GRAPHICAL ABSTRACT

Highlights

• HFD induces tau phosphorylation and renal injury in NOX5 pod+ mice.

• Tau pSer202/Thr205 is elevated in both mouse and human DN kidneys.

• ER stress and TGF-β-mediated fibrosis promote tau phosphorylation.

• Tau phosphorylation and ER stress are potential therapeutic targets.

INTRODUCTION

Diabetic nephropathy (DN), a major cause of renal dysfunction, is a leading contributor to morbidity and mortality [1]. Microtubules, composed of α- and β-tubulin heterodimers, are essential components of the cytoskeleton. Tau, a microtubule-associated protein (MAPT) that is highly expressed in neuronal axons, where it stabilizes microtubules and regulates their assembly and organization [2,3]. In tauopathies such as Alzheimer’s disease (AD) and Parkinsonism, tau becomes abnormally hyperphosphorylated, resulting in cytoskeletal disruption, retrograde neurodegeneration, and neuronal cell death [4,5].

Recent studies have revealed that tau is expressed not only in the brain, but also in peripheral organs such as the kidney, liver, and skeletal muscle at levels comparable to those found in the brain [6-8]. The detection of both tau and amyloid β in the pancreas of individuals with type 2 diabetes by Miklossy et al. [9] suggests overlapping molecular features between metabolic and neurodegenerative disorders.

In the kidneys, microtubules are essential for maintaining cell polarity and tubular epithelial cell function [10,11]. Tau has also been implicated in renal physiology; Valles-Saiz et al. [12] showed that tau expression alters glomerular morphology in podocytes. Phosphorylated tau, similar to its role in neurons, contributes to microtubule instability in podocytes, leading to cell body shrinkage and foot process effacement in a mouse model of chronic kidney disease [13].

Emerging evidence suggests that metabolic stress, endoplasmic reticulum (ER) stress, and transforming growth factor β (TGF-β) signaling can promote tau phosphorylation [14,15]. These findings support the hypothesis that tau dysregulation may contribute to cytoskeletal instability, cellular dysfunction, and fibrotic remodeling in the diabetic kidney.

The ER stress caused by the accumulation of misfolded proteins and oxidative stress, is a known contributor to AD pathogenesis [16]. It is also implicated in kidney disease such as DN, renal fibrosis, and ischemia-reperfusion injury. However, the interplay between tau phosphorylation and ER stress in renal pathology remains poorly understood.

This study aimed to evaluate serum and urinary tau levels as potential diagnostic markers for DN, and to investigate the role of phosphorylated tau and its interaction with ER stress in high-fat diet (HFD)-induced kidney injury. These findings may offer novel insights into the pathogenesis and therapeutic targeting of DN.

METHODS

Human kidney samples

Human kidney tissues used in this study were obtained with approval from the Institutional Review Board (IRB) of Soonchunhyang University Cheonan Hospital (IRB No. 2023-12-02 and 2024-03-035, Cheonan, Korea). As this was a retrospective study, the requirement for informed consent was waived. Control kidney tissues (n=5) were derived from patients undergoing partial or radical nephrectomy for urothelial carcinoma or renal cell carcinoma; only non-neoplastic cortical regions, confirmed to be free of tumor involvement, were selected for analysis and embedded in paraffin blocks. DN samples (n=4) were obtained via renal biopsy and were histologically confirmed as DN. All specimens were independently reviewed and validated by a pathologist (Ji-Hye Lee) to confirm the final diagnoses.

Animal experiments

Mice expressing the renal podocyte-specific human NADPH oxidase 5 (NOX5 pod⁺ mice) were kindly provided by Dr. Christopher R.J. Kennedy [17]. Eight-week-old NOX5 pod⁺ mice were housed in cages placed in a room maintained at 12-hour light/dark cycles and ambient temperatures (22°C–24°C). The mice were randomly divided into the following six groups: group I, NOX5 pod⁺ mice fed a regular diet (RD) for 3 weeks; group II, NOX5 pod⁺ mice fed an HFD (60% kcal from fat) for 3 weeks; group III, NOX5 pod⁺ mice fed an RD for 6 weeks; group IV, NOX5 pod⁺ mice fed an HFD for 6 weeks; group V, NOX5 pod⁺ mice fed an RD for 12 weeks; and group VI, NOX5 pod⁺ mice fed an HFD for 12 weeks. Food intake and body weight were recorded weekly. After lethal anesthesia, serum and tissues from the mice were collected and stored for further studies. All experiments were approved by the Institutional Animal Care and Use Committee of Yonsei University Wonju College of Medicine (YWC-200515-1) (Wonju, Korea).

