Lysyl oxidase-like 2 inhibition ameliorated podocyte fibrosis by inhibiting transforming growth factor-beta signaling under high-glucose conditions

Nara Jeon; Beom Jin Lim; Hoon Young Choi

doi:10.23876/j.krcp.25.097

Kidney Res Clin Pract > Epub ahead of print

Jeon, Lim, and Choi: Lysyl oxidase-like 2 inhibition ameliorated podocyte fibrosis by inhibiting transforming growth factor-beta signaling under high-glucose conditions

Original Article

Kidney Research and Clinical Practice 2025;j.krcp.25.097.

Published online: September 30, 2025

DOI: https://doi.org/10.23876/j.krcp.25.097

Lysyl oxidase-like 2 inhibition ameliorated podocyte fibrosis by inhibiting transforming growth factor-beta signaling under high-glucose conditions

Nara Jeon¹

, Beom Jin Lim^1,^*

, Hoon Young Choi^2,^3,^*

¹Department of Pathology, Yonsei University College of Medicine, Seoul, Republic of Korea

²Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea

³Division of Nephrology, Gangnam Severance Hospital, Yonsei University Health System, Seoul, Republic of Korea

Correspondence: Hoon Young Choi Department of Internal Medicine, Yonsei University College of Medicine, 211 Eonju-ro, Gangnam-gu, Seoul 06273, Republic of Korea. E-mail: hychoidr@yuhs.ac

Beom Jin Lim Department of Pathology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea. E-mail: BJLim@yuhs.ac

^*Hoon Young Choi and Beom Jin Lim contributed equally to this study as co-corresponding authors.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial and No Derivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/) which permits unrestricted non-commercial use, distribution of the material without any modifications, and reproduction in any medium, provided the original works properly cited.

Abstract

Background

Lysyl oxidase-like 2 (LOXL2) has been implicated in tissue fibrosis; however, its role in diabetic podocyte injury remains unclear. This study aimed to investigate the contribution of LOXL2 to fibrotic signaling in podocytes under high-glucose conditions and identify its downstream molecular pathways.

Methods

LOXL2 expression was examined in the glomeruli of patients with diabetes using immunofluorescence staining. Human podocytes were cultured under normal or high-glucose conditions, and LOXL2 was silenced using short hairpin RNA. The gene and protein expression of fibrotic markers, autophagy-related proteins, and key signaling molecules were assessed using quantitative real-time polymerase chain reaction and western blotting.

Results

LOXL2 expression was markedly elevated in the glomeruli of patients with diabetes, particularly in podocytes. In vitro, high-glucose levels significantly upregulated LOXL2 and TGF-β messenger RNA expression in podocytes. LOXL2 knockdown suppressed TGF-β expression and reduced the protein levels of collagen I and α-smooth muscle actin. Furthermore, the phosphorylation of Smad2 and expression of Smad4 decreased in LOXL2-deficient cells, indicating that LOXL2 promotes fibrosis via the transforming growth factor-beta (TGF-β)/Smad pathway. In contrast, the LOXL2 knockdown did not significantly affect the expression of autophagy markers (p62, Beclin-1, and LC3A/B) or activation of the p38 MAPK pathway.

Conclusion

LOXL2 is upregulated in diabetic podocytes and contributes to hyperglycemia-induced fibrosis by activating the TGF-β/Smad signaling pathway. These findings suggest that LOXL2 may serve as a potential therapeutic target for preventing or attenuating podocyte injury in patients with diabetic nephropathy.

