of mice were clamped with an atraumatic vascular clip for 35 min after 10 days. (B and C) Graphical representation of Scr and BUN levels in different experimental groups. (D to F) Representative images of immunohistochemical staining and quantification of MCM4 and NGAL expression in different groups. Graphical representation of the tubular damage score in different groups. Scale bar: 50 μm. (G and H) Representative western blotting and quantification of MCM4, NGAL, and Bax in different groups. (I and J) Representative images depicting TUNEL-positive cells and Ki67-positive cells in different treatment groups. Scale bar: 50 μm. N = 6 for each group. All data were represented as mean ± SEM. ***P < 0.001, ^###P < 0.001.

In vivo overexpression of MCM4 by intrarenal AAV gene delivery protects against cisplatin-induced AKI

To clarify the therapeutic potential of targeting MCM4 in a cisplatin-induced AKI model, we delivered an AAV to overexpress MCM4 (AAV-OE-MCM4) into the mouse kidney (aimed at renal proximal tubules) via intraparenchymal injection (Fig. 4A). The restoration of MCM4 expression inhibited the increase of Scr and BUN induced by cisplatin treatment (Fig. 4B and C), attenuated the severity of histological injury (Fig. 4D and E), and decreased the expression of NGAL and Bax (Fig. 4D-H). Additionally, IF staining revealed that overexpression of MCM4 declined the proportion of TUNEL-positive cells and restored the percentage of Ki67-positive cells upon cisplatin stimulations (Fig. 4I and J). Collectively, these findings demonstrate that in vivo overexpression of MCM4 significantly alleviates cisplatin-induced renal injury, further confirming its protective role in the progression of AKI.

Fig. 4.

Intrarenal AAV-Mediated overexpression of MCM4 protects against cisplatin-induced AKI in vivo. (A) Experimental design: Intrarenal delivery of AAV-OE-Ctrl or AAV-OE-MCM4 into the kidneys of mice via intraparenchymal injections (indicated by the red arrow). Cisplatin injection was performed at the time indicated by the blue arrows. (B and C) Graphical representation of Scr and BUN levels in different experimental groups. (D to F) Representative images of immunohistochemical staining and quantification of MCM4 and NGAL expression in different groups. Graphical representation of the tubular damage score in different groups. Scale bar: 50 μm. (G and H) Representative western blotting and quantification of MCM4, NGAL, and Bax in different groups. (I and J) Representative images depicting TUNEL-positive cells and Ki67-positive cells in different treatment groups. Scale bar: 50 μm. N = 8 for each group. All data were represented as mean ± SEM. ***P < 0.001, ^###P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

MCM4 deficiency inhibits cell proliferation and DNA synthesis, promotes G1 phase arrest and cell apoptosis in cisplatin-treated TECs

In vitro, MCM4 expression in NRK-52E cells was knocked down using MCM4-siRNA (si-MCM4). The MTT assay indicated that MCM4 deficiency heightened TEC sensitivity to cisplatin (Fig. 5A) and reduced their viability (Fig. 5B). Silencing MCM4 expression led to a dose-dependent reduction in both the number and size of cell colonies in cisplatin-treated NRK-52E cells (Fig. 5C and D). Immunoblotting analysis indicated that MCM4 deficiency exacerbated cisplatin-induced injury in TECs, demonstrated by elevated NGAL and Bax expression (Fig. 5E and F). EdU incorporation assay, a sensitive method for detecting and quantifying DNA synthesis, indicated that loss of MCM4 inhibited TECs regeneration after injury by diminishing DNA synthesis of TECs (Fig. 5G and H). Consistently, flow cytometry analysis indicated that MCM4 deficiency induced in a decreased percentage of S-phase cells and an increased ratio of G0/G1 phase cells in response to cisplatin stimulation (Fig. 5I and J). Furthermore, MCM4 deficiency increased the ratio of cisplatin-induced apoptotic cells (Fig. 5K and L). Moreover, overexpression of MCM4 inhibited cisplatin-induced TECs injury (Additional file1: Fig. S3).

Fig. 5.