Western blot analysis

The sliced renal cortex was homogenized to extract proteins in a protein lysis buffer (Elpis Biotech, Daejeon, Korea) with a phosphatase inhibitor (GenDepot, Barker, TX, USA). Proteins were separated by 8% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membranes were incubated with primary antibodies (Supplementary Table 1) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, Danvers, MA, USA). The blots were visualized using a chemiluminescence UVP BioSpectrum 600 imaging system (UVP LLC, Upland, CA, USA) and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Protein dot blot analysis

A 1 μL cell lysate was pipetted on to a dry nitrocellulose membrane to form a dot. Membranes were blocked in 5% skimmed milk in tris-buffered saline with 1% Tween 20 detergent (TBST) for 1 hour at room temperature, and blotted in 5% skimmed milk in TBST for 1 hour. Blotted membranes were imaged using a chemiluminescence UVP BioSpectrum 600 imaging system and quantified using Image J software. Identical circles were centered on each dot, and the signal volume was computed using the software.

Glucose tolerance test

After 8 hours of fasting, mice received an intraperitoneal injection of glucose (1 g/kg). Blood glucose levels were measured from tail vein samples at 0, 15, 30, 60, 90, and 120 minutes using an Auto-Check device (Diatech Korea Co., Seoul, Korea). Assessment of urine albumin and creatinine For 24 hours urine collection, the mice were individually housed in metabolic cages for 24 hours and urine was collected. Urinary albumin and creatinine (ACR) levels were measured using Exocell Nephrat II and The Creatinine Companion, respectively, in accordance with the manufacturer’s instructions (Exocell Inc., Doylestown, PA, USA), and ACR was calculated.

Assessment of the phosphorylated tau level in serum and urine

To confirm the change in the expression of phosphorylated tau due to the HFD, it was measured according to the manufacturer’s instructions using an enzyme-linked immunosorbent assay (MyBioSource Inc., San Diego, CA, USA) in serum and 24 hours urine.

Cell cultures

Mouse and human renal mesangial cells were cultured in standard growth media. After 2 hours of serum starvation, cells were treated with 30 mM high glucose (HG), 100 μM hydrogen peroxide (H₂O₂), or 5 ng/mL recombinant mouse TGF-β (Abcam, Cambridge, UK). ER stress was induced using 500 nM thapsigargin (Tg; Sigma-Aldrich, St. Louis, MO, USA), and cells were also treated with 100 nM 4-phenylbutyric acid (4PBA; ER stress inhibitor, Sigma-Aldrich) and 10 μM thousand and one amino acid kinases (TAOKs; Cayman Chemical, Ann Arbor, MI, USA) inhibitor for 24 hours.

Immunocytochemical staining

Immunocytochemistry was performed on mouse and human renal mesangial cells treated with HG (30 mM), H₂O₂ (100 μM), or TGF-β (5 ng/mL). After fixation with 4% paraformaldehyde, cells were incubated with antibodies against pTau Ser202 and Thr205, E-cadherin, and collagen type 1, followed by 4ʹ,6-diamidino-2-phenylindole (DAPI) nuclear staining. Images were captured using a Zeiss LSM 800 confocal microscope (LSM800; Carl Zeiss, Jena, Germany).

Expression of pTau Ser202 and Thr205 in mouse and human kidney

Paraffin-embedded mouse and human kidney tissues were stained for pTau Ser202 and Thr205 using a commercial immunohistochemistry staining kit. Tissue sections (2.5-μm thickness) were cut, deparaffinized in xylene, and hydrated using an ethanol-deionized water series. Endogenous peroxidase activity was blocked using 3% H₂O₂ in methanol for 15 minutes.

Sections were washed and stained with antibody against pTau Ser202 and Thr205 (1:400, Invitrogen, Waltham, MA, USA) and total tau (1:500, Santa Cruz, Santa Cruz, CA, USA). Primary antibody binding was visualized using 3,3ʹ-diaminobenzidine (DAB; Sigma-Aldrich) as a chromogen, followed by hematoxylin counterstaining and mounting. The tissue sections were examined using an optical microscope equipped with a charge-coupled device camera (Pulnix, Sunnyvale, CA, USA).