Keywords: Diabetic nephropathies, Fibrosis, LOXL2, Podocytes, Transforming growth factor beta

Introduction

Diabetic nephropathy (DN) is a major complication of diabetes and a leading cause of chronic kidney disease (CKD), characterized by alterations that include mesangial expansion, thickening of the glomerular basement membrane (GBM), and accumulation of extracellular matrix (ECM) proteins, culminating in glomerular sclerosis [1–6]. Podocytes, which are critical for maintaining the structural and functional integrity of the GBM, are vulnerable to diabetes [3,4,7]. Podocyte depletion, structural changes, and functional damage drive the progression of DN by compromising the filtration barrier and contributing to albuminuria. Central to these processes is the hyperglycemic milieu, which induces reactive oxygen species production and activates profibrotic pathways, particularly transforming growth factor-beta (TGF-β) signaling [1,8–12]. The TGF-β/Smad signaling pathway plays a pivotal role in ECM remodeling and fibrosis in DN. Hyperglycemia induces the upregulation of TGF-β1 in renal cells, including podocytes, mesangial cells, and tubular epithelial cells [4,10–12]. Other pathways, including p38 mitogen-activated protein kinase (p38 MAPK) activation and impaired autophagy, have also been implicated in podocyte injury under diabetic conditions [13–17]. However, their specific contribution to chronic fibrotic remodeling in podocytes remains less clearly defined.

Among ECM remodeling enzymes, the lysyl oxidase (LOX) family, particularly lysyl oxidase-like 2 (LOXL2), is intricately linked to the profibrotic effects of the TGF-β/Smad signaling pathway. LOXL2, an enzyme responsible for cross-linking collagen and elastin, stabilizes the ECM and promotes its pathological accumulation. TGF-β signaling directly upregulates LOXL2 expression, enhancing ECM stiffness and fibrosis [18,19]. Some studies have suggested that LOXL2 could modulate intracellular stress pathways, including p38 MAPK activation and autophagy [20,21], although the functional significance of these interactions in chronic diabetic settings remains uncertain.

This study aimed to elucidate the molecular and cellular changes in podocytes exposed to high-glucose conditions, with particular emphasis on the roles of TGF-β signaling, Smad pathway activation, and LOXL2. The potential involvement of p38 MAPK activation and autophagy impairment as secondary pathways was also explored. By exploring these pathways in the context of hyperglycemia, targeting the TGF-β/Smad pathway and LOXL2 represents a promising strategy for mitigating fibrosis in DN [22].

Methods

Ethics statement

The study protocol was approved by the Institutional Review Board of the Gangnam Severance Hospital, Yonsei University College of Medicine (IRB No. 3-2016-0234).

Cell culture

Conditionally immortalized human podocytes were produced by transfection with the temperature-sensitive simian virus 40 (SV40) T gene [23,24]. To establish stable LOXL2 knockdown in human podocytes, the cells were transduced with LOXL2-specific small hairpin RNA (shRNA) lentiviral particles (Santa Cruz Biotechnology). Before transduction, the cells were incubated in a medium containing 5 µg/mL polybrene (Santa Cruz Biotechnology) to enhance the viral transduction efficiency. Subsequently, LOXL2 shRNA and control shRNA lentiviral particles were added at a multiplicity of infection of 2 and incubated overnight at 33 °C. Transduced cells were selected using a medium supplemented with 4 µg/mL puromycin dihydrochloride (Santa Cruz Biotechnology). For clonal enrichment, cells were further selected with 4 µg/mL puromycin and maintained in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), antibiotics, and puromycin.

Podocyte proliferation was supported at 33 °C in the RPMI 1640 medium with insulin-transferrin-selenium (ITS) (Gibco). Differentiation was induced by transferring the cells to an ITS-free RPMI 1640 medium at 37 °C for 14 days, during which morphological changes characteristic of podocytes were observed. Differentiation was confirmed by synaptopodin expression using immunofluorescence staining and western blot analysis. Before the experiments, the cells were serum-starved overnight to synchronize their metabolic state. For glucose treatment experiments, differentiated podocytes were exposed to high glucose (30 mM D-glucose) for 48 hours.