MCM4 deficiency inhibits cell proliferation and DNA synthesis, while promoting G1 phase arrest and cell apoptosis in TECs with cisplatin treatment. (A) Quantitative colorimetric MTT assay showing the sensitivity of NRK-52E cells with MCM4 deficiency to cisplatin. (B) Quantitative colorimetric MTT assay showing cell viability of NRK-52E cells with MCM4 deficiency following cisplatin treatment. (C and D) Colony formation assay and summarized data demonstrating the proliferation ability of NRK-52E cells in response to cisplatin. (E and F) Representative western blotting and quantification of MCM4, NGAL and Bax in different experimental groups. (G and H) EdU incorporation cells assay depicting changes in mitogen-mediated DNA synthesis in NRK-52E in different experimental groups. Scale bar: 50 μm. (I and J) Representative flow cytometry histograms and quantitative data showing the distribution of cells in different phases in cell cycle of NRK-52E cells in different experimental groups. (K and L) Representative flow cytometric histograms and quantitative data showing the percentage of apoptotic cells in different experimental groups. N = 6 for each group. All data were represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P, ^##P < 0.01, ^###P < 0.001.

Inhibition of the p53/p21 signaling rescues the functional defect in MCM4-deficient TECs

Considering that the p53 signaling pathway is a key regulatory pathway involved in cell proliferation and apoptosis [22], it may be a potential mechanism underlying the role of MCM4 in TECs. Compared to cisplatin-treated Cre^-/MCM4^fl/fl mice, the induction of p53 and p21 were further upregulated in cisplatin-treated Cre⁺/MCM4^fl/fl mice (Fig. 6A and B), while were decreased by overexpression of MCM4 (Fig. 6C and D). Consistent with in vivo observations, in vitro studies also demonstrated that cisplatin-induced p53 and p21 were further upregulated by MCM4 knockdown (Fig. 6E and F), whereas decreased by MCM4 overexpression (Additional file1: Fig. S4A and B). And co-immunoprecipitation assays further confirmed the interaction between MCM4 and p53 (Additional file1: Fig. S4C). These results indicate that MCM4 deficiency plays a role in activating the p53/p21 pathway during cisplatin-induced AKI. To clarify the interaction between MCM4 and p53, we used PFT-α, a p53 inhibitor, which significantly suppressed the MCM4 deficiency-induced increase in p53 and p21 levels (Fig. 6G and H). As shown in Fig. 6I-O, inhibition of p53 remarkedly rescued MCM4 deficiency-induced cytotoxicity, cell cycle arrest, and impaired proliferative ability. Overall, our results reveal that loss of MCM4 results in the aberrant activation of p53/p21 signaling during AKI development.

Fig. 6.

Inhibition of the p53/p21 signaling pathway rescues the functional defect in MCM4-deficient TECs. (A to D) Representative western blotting and quantification of p53 and p21 in different experimental groups. A and B: tubule-specific MCM4 knockout mice. C and D: mice with AAV-OE-MCM4 delivery. (E to H) Representative western blotting and quantification of p53 and p21 in different experimental groups. G and H: NRK-52E cell were pretreated with PFT-α before cisplatin treatment. (I) Quantitative colorimetric MTT assay representing the ability of cell proliferation in different experimental groups. (J and K) EdU incorporation assay showing the changes in mitogen-mediated DNA synthesis in different experimental groups. Scale bar: 50 μm. (L and M) Representative flow cytometry histograms and quantitative data demonstrating the distribution of cells in different phases of the cell cycle in different experimental groups. (N and O) Representative flow cytometry histograms and quantitative data showing the percentage of apoptotic cells in different experimental groups. N = 6 for each group. All data were represented as mean ± SEM. **P < 0.01, ***P < 0.001, ^##P < 0.01, ^###P < 0.001.

MCM4 deficiency contributes to the activation of p53/p21 signaling by regulating 53BP1 expression

Next, we investigated the potential mechanism through which MCM4 activates the p53/p21 signaling pathway. Recent studies have established that 53BP1 is a p53-interacting protein that regulates p53 signaling activation [23], [24]. However, the involvement of 53BP1 in regulating the p53 pathway during AKI development remains unknown. We observed the induction of 53BP1 expression in cisplatin-induced AKI mice by IHC staining, suggesting its potential role in AKI (Fig. 7A and B). Silencing 53BP1 expression significantly decreases cisplatin-induced p53 and p21 levels (Fig. 7C and D), indicating the importance of 53BP1 in regulating p53/p21 signaling. We hypothesized that MCM4 may influence the activation of p53/p21 signaling through the regulation of 53BP1. In animal studies, compared to cisplatin-treated Cre^-/MCM4^fl/fl mice, the induction of 53BP1 was further increased in cisplatin-treated Cre⁺/MCM4^fl/fl mice (Fig. 7E and F), however, it was decreased by overexpression of MCM4 (Fig. 7G and H). In vitro studies showed that MCM4 deficiency increased cisplatin-induced 53BP1 expression, while its overexpression reduced it (Additional file1: Fig. S5A and B). 53BP1 deficiency inhibited the increase in p53 and p21 levels caused by MCM4 knockdown (Fig. 7K and L). The findings suggest that MCM4 partially mediates 53BP1 expression in cisplatin-induced AKI.