Statistics

All data are expressed as the mean±standard error of the mean. Statistical analyses were performed using Student’s t-test for comparisons between two groups. For experiments involving more than two groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used. Differences were considered statistically significant at P<0.05.

RESULTS

Phosphorylation of tau contributes to renal damage

To assess the link between tau phosphorylation and renal damage, levels were measured in wild type and NOX5 pod⁺ mice after 12 weeks of HFD. Phosphorylation was higher in HFD-fed mice compared to RD-fed controls, and further elevated in NOX5 pod⁺ mice under both diets (Fig. 1A). In mesangial cells, HG or H₂O₂ treatment for 24 hours increased tau phosphorylation (Fig. 1B). Time-dependent HG exposure elevated tau phosphorylation at Ser202 and Thr205 and α-smooth muscle actin (α-SMA) expression (Fig. 1C-F), indicating a role of tau phosphorylation in renal injury.

Phosphorylated Tau Ser202 and Thr205 are increased in kidney tissues from patients with diabetic nephropathy

Immunohistochemistry for pTau Ser202 and Thr205 was performed in kidneys from control and DN patients. In control human kidney tissues, glomerular cells showed no detectable staining for pTau Ser202 and Thr205. Compared to controls, expression of pTau Ser202 and Thr205 was increased in DN glomeruli, predominantly localized in the nuclei of glomerular cells (Fig. 2A and B). Correlation analysis was performed between the number of pTau Ser202 and Thr205 positive cells and clinical parameters including estimated glomerular filtration rate and proteinuria (Fig. 2C and D). Proteinuria showed a statistically significant positive correlation with the number of pTau Ser202 and Thr205 positive cells, indicating that higher urinary protein levels were associated with increased pTau Ser202 and Thr205 accumulation in glomerular cells (Fig. 2E). These results suggest that pTau Ser202 and Thr205 accumulation in the nuclei of glomerular cells may contribute to cellular injury or dysfunction in DN, particularly in relation to proteinuria severity.

HFD induces early metabolic changes and increases phosphorylated tau levels in NOX5 pod⁺ mice

To investigate how HFD affects metabolism overtime, we monitored body weight, glucose tolerance, and tau phosphorylation in mice for 12 weeks. HFD-fed mice showed increased body weight at 3 and 6 weeks; however, this gain was not observed at 12 weeks (Supplementary Fig. 1A, E, and I). Glucose intolerance progressively worsened during the study periods, as reflected by increasing area under the curve values (Supplementary Fig. 1B, F, and J). Notably, tau phosphorylation levels detected by enzyme-linked immunosorbent assay (ELISA), along with phosphorylation at Ser202 and Thr205 measured by Western blot, increased significantly at 3 and 6 weeks, but returned to baseline by 12 weeks (Supplementary Fig. 1C-L). These finding suggest that elevated serum and urinary phosphorylated tau, as detected by ELISA, may serve as early markers of HFD-induced metabolic and renal stress.

Progressive renal damage and fibrotic changes induced by HFD in NOX5 pod⁺ mice

HFD-fed mice showed increased urinary ACR at 6 and 12 weeks, indicating renal dysfunction (Fig. 3A). Electron microscopy revealed glomerular basement membrane (GBM) thickening and reduced slit diaphragm length, especially at 6 weeks (Fig. 3B-D). Podocyte marker nephrin and endothelial cell-selective adhesion molecule (ESAM) expression were significantly decreased at 6 and 12 weeks, while α-SMA expression was elevated (Fig. 3E-H). These findings indicate that HFD induces renal structural and fibrotic changes, and the link between these alterations and tau phosphorylation warrants further investigation.

Changes in tau phosphorylation residues in DN in NOX5 pod⁺ mice

In the 6-week HFD group, phosphorylation changes were observed at multiple residues of the tau protein, particularly phosphorylation at Ser202 and Thr205 (Fig. 4A). This trend was further verified in the glomeruli of mice fed HFD for 3, 6, and 12 weeks, suggesting that expression increased over time (Fig. 4B). When examining phosphorylation of the tau protein extracted from the kidney cortex, significant increases were observed at Ser202 and Thr205 after 6- and 12-weeks of HFD (Fig. 4C and D). Conversely, phosphorylation at Ser404 showed a decrease after 6-weeks of HFD (Fig. 4C and E). Additionally, phosphorylation at Ser262 was decreased notably after 12-weeks of HFD (Fig. 4C and F). These results suggest that the increased phosphorylation of Ser202 and Thr205 induced by HFD may significantly contribute to kidney damage.