RNA isolation and real-time polymerase chain reaction

Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions to ensure RNA purity and integrity are suitable for downstream applications. RNeasy lysis buffer with β-mercaptoethanol deactivated RNases, and lysates were passed through QIAshredder columns (homogenization column; Qiagen) for disruption and removal of debris. Ethanol was added to the lysate before binding RNA to RNeasy spin columns, followed by washing with RNeasy wash buffer 1 (Qiagen) and RNA purification buffer (Qiagen) and elution in RNase-free water. RNA quality was confirmed using NanoDrop spectrophotometry (Thermo Fisher Scientific), ensuring A260/A280 and A260/A230 ratios of >1.8. For complementary DNA (cDNA) synthesis, 1 μg of RNA was reverse transcribed using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative real-time polymerase chain reaction (qPCR) was performed on a LightCycler 96 System (Roche Diagnostics) using FastStart DNA Probes purchased from Thermo Fisher Scientific and used according to the manufacturer’s instructions. Thermal cycling included 40 cycles at 95 °C for 15 seconds and 60 °C for 1 minute. Gene expression levels were calculated using the 2^–ΔΔCt method, with 18S ribosomal RNA as the internal control. Melt curve analysis confirmed amplification specificity, and the results were validated through triplicate biological replicates. The primer sequences used for qPCR are listed in Supplementary Table 1 (available online).

Western blot analysis

Protein extraction for western blotting was conducted using radioimmunoprecipitation assay buffer (Millipore) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Homogenized samples were lysed on ice and centrifuged at 17,500 ×g for 20 minutes, and the supernatants were collected. Protein concentration was determined using the bicinchoninic acid assay, with absorbance measured at 562 nm. Proteins were denatured with NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen, Thermo Fisher Scientific) and a reducing agent and heated at 70 °C for 10 minutes. Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore) using a wet-transfer system. Membranes were blocked with 5% skim milk in Tris-buffered Saline with Tween 20 (TBST) (BIOSESANG) to prevent nonspecific binding, followed by overnight incubation at 4 °C with primary antibodies diluted in TBST with bovine serum albumin or skim milk (Becton, Dickinson and Company). After washing, horseradish peroxidase-conjugated secondary antibodies were applied for 1 hour at room temperature. Protein bands were visualized with enhanced chemiluminescence reagent (Cytiva) and captured using the Amersham ImageQuant 800 system (Cytiva). Band intensities were quantified using ImageJ software (NIH) [25] and normalized to β-actin as a loading control. Relative protein expression levels were expressed as fold changes, and experiments were performed in triplicate to ensure reproducibility. Antibody information is provided in Supplementary Table 2 (available online).

Immunofluorescence staining

Paraffin-embedded human kidney tissues were sectioned (4 μm), deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer (pH 6.0). Sections were incubated with primary antibodies overnight at 4 °C, followed by DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) counterstaining. Slides were mounted with anti-fade medium and imaged using an Axio Imager M2 microscope (Zeiss).

Statistical analysis

Data are expressed as mean ± standard error of the mean. Differences between groups were analyzed by the Mann-Whitney test or Kruskal-Wallis test using IBM SPSS version 27.0 (IBM Corp.). A p-value <0.05 was considered statistically significant.

Results

Transforming growth factor-beta/Smad pathway in high-glucose conditions

LOXL2 expression was confirmed in human kidney glomeruli, as observed using immunofluorescence analysis. Synaptopodin staining (red) highlights podocytes, whereas LOXL2 staining (green) shows its localization in the glomerular structure. In the diabetic patient group, LOXL2 expression was significantly elevated compared to that in the normal control group, as indicated by the intensified green fluorescence and distinct localization within the glomeruli (arrows). This result demonstrates that LOXL2 is actively expressed in the glomeruli, particularly under diabetic conditions (Supplementary Fig. 1, available online).