Fig. 7.

Regulation of p53/p21 Signaling by 53BP1 in MCM4-Functioned AKI. (A and B) Representative images of immunohistochemical staining and quantification of 53BP1 expression in sham and cisplatin treatment. Scale bar: 50 μm. (C and D) Representative western blotting and quantification of 53BP1, p53 and p21 in different experimental groups. (E to H) Representative images of immunohistochemical staining and quantification of 53BP1 expression in different experimental groups. E and F: tubule-specific MCM4 knockout mice. G and H: mice with AAV-OE-MCM4 delivery. Scale bar: 50 μm. (I and J) Representative western blotting and quantification of 53BP1 in different experimental groups. (K and L) Representative western blotting and quantification of MCM4, 53BP1, p53, and p21 in different experimental. (M) Graphical representation of luciferase activity in NRK-52E cells by using a dual luciferase reporter assay. (N and O) Representative western blotting and quantification of c-jun phosphorylation in different experimental groups. (P and Q) Representative western blotting and quantification of 53BP1, p53, and p21 in different experimental groups. N = 6 for each group. All data were represented as mean ± SEM. **P < 0.01, ***P < 0.001, ^###P < 0.001.

We utilized bioinformatics analysis via the JASPAR online transcription factor prediction tool to identify potential transcription factors that may bind to the 53BP1 promoter, aiming to understand MCM4′s regulatory role in 53BP1 expression. c-Jun was identified as one of the predicted TFs. Dual-luciferase reporter assays confirmed the prediction, showing that co-transfecting c-Jun overexpressing plasmids with the pGL3-53BP1 promoter significantly enhanced luciferase activity in NRK-52E cells (Fig. 7M). Immunoblotting assay showed that the phosphorylation of c-Jun was further increased by MCM4 deficiency (Fig. 7N and O), whereas decreased by MCM4 overexpression (Additional file1: Fig. S5C and D). Furthermore, the inhibition of c-Jun by T5224, a specific AP-1 inhibitor, partially suppressed the elevation of 53BP1, p53, and p21 caused by MCM4 knockdown in cisplatin-treated NRK-52E cells (Fig. 7P and Q). These findings corroborate our hypothesis that MCM4 deficiency enhances 53BP1 expression by modulating c-Jun activity, consequently activating the p53/p21 pathway.

Tubule-specific loss of MAD2B exacerbates renal injury and decreases MCM4 expression

Previous studies have demonstrated the biological functions of MAD2B in the pathogenesis of diabetic nephropathy, crescentic glomerulonephritis, tubulointerstitial fibrosis, and AKI [25], [26]. Therefore, we wondered if there was an interaction between MAD2B and MCM4. We investigated the association between MAD2B and MCM4 through immunofluorescence staining, revealing their co-localization in mouse renal tubules (Fig. 8A) and NRK-52E cells (Fig. 8B). Co-immunoprecipitation assays further confirmed the interaction between MAD2B and MCM4 (Fig. 8C). To further investigate the relationship between MAD2B and MCM4, we used tubule-specific MAD2B knockout mice (Cre⁺/MAD2B^fl/fl) and lentivirus targeting MAD2B (LV-MAD2B) to knock down MAD2B expression. Cisplatin-treated Cre⁺/MAD2B^fl/fl mice exhibited elevated Scr and BUN levels, along with increased NGAL, Bax, p53, and p21, and reduced MCM4 levels, compared to cisplatin-treated Cre^-/MAD2B^fl/fl mice (Fig. 8D to K). Consistently, silencing MAD2B decreased the expression of MCM4 in TECs (Fig. 8L and M), suggesting the regulation of MAD2B on MCM4 expression. Prior research indicates that E2F1 modulates MCM transcription levels, influencing MCM-mediated biological functions [27], [28]. MAD2B deficiency decreased the expression of cell cycle-related proteins including CDK4, Cyclin D1, and E2F1, and the phosphorylation of Rb. These findings suggest that MAD2B knockdown inhibited the activity of CDK4/Cyclin D1 complex, leading to the decrease in Rb phosphorylation and E2F1 expression, thereby suppressing MCM4 expression.