Inhibition of tau phosphorylation in renal mesangial cells

To evaluate the role of inhibition of tau phosphorylation, renal mesangial cells were treated with tau siRNA or a TAOK inhibitor. Tau siRNA or TAOK inhibitor (Fig. 5A-C) inhibited TGF-β-induced tau phosphorylation and E-cadherin reduction (Fig. 5C and D). The extent of inhibition of tau phosphorylation by dehydrozingerone (DHZ), which had shown improvement in DN [18], was examined. Western blot and immunofluorescence analyses revealed that, DHZ significantly inhibited the phosphorylation of tau at Ser202 and Thr205 residues. Additionally, DHZ significantly reduced the expression of α-SMA and collagen induced by TGF-β in renal mesangial cells (Fig. 5E-I). These results suggest that regulating the phosphorylation of tau at Ser202 and Thr205 residues could be an important strategy for resolving kidney damage.

Bidirectional crosstalk between ER stress and tau phosphorylation in DN pathogenesis

ER stress from diabetes can promote tau phosphorylation, contributing to both AD and DN, while phosphorylated tau further aggravates ER stress [4], forming a vicious cycle [5]. To explore this interaction, changes in ER stress and tau phosphorylation were assessed following inhibition of each pathway. ER stress markers were significantly elevated in HFD-fed mice, especially at 6 weeks, with increases also observed at 3 and 12 weeks (Fig. 6A-G). In mesangial cells, Tg-induced tau phosphorylation was reduced by both TAOK and ER stress inhibitors, which also suppressed ER stress (Fig. 6H-N). These findings suggest a bidirectional relationship between ER stress and tau phosphorylation in DN.

DISCUSSION

This study investigated changes in tau phosphorylation at specific residues in HFD-induced DN model and confirmed that the interaction between tau and ER stress contributes to the pathogenesis of DN. The MAPT gene encodes the microtubule-associated protein tau, which has been extensively studied in the brain [19]. Tau stabilizes microtubules and supports intracellular transport in neuron [20]. Under physiological conditions, tau maintains a basal phosphorylation level; however, in disease states, tau becomes hyperphosphorylated, dissociates from microtubules and forms neurofibrillary tangles, ultimately disrupting cellular function and leading to cell death [21]. Although tau is highly expressed in the brain, it is also present in other organs, including heart, muscles, lungs, and kidneys [22]. However, its role in non-neuronal tissues remains poorly understood. Previous studies have shown that reactive oxidative stress (ROS), mitochondrial dysfunction, and impaired autophagy also contribute to tau hyperphosphorylation. Elevated reactive oxygen species exacerbate glomerular injury and may promote aberrant kinase activation, while defective mitophagy leads to accumulation of damaged mitochondria and increased cellular stress. Impaired autophagy further prevents clearance of misfolded proteins and dysfunctional organelles, compounding cellular injury. Given the intricate crosstalk among these stress pathways and tau modification, it is unlikely that tau phosphorylation functions as an isolated driver of DN. Rather, our data suggest that tau dysregulation operates in concert with broader cellular stress responses to exacerbate renal damage. The kidneys are structurally dependent on microtubules, particularly in specialized cells such as podocytes and mesangial cells, which are essential for filtration and glomerular support [12,23]. In this study, HFD-induced kidney injury led to podocyte damage, increased GBM thickness, and albuminuria. Moreover, mesangial cells, which rely on microtubules for structural integrity, intracellular transport, and proliferation, showed increased tau phosphorylation in response to fibrotic stress. Interestingly, although HFD-fed mice exhibited significant weight gain at 3 and 6 weeks, this difference was no longer apparent at 12 weeks when compared to RD-fed controls. This may be attributed to the metabolic characteristics of the friend virus B NIH Jackson (FVB/N) mouse strain, which has been reported to develop increased adiposity and insulin resistance even under standard diet conditions. As a result, RD-fed FVB mice may gradually gain weight over time, potentially obscuring the differential impact of HFD at later time points [24].