To assess whether hyperglycemia directly induces LOXL2 and fibrogenic signaling in podocytes, human podocytes were treated with high glucose (30 mM) or normal glucose (5.5 mM) concentrations, and qPCR was performed. As shown in Fig. 1, high-glucose treatment significantly upregulated both LOXL2 and TGF-β messenger RNA (mRNA) expression, suggesting that hyperglycemia enhances fibrotic gene expression in the podocytes. To investigate whether LOXL2 regulates TGF-β expression, LOXL2 was silenced using shRNA in human podocytes. As shown in Fig. 2A, TGF-β mRNA expression was significantly reduced in LOXL2 knockdown cells, indicating that LOXL2 contributes to the transcriptional regulation of TGF-β. In parallel, western blot analysis (Fig. 2B) revealed that LOXL2 knockdown also reduced the protein levels of the fibrotic markers α-smooth muscle actin (α-SMA) and collagen I, supporting a key role for LOXL2 in promoting podocyte fibrosis. These findings were further validated under high-glucose conditions (Fig. 3), where LOXL2 and TGF-β mRNA levels were examined in both control and LOXL2 shRNA-transduced cells. While high-glucose levels significantly increased LOXL2 and TGF-β mRNA levels in control cells, this induction was markedly attenuated in LOXL2 knockdown human podocytes, confirming that LOXL2 mediates the high glucose-induced upregulation of TGF-β. To elucidate downstream signaling, we examined the activation of Smad2 and Smad4 using western blot analysis (Fig. 4). High-glucose levels increased the phosphorylation of Smad2 and expression of Smad4 in control cells, but these responses were significantly suppressed in LOXL2-deficient podocytes, suggesting that LOXL2 promotes the activation of the canonical TGF-β/Smad signaling pathway.

Autophagy pathway and p38 mitogen-activated protein kinase in the high-glucose condition

To determine whether LOXL2 affects autophagy, we analyzed the protein levels of p62, Beclin-1, and LC3A/B. No significant differences were observed in the expression of these autophagy-related proteins between the control and LOXL2 knockdown groups, indicating that LOXL2 does not play a key role in the regulation of autophagy under these experimental conditions (Fig. 5).

To further assess the role of LOXL2 under hyperglycemic conditions, quantitative densitometric analysis of total p38 and phosphorylated p38 (p-p38) protein levels was performed and statistically compared using unpaired t tests. For total p38 expression, no significant differences were observed between the control shRNA group and the LOXL2 knockdown group under both normal glucose and high-glucose conditions (p = 0.93 and p = 0.81, respectively). Similarly, p-p38 levels showed a tendency to increase in the LOXL2 knockdown group under normal glucose (mean = 1.39 vs. 1.00 in control), but this change did not reach statistical significance (p = 0.12). No significant difference in p-p38 expression was detected under high-glucose conditions either (p = 0.92). These results suggest that neither total p38 nor p-p38 levels are significantly altered by LOXL2 knockdown, supporting the notion that LOXL2 exerts its fibrotic effects predominantly through the TGF-β/Smad signaling pathway rather than via the p38 MAPK cascade (Fig. 6).

Discussion

This study investigated the role of LOXL2 in DN progression using a hyperglycemic podocyte model. The findings revealed that LOXL2 is critically involved in mediating fibrotic changes through its interaction with the TGF-β/Smad signaling pathway. This may contribute to GBM thickening and glomerular sclerosis progression. The observed increase in LOXL2 and TGF-β mRNA expression in human podocytes exposed to high-glucose conditions aligns with previous studies, suggesting that hyperglycemia induces profibrotic signaling pathways in podocytes. Knockdown of LOXL2 successfully attenuated these effects, reducing TGF-β expression and the downstream activation of Smad2 and Smad4, which are central mediators of fibrosis. These findings underscore the importance of LOXL2 in pathological ECM remodeling in diabetic kidneys [26].