Fig. 8.

Tubular-specific loss of MAD2B exacerbates renal injury and decreases MCM4 expression. (A) Representative IF staining showing the colocalization of MAD2B (red) and MCM4 (green) in the mouse kidney. Scale bar: 50 μm. (B) Representative confocal microscopic images showing the colocalization of MAD2B (red) and MCM4 (green) in NRK-52E cells. Scale bar: 6.25 μm. (C) Representative western blotting images of immunoprecipitation depicting cisplatin-induced enhancement of the interaction of MAD2B and MCM4. (D) Graphical representation of Scr and BUN levels in different experimental groups. (E and F) Representative images of immunohistochemical staining and quantification of MAD2B, MCM4, and NGAL in different experimental groups. Scale bar: 50 μm. (G) Graphical representation of the tubular damage score in different experimental groups. (H and I) Representative western blotting and quantification of MAD2B, MCM4, and NGAL in different experimental groups. (J and K) Representative western blotting and quantification of p53, p21, and Bax in different experimental groups. (L and M) Representative western blotting and quantification of MAD2B, MCM4, and NGAL in different experimental groups. (N and O) Representative western blotting and quantification of CDK4, cyclin D1, and E2F1 in different experimental groups. D to K: N = 8 for each group. L to O: N = 6 for each group. All data were represented as mean ± SEM. ***P < 0.001, ^###P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Discussion

Although numerous studies have linked MCM4 to cell proliferation, cell cycle, and DNA damage response [11], [12], [15], [16], [29], less research has focused on its role in kidney diseases. However, the function and underlying mechanism of MCM4 in AKI remain unknown. In this study, we uncovered that MCM4 is significantly upregulated in renal tubules in AKI mouse models. Surprisingly, the upregulation of MCM4 in PTCs was adaptive during AKI development, as it mitigated tubular cells injury and promoted the regeneration of tubular cells. This response was observed in vivo in two unrelated AKI models using tubule-specific MCM4 knockout mice, as well as in cultured TECs treated with cisplatin. This protective function of MCM4 was further confirmed in cisplatin-treated AKI mice by overexpressing MCM4 in the kidneys via intrarenal AAV delivery. Moreover, we demonstrated the potential function of 53BP1 in AKI and uncovered that MCM4 mediates the expression of 53BP1 by regulating c-Jun activity, providing a new regulatory mechanism for the activation of p53/p21 signaling during AKI development.

Previously, substantial studies reported that MCM4 exerted an essential role in the regulation of cell proliferation, cell cycle, and cell death [11], [15], [30], [31]. It has been reported that MCM4 expression and activity are regulated by the cell cycle, accumulating in the G1 phase, activating in the S phase, and degrading in the M phase, ensuring that replication occurs only at the appropriate stage [11], [31]. Therefore, MCM4 deficiency was mainly induced the G0/G1 cell cycle arrest [32], [33], [34]. Multi-omics analysis also indicated the enrichment in cell cycle of the interacting protein of MCM [35]. Besides, the phosphorylation of MCM4 also closely associated with DNA synthesis during DNA replication, resulted in a stagnation of cell cycle progression [36]. Moreover, the upregulation of MCM4 was often observed in a variety of carcinomas, which might relate to the malignant proliferation of tumor cells [17], [37], [38]. As a result, MCM4 has been identified as a potential novel proliferation marker [17], [18], with more sensitivity than Ki67 and PCNA, providing clinical value and clinicopathological significance for predicting the occurrence and prognosis of tumors. In addition, the involvement of MCM4 in cell apoptosis also deserved attention. Overexpression of MCM4 markedly inhibited the ratio of cell apoptosis in tumor cells, with the improvement of proliferative ability, acting as a biomarker for prognosis [32], [39]. And the activation of MCM4 in DNA damage checkpoint also had an effect on apoptosis in HT29 cells [16]. As expected, our results also exhibited the similar changes that deficiency of MCM4 obviously inhibited the proliferative ability of TECs, accompanied with the G1-phase cell cycle arrest and apoptosis. These findings suggest that the functional role of MCM4 in the regulation of cell proliferation, cell cycle, and cell apoptosis is not limit to tumors cells, which may be applicable to all cells with proliferative capacity, just like TECs. However, there is few evidences exhibited the role of MCM4 in disease except for tumors. Therefore, this assumption needs further investigations.