Among the various phosphorylation residues of tau, Ser202 and Thr205 were significantly phosphorylated, particularly at 6 and 12 weeks following HFD. In contrast, Ser262 and Thr404 decreased. This may be explained by findings from AD, which show that tau phosphorylation is regulated in a residue-specific and stage-dependent manner. Ser202 and Thr205 and Thr231 are markedly increased at later disease stages, while Ser262 and Ser396 show only minimal changes [25]. These findings suggest that different phosphorylation sites on tau may contribute differently to disease progression, and such site-specific regulation may also apply in the context of DN.

To enhance the clinical utility of phosphorylated tau as a diagnostic biomarker for DN, future studies should incorporate larger sample sizes and longitudinal assessments across various disease stages. A recent study has explored the use of phosphorylated tau levels in the serum or urine as diagnostic markers for diseases [26]. In this study, we aimed to determine whether phosphorylated tau levels in the serum and urine could be used for the early diagnosis of DN. However, the results were not consistent with those from tissue analysis. This may be attributed to the limited number of animals used and the inability to assess specific tau phosphorylation sites. While our ELISA-based assay indicated a general increase in phosphorylated tau during early disease stages, it did not distinguish between specific phosphorylation residues. In contrast, Western blot analysis clearly showed that phosphorylation at Ser202 and Thr205 was elevated in both kidney tissue and mesangial cells under fibrotic and metabolic stress. These findings suggest that site-specific phosphorylated tau, such as Ser202 and Thr205, may serve as a more precise and reliable early biomarker for DN. Future studies should focus on validating these specific residues in expanded study populations and across various stages of disease progression.

ER stress is a condition in which the ER within a cell experiences stress that leads to a decline in its function. Stress can result from abnormal protein folding, calcium imbalance, and other environmental factors. It has a significant impact on cellular health, and cell death caused by ER stress can accelerate the onset and progression of diseases, such as AD and DN. In AD, tau phosphorylation and ER stress are closely connected, and their exacerbation not only increases the accumulation of β-amyloid plaques, a hallmark of the disease, but also leads to neuronal cell death, thereby contributing to the progression of AD [27,28]. Increasing evidence indicates that diabetic conditions such as metabolic stress, ER stress, and TGF-β signaling can induce tau phosphorylation [15,29,30]. Therefore, in DN, ER stress may be associated with abnormal phosphorylation of the MAPT, tau. This can affect the structural stability of kidney cells and accelerate the impairment of kidney function.

In this study, TGF-β- and Tg-induced fibrosis and ER stress in mesangial cells increased tau phosphorylation at Ser202 and Thr205 (Figs. 5 and 6), which was reduced by the ER stress inhibitor 4PBA and the TAOK inhibitor. Interestingly, tau phosphorylation exhibited a biphasic response to TGF-β, 5 ng/mL increased phosphorylation, while 10 and 20 ng/mL decreased it. This could be due to apoptosis or microtubule disruption, as supported by the increased expression of apoptotic markers (Supplementary Fig. 2). These findings highlight the complex, context dependent regulation of tau in DN. Moreover, ER stress and hyperphosphorylated tau appear to form a vicious cycle, each exacerbating the other and promoting cellular damage, a mechanism also noted in AD [27,31]. Further studies are needed to elucidate the time- and stage-specific dynamics of this interaction in DN.

To further explore the mechanism, we employed the NOX5 pod⁺ transgenic mouse, which is specifically engineered to overexpress NOX5 in podocytes. NOX5 has recently been identified as a significant target in studies on kidney damage [32]. Since NOX5 is not present in rodents, this humanized model enables the investigation of NOX5-related pathogenesis in DN. Increased NOX5 expression was associated with greater tau phosphorylation in these mice, suggesting that NOX5-derived ROS may activate tau-related kinases such as cyclin-dependent kinase 5 (CDK5), mitogen-activated protein kinases (MAPKs), casein kinase 1 (CK1), and calcium/calmodulin-dependent protein kinase II (CaMKII), thereby promoting tau phosphorylation [33-37]. Additionally, our analysis revealed that phosphorylated tau at Ser202 and Thr205 is increased in glomerular cells of patients with DN, with predominant nuclear localization. This accumulation was not observed in control kidney tissues. Importantly, the number of pTau-positive cells showed a positive correlation with proteinuria levels, suggesting that nuclear pTau may be associated with glomerular injury and urinary protein leakage. Together, these findings suggest that site-specific tau phosphorylation, particularly at Ser202 and Thr205, may contribute to the progression of DN by promoting proteinuria-related damage. However, other mechanisms such as oxidative stress, mitochondrial dysfunction, and impaired autophagy are also known contributors to both DN pathology and tau dysregulation. Given the complex interplay between these pathways, tau phosphorylation likely acts in concert with broader cellular stress responses [38-40]. Given the complex interplay between these pathways, tau phosphorylation likely acts in concert with broader cellular stress responses. Furthermore, our findings reveal a dynamic and potentially bidirectional interaction between ER stress and tau phosphorylation, which may amplify kidney injury under diabetic conditions. Targeting the tau–ER stress axis may therefore represent a promising therapeutic strategy for mitigating renal damage in DN. To further explore the chronic impact of tau pathology in DN, we propose a two-step strategy for future investigation. First, we plan to examine the longitudinal progression of kidney damage in tau transgenic mouse models of diabetes (e.g., HFD-induced or streptozotocin-induced). This will help determine whether tau pathology contributes to chronic renal dysfunction in diabetic patients. Second, based on these results, we will evaluate whether long-term pharmacological inhibition of tau in these models can mitigate or reverse kidney injury. This will help to determine the therapeutic potential of targeting tau in DN.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: E.S.L., J.S.K., S.H.J., C.H.C.

Acquisition, analysis, or interpretation of data: E.S.L., S.W.L., J.H.L., E.Y.L., C.H.C.

Drafting the work or revising: E.S.L., S.H.J., E.Y.L., C.H.C.

Final approval of the manuscript: all authors

FUNDING

This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) fun ded by the Ministry of Education (NRF-2019R1I1A1A01042030 and NRF-2022R1I1A1A01068782

ACKNOWLEDGMENTS

We thank Dr. Christopher R. J. Kennedy for providing mice expressing the renal podocyte-specific human NADPH oxidase 5 (NOX5 pod⁺ mice).

Fig. 1.

Tau phosphorylation in kidney tissue and renal mesangial cells. (A) Renal tau phosphorylation levels after 12 weeks of high-fat diet (HFD) in wild-type (WT) and NADPH oxidase 5 (NOX5) pod⁺ mice. (B) High glucose (HG; 30 mM) and H₂O₂ (100 μM) induces pTau^{Ser202, Thr205} expression in renal mesangial cells. (C-F) Time-dependent increase in tau phosphorylation under hyperglycemic conditions confirmed by immunofluorescence and Western blot analysis. Scale bar=50 μm. DAPI, 4ʹ,6-diamidino2-phenylindole; RD, regular diet; CON, control; α-SMA, α-smooth muscle actin. ^aP<0.05, vs. RD or CON; n=3 per group.

Fig. 2.

Increased expression of tau proteins in glomeruli of patients with diabetic nephropathy (DN). (A) Immunohistochemistry (IHC) staining for pTau Ser202 and Thr205 was performed using archived kidney tissue samples. Representative IHC images show increased expression of pTau Ser202 and Thr205 in glomeruli from DN patients compared to controls. Enlarged images correspond to the red dotted boxes in the pTau Ser202 and panels. Red arrows indicate glomerular cells, confirming that pTau Ser202 and Thr205 are localized in the nuclei of glomerular cells in the DN group. Scale bar 100 μm. (B) Quantification graphs were generated from 2 to 6 images per slide, obtained from control (n=5) and DN (n=4) samples. (C, D) To confirm DN diagnosis, clinical data including estimated glomerular filtration rate (eGFR) and proteinuria (urine protein-to-creatinine ratio [uPCR]) levels were reviewed. As expected, DN patients showed significantly decreased eGFR and increased urine protein levels compared to controls. (E) A positive correlation was observed between the number of pTau Ser202 and Thr205 positive cells and proteinuria. Data are presented as mean±standard error. Cr, creatinine. ^aP<0.05, ^bP<0.01, ^cP<0.001 vs. control.

Fig. 3.

Assessment of high-fat diet (HFD)-induced kidney damage in NADPH oxidase 5 (NOX5) pod⁺ mice. (A) Kidney injury following 3, 6, and 12 weeks of HFD feeding was assessed by measuring the urinary albumin and creatinine (uACR). (B, C) Renal ultra-structures of EM samples were analyzed using a transmission electron microscope to evaluate glomerular basement membrane (GBM) thickness and (B, D) slit pore numbers, with images captured at 15,000×magnification. Scale bar 5 μm. (E-H) Protein expression levels of nephrin, endothelial cell-selective adhesion molecule (ESAM), and α-smooth muscle actin (α-SMA) were analyzed by Western blotting. Cr, creatinine; RD, regular diet. ^aP<0.05, vs. RD at the corresponding time point; n=4–6 per group.

Fig. 4.

Site-specific changes in tau phosphorylation in diabetic nephropathy (DN) observed in NADPH oxidase 5 (NOX5) pod⁺ mice. (A) In NOX5 pod⁺ mice fed a high-fat diet (HFD) for 6 weeks, site-specific tau phosphorylation in kidney tissue was assessed by dot blot analysis. (B) Phosphorylation levels at specific tau residues (Ser202, Thr205) were further evaluated by immunohistochemistry and (C-F) Western blotting in kidney tissues from mice fed an HFD for 3, 6, and 12 weeks. Scale bar=20 μm. RD, regular diet. ^aP<0.05, vs. RD at the corresponding time point; n=6 per group.

Fig. 5.

Effects of tau phosphorylation inhibition on renal mesangial cell injury. (A) Expression of pTau Ser202 and Thr205 following tau siRNA transfection. (B-D) Western blot analysis of transforming growth factor β (TGF-β) induced phosphorylation at Ser²⁰², Thr²⁰⁵ and Ser¹⁹⁹, and its inhibition by a thousand and one amino acid kinase (TAOK) inhibitor. (E) Immunofluorescence analysis of E-cadherin and pTau^{Ser202, Thr205} expression in mesangial cells after TGF-β stimulation, and their modulation by tau siRNA and TAOK inhibitor. Scale bar=20 μm. (F-J) The potential of dehydrozingerone (DHZ), a substance with therapeutic effects on diabetic nephropathy, to improve the TGF-β induced tau phosphorylation of Ser202, Thr205, and Ser199 and fibrosisrelated marker such as α-smooth muscle actin (α-SMA) and collagen type I were evaluated using Western blot and immunofluorescence staining in renal mesangial cells. Scale bar=50 μm. Con, control; Veh, vehicle; DAPI, 4ʹ,6-diamidino-2-phenylindole; COL1, collagen type 1. ^aP<0.05 vs. regular diet at the corresponding time point, ^bP<0.05 vs. TGF-β; n=3/group.

Fig. 6.

Endoplasmic reticulum (ER) stress–tau interaction in kidney and mesangial cells. (A-G) Temporal changes in ER stress-related markers were evaluated in kidney tissues of NADPH oxidase 5 (NOX5) pod⁺ mice fed high-fat diet (HFD). (H) To further elucidate the relationship between ER stress and tau phosphorylation, renal mesangial cells were treated with 500 nM thapsigargin (Tg), an ER stress inducer, and changes in tau phosphorylation were assessed. (I-N) Tau phosphorylation was assessed after treatment with 1 μM 4-phenylbutyric acid (4PBA) to evaluate the role of ER stress. The expression of ER stress markers was also examined following treatment with a thousand and one amino acid kinase (TAOK) inhibitor (10 μM). RD, regular diet; PERK, protein kinase R (PKR)-line endoplasmic reticulum kinase; eIF2α, eukaryotic initiation factor 2 alpha; ATF6, activating transcription factor 6; XBP1, X-box binding protein1; CHOP, C/EBP homologous protein; C-caspase3, Cleaved caspase3; CON, control; Veh, vehicle; Bip, binding immunoglobulin protein. ^aP<0.05 vs. RD at the corresponding time point or veh of CON, ^bP<0.05 vs. Veh-treated of 4PBA or TAOK-i; n=2/group.

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Lee ES, Kang JS, Lee SW, Jo SH, Lee JH, Lee EY, Chung CH. Interactions between Tau Phosphorylation and Endoplasmic Reticulum Stress in Diabetic Nephropathy. Diabetes Metab J. 2025 Nov 11. doi: 10.4093/dmj.2024.0618. Epub ahead of print.

Received: Oct 07, 2024; Accepted: Jul 18, 2025

DOI: https://doi.org/10.4093/dmj.2024.0618.

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