The involvement of LOXL2 in TGF-β-mediated signaling was further supported by the reduction in fibrosis markers, such as α-SMA and collagen I, following LOXL2 inhibition [27,28]. These markers are hallmarks of myofibroblast activation and ECM deposition, which contribute to the loss of renal function in DN [9,29]. The ability of LOXL2 to regulate these markers highlights its role as a key driver of podocyte fibrosis [30]. The data also suggests that LOXL2 may amplify TGF-β signaling by enhancing ECM stiffness and providing mechanotransductive feedback, perpetuating the fibrotic response. This feedback loop may create a self-reinforcing cycle of TGF-β activation and ECM remodeling, which exacerbates glomerulosclerosis and renal dysfunction in diabetic conditions [3,29].

Compared to previous studies, this study presents key advantages that deepen our understanding of LOXL2’s role in kidney fibrosis, especially in the context of DN. Cosgrove et al. [30] used Alport mice and demonstrated that pharmacologic inhibition of LOXL2 with a small molecule reduced interstitial and glomerular fibrosis, normalized GBM structure, and decreased albuminuria and BUN levels, highlighting LOXL2’s systemic role in promoting renal fibrosis and glomerulosclerosis in a genetic model of CKD. Stangenberg et al. [31] reported that LOXL2 inhibition ameliorates glomerulosclerosis and albuminuria in a streptozotocin-induced diabetic model, primarily through modulation of ECM deposition. Building upon these findings, this study is uniquely focused on podocyte-specific mechanisms of LOXL2 in a high-glucose environment, closely mimicking diabetic kidney disease at the cellular level. This study is the first to clearly demonstrate increased LOXL2 expression specifically in human podocytes under high-glucose conditions and link this expression to the upregulation of TGF-β1 and downstream fibrotic markers, such as phosphorylated Smad2. Moreover, using lentiviral gene silencing techniques in human immortalized podocytes, this study provides direct mechanistic evidence that LOXL2 mediates TGF-β1 signaling and collagen production. This human cell-based experimental approach bridges the gap between animal models and human diseases, offering strong translational relevance. Unlike the systemic inhibition models used in previous studies, the podocyte-specific analysis in this study allowed for a more precise investigation of LOXL2’s localized effects in glomerular pathology. Therefore, this study complements the existing literature and extends it by uncovering a novel podocyte-centric mechanism by which LOXL2 contributes to diabetic kidney disease, suggesting that targeted modulation of LOXL2 in glomerular cells may offer a promising therapeutic approach in DN. The data also suggests that LOXL2 may amplify TGF-β signaling by enhancing ECM stiffness and providing mechanotransductive feedback, perpetuating the fibrotic response. This feedback loop may create a self-reinforcing cycle of TGF-β activation and ECM remodeling, exacerbating glomerulosclerosis and renal dysfunction in diabetic conditions. Compared with previous studies on LOXL2’s role in fibrotic diseases [30], this study highlights distinct and novel findings regarding the TGF-β/Smad pathway and its downstream effects in human podocytes under hyperglycemic conditions.

Our data show that LOXL2 does not meaningfully influence autophagy-related proteins (p62, Beclin-1, LC3A/B) under hyperglycemic stress, suggesting a pathway-specific effect limited to TGF-β/Smad rather than a broad impact on other stress response mechanisms. Despite the established relationship between fibrosis and autophagy in CKD [15,16], this study found no significant changes in autophagy-related proteins, such as p62, Beclin-1, and LC3A/B, in LOXL2-inhibited podocytes. These results suggest that LOXL2’s role in DN may be primarily limited to ECM remodeling and TGF-β/Smad signaling rather than autophagy regulation. This finding contrasts with other studies that have implicated autophagy as a protective mechanism in podocytes under stress, indicating that LOXL2-mediated fibrosis may operate independently of autophagy.

p38 MAPK is activated by hyperglycemia and oxidative stress, leading to podocyte injury and apoptosis [13,32]. Our study revealed that the p38 MAPK pathway did not play a significant role in the fibrotic response of podocytes under high-glucose conditions in the context of LOXL2 signaling. This finding contrasts with some previous reports in other renal injury models, where p38 MAPK activation was associated with glomerular damage [33–35]. These results refine our understanding of the molecular mechanisms underlying diabetic podocytopathy and highlight LOXL2 as a selective regulator of established fibrotic signaling rather than a broad activator of stress response cascades. The discrepancy may be attributed to differences in the exposure duration of high-glucose condition. This study focused on the chronic effects of prolonged high-glucose exposure, modeling later-stage fibrotic changes rather than acute stress responses. It is plausible that p38 MAPK activation diminishes over time, giving way to sustained activation of profibrotic pathways such as TGF-β/Smad signaling. Thus, the lack of significant p38 MAPK activation observed in our study likely reflects a temporal shift from early protective mechanisms toward chronic fibrotic remodeling during prolonged hyperglycemia [13,14]. Therefore, targeting LOXL2 may offer a more focused antifibrotic strategy that minimizes off-target effects on stress-related pathways, such as p38 MAPK, which could be advantageous in preserving podocyte viability and function in DN.

While this study provides valuable insights into the role of LOXL2 in DN, it has limitations. First, the experiments were conducted using in vitro models of hyperglycemia in podocytes, which may not fully capture the complexity of DN in vivo. The podocyte-specific effects of LOXL2 observed in this study require validation in animal models to confirm their relevance in the context of an intact renal microenvironment. Second, this study focused primarily on fibrotic and stress signaling pathways and did not explore other potential downstream effects of LOXL2 inhibition, such as immune or inflammatory responses. Third, although autophagy-related proteins were analyzed, the methodology may not have captured subtle or dynamic changes in autophagic flux. More comprehensive autophagy assays, such as live-cell imaging or electron microscopy, could provide a clearer picture of LOXL2’s role in autophagy regulation. Finally, this study did not evaluate the long-term effects of LOXL2 inhibition, which are essential for understanding its therapeutic potential and safety profile in CKD.

Despite these limitations, our findings highlight the critical role of LOXL2 in driving fibrotic and stress responses in diabetic podocytes, offering promising directions for therapeutic development [36]. Future studies should address these limitations by extending investigations to in vivo models, examining broader signaling pathways, and evaluating the long-term outcomes of LOXL2 inhibition. Such efforts could pave the way for novel and effective treatments for DN.

Supplementary Materials

Supplementary data are available at Kidney Research and Clinical Practice online (https://doi.org/10.23876/j.krcp.25.097).

j-krcp-25-097-Supplementary-Table-1.pdf

j-krcp-25-097-Supplementary-Table-2.pdf

j-krcp-25-097-Supplementary-Fig-1.pdf

Notes

Conflicts of interest

All authors have no conflicts of interest to declare.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (IRIS no. RS-2016-NR015228, RS-2020-NR053581). This study was also supported by a research grant from the Gangnam Severance Hospital, Yonsei University College of Medicine and a faculty research grant from Yonsei University College of Medicine for 2012 (6-2012 0036).

Acknowledgments

The authors would like to thank Professor Jun Oh at the University of Hamburg for kindly providing the immortalized human podocyte cell line used in this study.

Data sharing statement

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

Authors’ contributions

Conceptualization: All authors

Data curation: BJL, HYC

Formal analysis, Project administration: HYC

Investigation: All authors

Methodology: NJ, BJL

Supervision, Validation: BJL

Writing–original draft: NJ, HYC

Writing–review & editing: HYC

All authors read and approved the final manuscript.

Figure 1.

Upregulation of LOXL2 and TGF-β expression in podocytes under high-glucose conditions.

(A) Quantitative real-time-PCR analysis showed that LOXL2 messenger RNA (mRNA) expression was significantly increased in podocytes treated with high glucose (30 mM) compared to normal glucose (5.5 mM) conditions. (B) TGF-β mRNA levels were also significantly elevated in response to high glucose. Data are presented as mean ± standard error of the mean from three independent experiments. ^*p < 0.05.

Figure 2.

LOXL2 knockdown reduces the expression of fibrotic markers in human podocytes (H. podocytes).

(A) TGF-β messenger RNA (mRNA) levels were significantly suppressed in H. podocytes transduced with LOXL2 short hairpin RNA (shRNA). (B) Western blot analysis of collagen I and α-smooth muscle actin (α-SMA) protein expression in H. podocytes transduced with control or LOXL2 shRNA. β-actin was used as a loading control. Quantification of protein levels showed that LOXL2 knockdown significantly reduced collagen I and α-SMA expression. Bar graphs represent protein expression levels normalized to β-actin protein expression, quantified using ImageJ (NIH). Data are presented as mean ± standard error of the mean from three independent experiments. ^*p < 0.05.

Figure 3.

Effect of LOXL2 knockdown on LOXL2 and TGF-β mRNA expression in high glucose-treated human podocytes (H. podocytes).

(A) Quantitative real time polymerase chain reaction analysis showed that LOXL2 mRNA levels were significantly elevated under high-glucose conditions in control short hairpin RNA (shRNA)-transduced H. podocytes, but this upregulation was effectively suppressed in LOXL2 shRNA-transduced cells. (B) TGF-β mRNA expression was increased in high glucose-treated control cells, whereas LOXL2 knockdown markedly attenuated this induction. Data are presented as mean ± standard error of the mean from three independent experiments. mRNA, messenger RNA. ^*p < 0.05.

Figure 4.

Effects of LOXL2 knockdown on Smad2/4 signaling in high glucose-treated human podocytes (H. podocytes).

Protein expression of Smad2, phosphorylated Smad2 (p-Smad2), and Smad4 was evaluated by Western blot in H. podocytes transduced with control or LOXL2 short hairpin RNA (shRNA) under normal and high-glucose conditions. β-actin was used as a loading control. Bar graphs represent the quantification of Smad2 and Smad4 protein levels normalized to β-actin. LOXL2 knockdown attenuated the high glucose-induced upregulation of Smad2 and Smad4 expression. Bar graphs represent protein expression levels normalized to β-actin protein expression, quantified using ImageJ (NIH). Data are presented as mean ± standard error of the mean from three independent experiments. ^*p < 0.05.

Figure 5.

Effect of LOXL2 knockdown on autophagy-related protein expression in human podocytes (H. podocytes) under high-glucose conditions.

Protein expression levels of p62, Beclin-1, and LC3 A/B were assessed by western blot in H. podocytes transduced with control or LOXL2 short hairpin RNA (shRNA) under normal and high-glucose conditions. β-actin was used as a loading control. Quantitative analysis showed no significant changes in the expression of p62, Beclin-1, or LC3 A/B with either high-glucose treatment or LOXL2 knockdown. Bar graphs represent protein expression levels normalized to β-actin protein expression, quantified using ImageJ (NIH). Data are presented as mean ± standard error of the mean from three independent experiments.

Figure 6.

Effect of LOXL2 knockdown on p38 MAPK signaling in human podocytes (H. podocytes) under high-glucose conditions.

Western blot analysis of total p38 and phosphorylated p38 (p-p38) protein levels in H. podocytes transduced with control or LOXL2 short hairpin RNA (shRNA), under normal and high-glucose conditions. β-actin was used as a loading control. Quantitative analysis showed no significant changes in total p38 expression, while a slight increase in p-p38 levels was observed in the LOXL2 knockdown group under high-glucose conditions. Compared to the control shRNA under normal glucose, neither LOXL2 knockdown under normal glucose (p = 0.93) nor high glucose (p = 0.81) showed statistically significant changes. For p-p38, a trend toward increased phosphorylation was observed in LOXL2 shRNA-treated cells under normal glucose compared to control (p = 0.12), but this did not reach statistical significance. Similarly, no significant difference was detected under high-glucose conditions (p = 0.92). Bar graphs represent protein expression levels normalized to β-actin protein expression, quantified using ImageJ. Data are presented as mean ± standard error of the mean from three independent experiments. MAPK, mitogen-activated protein kinase.

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