Research indicates that cell cycle arrest primarily triggers AKI onset and subsequent renal injury repair, with p53 being pivotal in cell cycle regulation [6], [40]. P21, a key downstream effector of p53, modulates cyclin-dependent kinase activity and is involved in cell cycle regulation [41]. During AKI, where kidney cells are damaged by ischemia, hypoxia, or exposure to toxins, p53 prevents the replication of damaged DNA and the transmission of genetic errors by blocking the cell cycle [40]. And p53-induced apoptosis helps to clear cells beyond repair, but excessive apoptosis may lead to further deterioration of kidney function [42], [43]. In addition, during AKI recovery, the level and activity of p53 are declined, allowing surviving cells to re-enter the cell cycle and promote tissue repair and regeneration [6], [44]. That is to say, p53 clears damaged cells by blocking cell cycle and inducing apoptosis in the early stage of AKI, while the decreased activity of p53 promotes cell proliferation and tissue repair during the recovery period of AKI [6], [44]. This dynamic balance of p53 activity is critical to the progression and recovery of AKI. Based on these researches, we hypothesized whether p53 was involved in MCM4-induced AKI by regulation cell proliferation, cell cycle, and cell apoptosis, and we also aimed to identify novel targets that modulate p53 pathway in AKI. Gene enrichment analysis identified MCM4′s role in regulating DNA replication, the cell cycle, the p53 pathway, and Notch signaling [45]. Furthermore, the transcriptome analysis of the gain-of-function p53 mutant revealed elevated MCM4 protein levels, despite unchanged MCM4 transcript quantities [46]. A recent study identified 415 genes were regulated by the p53-p21-RB signaling pathway, one of them being MCM4 [47]. In our study, we found that MCM4 negatively modulates the p53/p21 pathway. Inhibition of p53 activity partially reversed the effects on cell proliferation, cell cycle arrest, and apoptosis in MCM4-deficient cells, offering direct evidence that MCM4 could be a novel target in regulating the p53/p21 pathway. However, the relationship between p53 and MCM4 is complex, and their specific regulatory mechanisms require further investigation.

The p53-interacting protein, 53BP1, is essential for the functional effects of p53 [8]. Recently, studies have identified 53BP1 as a key component in the p53 pathway, influencing p53-dependent gene activation and cell fate determination [9], [24]. The 53BP1-USP28-p53 pathway plays a role in autosomal dominant polycystic kidney disease by regulating centrosomal integrity and the mitotic surveillance mechanism [48]. Besides polycystic kidney disease, 53BP1 has been implicated in hemodialysis and transplantation [48], [49]; however, studies on the function of 53BP1 in the development of AKI are limited. In our study, we firstly found the upregulation of 53BP1 in AKI mouse models and cisplatin-treated TECs. And we further declared the correlation between 53BP1 and p53/p21 signaling in the functional role of MCM4. Although significant c-jun-binding activity was identified in the 53BP1 promoter and MCM4 regulates c-jun activity, further research is required to elucidate the specific regulatory mechanisms.

MAD2B has been identified as a crucial regulator of cell proliferation in vascular smooth muscle, parietal epithelial, and glomerular endothelial cells [50], [51], [52]. Recently, we observed upregulation of MAD2B in cisplatin-induced AKI, and that the tubule-specific loss of MAD2B aggravated the severity of kidney injury by enhancing p53 signaling, indicating an adaptive response of MAD2B in PTCs during AKI. In our study, we also found a similar adaptative phenomenon, in which MCM4 was increased during AKI development, where it played a renoprotective role. Therefore, we hypothesized a connection between MAD2B and MCM4 during AKI development. As anticipated, MAD2B mediated the activity of CDK4/Cyclin D1 complex, then affected the phosphorylation level of Rb and the expression of E2F1, ultimately regulated the transcription of MCM4.

The MCM4/6/7 complex, part of the MCM2-7 complexes, acts as a replicative helicase and controls DNA replication [53]. Mutations in MCM4 disrupt the integrity of the MCM4/6/7 complex, leading to the dissociation into trimeric complexes and affecting the subsequent interactions of trimers [54]. MCM4 mutations disrupt its interaction with MCM6, destabilizing the MCM2-7 complex, which leads to deregulated DNA replication and abnormal chromosome structures [55]. And the disorder of DNA replication caused by MCM4 mutation might be associated with the disordered progression of the S phase [56]. In our study, MCM4 deficiency resulted in the inhibition of G1/S transition and DNA synthesis, potentially affecting the proliferative ability of tubular cells. In addition, MCM4 knockdown decreased the levels of MCM6 and MCM7 (Additional file1: Fig. S5), indicating that MCM4 deficiency disturbs the stability of MCM4/6/7 complex, impacting DNA helicase activity. Our prior research indicated that MCM6 depletion reduced MCM4 and MCM7 levels, underscoring the critical role of MCM4/6/7 complex stability [57]. These results may explain the effect of the loss of MCM4 on the inhibition of TECs proliferation, which may be attributed to the instability of MCM2-7 complex and thereby retarding DNA replication.

Recently, the role of MCM4 in kidney injury and repair has not been fully understood, but may be related to cell proliferation and DNA repair. After kidney injury, TECs need to proliferate to replace the damaged tissue in order to repair. MCM4, as a key factor in DNA replication, may support this process by promoting cell proliferation. In addition, kidney damage is often accompanied by DNA damage. MCM4 may help cells restore genomic stability by participating in DNA repair mechanisms. On the other hand, MCM4 may collaborate with other repair proteins to repair damaged DNA and prevent cell apoptosis or cancer. In our studies, the high expression of MCM4 accelerated the cell cycle progression and promoted the proliferation of TECs, indicating an important role of MCM4 during AKI development. In AKI-post renal repair progression, MCM4 may accelerate the recovery from injury by promoting the regeneration of TECs and replacing the injured cells, as well as the involvement of cell cycle regulation. Moreover, excessive proliferation may lead to fibrosis, and regulating MCM4 may help inhibit this process. At present, the functional role of MCM4 in AKI-post kidney repair is being studied by our research group. And the specific mechanisms of MCM4 in kidney injury and repair needs further investigation. Future studies can further explore the role of MCM4 in other kidney diseases.

In addition, it is currently unclear whether 53BP1 upregulation, induced by cisplatin administration, is causatively linked to worsening of AKI. The relationship between 53BP1 and p53 protein levels in AKI-induced renal injury is not yet understood. Investigating whether 53BP depletion induces p53 protein degradation is crucial, as 53BP1 silencing in renal cells results in reduced p53 protein expression. Therefore, it would be inspiring to explore the functional role of 53BP1 and its interaction with p53 during AKI development in future studies. Moreover, despite of careful inquiry, the specific role and underlying mechanism of MCM4 in the regulation of 53BP1 expression is poorly understood. Future proteomic studies of MCM4 and 53BP1 proteins might help solve this problem. Besides, no specific therapeutic strategy targeting MCM4 is currently undergoing clinical trials, and the clinical value and efficiency targeting MCM4 in the treatment of patients with AKI remains unclear.

Conclusion

In conclusion, our study firstly provides direct evidence that MCM4 plays an adaptive and renoprotective role in PTCs in AKI, preserving the proliferative ability of injured PTCs and limiting their apoptosis, thereby mitigating the severity of AKI development. Additionally, we have uncovered that the 53BP1-p53-p21 axis, mediated by MCM4, is an instrumental mechanism responsible for AKI development, which is a novel regulatory pathway. And the reno-protective role of MCM4 is partial mediated by MAD2B in the progression of AKI. Additional studies are required to clarify the regulatory mechanisms and investigate the therapeutic potential of targeting MCM4 and its related pathways in managing AKI.

Compliance with Ethics Requirements

All animal experiments were performed in compliance with the ethical guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (Approval No. IACUC 2854, approval date 2022).

Funding

This study was financially supported by the National Natural Science Foundation of China (82370728, 81974096, 82170773, 81974097, 82100729, 82200808, 82200841, 82300843, and 82300786), the National Key Research and Development Program of China (2024YFC3044900 and 2021YFC2500200), and the Key Research and Development Program of Hubei Province (2023BCB034).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: