POU2F2 activates the Akt/mTOR signalling pathway and enhances B lymphocyte function during diabetic kidney disease by promoting PIK3CD transcription

Abstract

Background

Phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta (PIK3CD) is the predominant isoform of the catalytic subunit of PI3K in lymphocytes. Based on comprehensive bioinformatics, this study explores the functions of PIK3CD in diabetic kidney disease (DKD) progression in mice and B lymphocyte activity.

Methods

Non‐obese diabetic (NOD) mice that spontaneously develop DKD were applied as animal models. Lentiviral vector‐mediated PIK3CD silencing was introduced in mice, followed by histological staining and biomarker assessments to evaluate DKD‐associated symptoms. The activity of the Akt/mTOR signalling pathway was determined by western blot analysis. Following bioinformatics analyses, the interaction between POU class 2 homeobox 2 (POU2F2) and PIK3CD in B lymphocytes was determined by chromatin immunoprecipitation and luciferase reporter assays. Their functions in B cell function were identified by MTT, flow cytometry and IgG deposition assessment.

Results

PIK3CD was found to be highly expressed in the kidney of DKD mice. Knockdown of PIK3CD inactivated the Akt/mTOR signalling, thus ameliorating tissue injury and fibrosis, enhancing the expression of AQP1 and decreasing urinary NGAL contents, UACR, BUN and β‐NAG/creatinine ratio. These effects were negated by the Akt‐specific activator SC79. POU2F2 was found to promote PIK3CD transcription by binding to its promoter, thus activating the Akt/mTOR pathway. Knockdown of POU2F2 suppressed B cell proliferation in vitro and decreased B cell population and IgG deposition in the mouse kidney. However, these trends were reversed upon additional PIK3CD overexpression.

Conclusion

This study demonstrates that POU2F2 mediates PIK3CD‐dependent Akt/mTOR signalling activation, thus enhancing B lymphocyte function and promoting DKD progression.

Summary at a Glance

This study demonstrates that POU2F2 mediates PIK3CD‐dependent Akt/mTOR signalling activation, thus enhancing B lymphocyte function and promoting DKD progression.

1. INTRODUCTION

The kidney stands out as a primary site of microvascular impairment in diabetes. ¹ Diabetic kidney disease (DKD) emerges as a prevalent microvascular complication affecting around 40% of individuals with diabetes. ² This condition significantly contributes to chronic kidney disease and end‐stage renal disease (ESRD), exerting a substantial toll on both mortality and morbidity in diabetic patients, with the cardiovascular risk associated with DKD exacerbating these outcomes. ² , ³ Clinically, DKD presents with elevated blood sugar levels, hypertension, albuminuria, declining glomerular filtration rate, mesangial lesions, glomerular damage, tubulointerstitial fibrosis and podocyte dysfunction. ⁴ , ⁵ Regrettably, therapeutic interventions for DKD remain limited, with progression to ESRD often deemed inevitable. ⁶

Uncovering the fundamental molecular mechanisms underpinning DKD pathogenesis holds immense promise for enhancing disease management. In the initial phase of our investigation, our research team conducted comprehensive transcriptome analyses using publicly available datasets, revealing phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta (PIK3CD) as a compelling gene displaying altered expression patterns in diabetic kidney tissue. PIK3CD, alternatively known as PI3Kdelta or p110delta, represents a prominent isoform of the catalytic subunits within Class I phosphatidylinositol 3‐kinases (PI3Ks). ⁷ This particular isoform predominates in leukocytes, contributing up to 60% of PI3K activity in B lymphocytes. ⁷ , ⁸ Given the pivotal roles of these cells in diabetes pathogenesis, attributed to their functions in antibody production, antigen presentation and immunomodulation, ⁹ we posit that dysregulated PIK3CD expression potentially correlates with B cell activity and the progression of DKD. Notably, the PI3K/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) signalling pathway has been implicated in crucial processes such as B cell activation, proliferation and survival. ¹⁰ , ¹¹ This underscores the rationale behind our hypothesis regarding the association between aberrant PIK3CD expression and DKD advancement.

Further bioinformatics analyses pinpointed POU class 2 homeobox 2 (POU2F2) as a promising transcription factor targeting PIK3CD. Intriguingly, POU2F2, also recognized as octamer‐binding protein 2, operates as a transcription factor playing pivotal roles in B cell proliferation and differentiation. ¹² Notably, a prior investigation by Yang et al. elucidated POU2F2's capacity to activate the PI3K/Akt signalling pathway in a manner reliant on 3‐phosphoinositide dependent protein kinase 1. ¹³ The prospect of POU2F2 exerting its influence on this signalling cascade through modulation of PIK3CD, thereby impacting B cell activity and the progression of DKD, presents an intriguing avenue for further exploration.

2. MATERIALS AND METHODS

2.1. Animals and treatment

The utilization of animals was sanctioned by the Animal Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University, in strict adherence to the Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD, USA). Fifty‐week‐old female non‐obese diabetic (NOD) Caj.Cg‐Ighm^tm1Cgn Tg (Igh‐VB1‐8/Igh‐6 m) 1Mjsk/FswJ mice (NOD.mIgM; Strain #: 005306), genetically predisposed to spontaneous autoimmune diabetes, were sourced from The Jackson Laboratory (Bar Harbour, Maine, USA). NODCaj.Cg‐Ighm^tm1Cgn Tg (Igh‐VB1‐8/Igh‐6) 2Mjsk/FswJ (NOD.s + mIgM; Strain #: 005309, The Jackson Laboratory), with a lesser predisposition to diabetes, were also obtained from The Jackson Laboratory for control purposes.

All mice were subjected to a regulated 12‐h light/dark cycle at 25°C and provided ad libitum access to water and food throughout the study. The blood glucose levels in mice were detected weekly using a blood glucose meter (FreeStyle Optium Neo H, Abbott Diabetes Care, Maidenhead, UK) Diabetes onset in NOD mice was established after two consecutive blood glucose readings exceeding 13.9 mmol/L (250 mg/dL). Mice diagnosed with diabetes were treated with sub‐therapeutic doses of insulin to maintain blood glucose levels between 13.9 and 22.2 mmol/L, ensuring their overall health.

After the diagnosis of diabetics, mice were immediately subjected to tail vein injection (10⁸ TU/ml, 100 μL/mice) of lentiviruses designed to knock down PIK3CD (TL515984V) and POU2F2 (TL509842V) or overexpress PIK3CD (MR211516L3V). These lentiviral constructs were provided by OriGene Technologies, Inc. (Rockville, MD, USA). After 4 weeks, mice were humanely euthanized via intraperitoneal injection of an excessive nembutal solution (150 mg/kg), and their kidneys were harvested for subsequent analysis. In addition, the Akt activator SC79 (T2274, Topscience Co. Ltd., Shanghai, China) was administered daily via intraperitoneal injection (40 mg/kg, dissolved in 10% DMSO [dimethyl sulphoxide]) following lentivirus treatment, with the control group receiving an equivalent volume of DMSO.

These procedures resulted in the following experimental groups: Ctrl, DKD, DKD + NC‐KD, DKD + PIK3CD‐KD, NC‐KD + DMSO, PIK3CD‐KD + DMSO, PIK3CD‐KD + SC79, NC‐KD + NC‐OE, POU2F2‐KD + NC‐OE and POU2F2‐KD + PIK3CD‐OE, each consisting of five mice.

2.2. Histological staining

The kidney tissue samples underwent fixation in 4% paraformaldehyde, followed by embedding in paraffin and subsequent sectioning. Tissue sections were stained using a picrosirius red staining kit (24 901, Polysciences Inc., Warrington, PA, USA) and haematoxylin and eosin (HE) staining kit (C0105S, Beyotime Biotechnology Co., Ltd., Shanghai, China), adhering strictly to the manufacturers' instructions. For picrosirius red staining, the prepared tissue sections were dewaxed and treated with phosphomolybdic acid for 2 min, followed by staining using the picrosirius red working solution for 60 min. After washing with deionised water, the sections were incubated with 0.1 N hydrochloride acid for 2 min and soaked in 70% ethanol for 45 s. The interstitial fibrosis degree was evaluated under polarized light. Histopathological alterations in the renal tubules, encompassing features such as tubular atrophy, dilation, epithelial cell detachment or vacuolar degeneration and the presence of tubular casts, were evaluated in ten random areas from HE‐stained sections to gauge tubular injury. The severity of the tubular injury was semi‐quantitatively graded on a scale ranging from 0 to 4: 0, normal; 1, <25%; 2, 25% ∼ 50%; 3, 50% ∼ 75%; 4, >75%. ¹⁴

For immunohistochemistry (IHC), sections were incubated with 5% goat serum at room temperature for 1 h to block nonspecific binding. They were then incubated overnight at 4°C with antibodies against PIK3CD (ab200372, 1:20, Abcam Inc., Cambridge, MA, USA), WT‐1 (NBP1‐31314, 1:500, Novus Biological Inc., Littleton, CO, USA), and POU2F2 (MA5‐42519, 1:50, Thermo Fisher Scientific, Rockford, IL, USA). The next day, the sections were incubated at 37°C for 2 h with goat anti‐rabbit immunoglobulin G (IgG) H&L (HRP) (ab6721, 1:1000, Abcam). Subsequently, the staining was developed using the DAB Kit (P0202, Beyotime) and observed under the microscope. The integrated optical density (IOD) was evaluated.

2.3. Cell culture and treatment

The B lymphocytes HCC1937 BL (CRL‐2337, American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium. When they reached 80%–90% confluence, they were detached and re‐suspended. Subsequently, 2 × 10⁵ cells were seeded into each well of a 24‐well plate. The cells were co‐incubated with lentiviral particles for 18 h, followed by further incubation in a medium without lentiviral particles for 72 h. Puromycin (1 μg/mL) was used to select clones with stable POU2F2 knockdown (POU2F2‐KD) or POU2F2‐KD along with PIK3CD overexpression (POU2F2‐KD + PIK3CD‐OE).

Additional materials and methods are presented in the S1 file.

2.4. Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 software (GraphPad Software Inc., CA, USA). Data are presented as the mean ± standard deviation. Differences between any two groups were compared using the unpaired t‐test. For comparisons among multiple groups, one‐way or two‐way analysis of variance (anova) was conducted, followed by Tukey's post hoc test. A p <.05 was considered statistically significant.

3. RESULTS

3.1. Bioinformatics analyses suggest the participation of PIK3CD in DKD progression

To identify possible drug targets for DKD treatment, we performed differential gene expression analysis using the GEO datasets to analyse genes involved in DKD‐related processes. Dataset GSE30529 was utilized to analyse transcriptomic differences between tubules of DKD kidney and control kidney; dataset GSE30528 was used to analyse transcriptomic differences between glomeruli of DKD kidney and glomeruli of control kidney; and dataset GSE142025 was employed to analyse transcriptomic differences between advanced stage DKD kidneys (Advanced_DN) and early stage DKD kidneys (Early_DN) (Figure 1A). Genes with an adjusted p <.05 from the selected datasets were intersected with DKD‐related genes downloaded from GeneCards using Jvenn, resulting in 454 intersections (Figure 1B). Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on these intersecting genes, revealing their significant involvement in immune‐related pathways. GO enrichment analysis indicated enrichment in biological processes such as leukocyte‐mediated immunity, positive regulation of cell activation and positive regulation of cell adhesion. Molecular function analysis showed that these intersecting genes mainly exerted immune receptor activity and extracellular matrix structural constituent functions. Cellular component analysis revealed their localization primarily to the external side of the plasma membrane and collagen‐containing extracellular matrix (Figure 1C). KEGG enrichment analysis indicated that these intersecting genes were predominantly involved in immune‐related pathways like the PI3K‐Akt signalling pathway and Phagosome (Figure 1D). Furthermore, an intersection analysis between proteins associated with immune‐related pathways in GO and KEGG enrichment analyses revealed five genes: SYK, PIK3CD, IL7R, KDR and ITGA4 (Figure 1E). PIK3CD and ITGA4 are the two genes whose roles in DKD have not been investigated. Among them, the relevance score of PIK3CD (23.1670455932617) with DKD, predicted by the GeneCards system (https://www.genecards.org/), was significantly higher than that of ITGA4 (17.0534858703613). Additionally, the logFC of PIK3CD in DKD was 0.47577613 (Tubuli of DKD kidney vs. Tubuli of control kidney), 0.6047169 (Glomerulus of DKD kidney vs. Glomerulus of control kidney) and 0.83202042 (Advanced_DN vs. Early_DN), indicating a gradual increase in PIK3CD expression with DKD progression. Therefore, PIK3CD was selected as the subject for further study. Notably, the expression of PIK3CD in the mouse model was investigated. The IHC assay revealed a significant increase in PIK3CD expression in the kidneys of DKD mice (Figure 1F). As anticipated, the DKD mice presented significantly higher glucose levels (Figure 1G) and lower body weight (Figure 1H) compared to control mice before they were euthanized.

FIGURE 1.

PIK3CD is a promising factor correlated with DKD progression. (A), DEGs between DKD kidney and control kidneys analysed using GSE30529, GSE30528 and GSE142025 datasets; (B), intersection analysis of DEGs between the three datasets; (C, D), Gene Ontology (GO) (C) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (D) enrichment analysis of the intersecting genes; (E), further intersection analysis of genes enriched in immune‐related functions and pathways; (F), PIK3CD expression in the kidney of mice with or without DKD determined by IHC; (G, H), blood glucose levels (H) and body weight (G) of mice in each group before animal sacrifice. Each group contained five mice. The difference was analysed by the unpaired t‐test (F, G, H). **p < .01, ****p < .0001. DKD, diabetic kidney disease; IHC, immunohistochemistry; PIK3CD, phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta.

3.2. PIK3CD knockdown ameliorates DKD‐like symptoms in mice

To explore the exact function of PIK3CD in DKD progression, DKD mice with stable PIK3CD were established by lentiviral vector injection. WB analysis revealed a significant increase in PIK3CD protein in the kidney of DKD mice, and this upregulation successfully blocked the PIK3CD‐KD administration (Figure 2A). Notably, the PIK3CD‐KD treatment significantly upregulated the blood glucose levels in mice (Figure 2B) while improving body weight (Figure 2C) of DKD mice before animal sacrifice. Additionally, insulin sensitivity, found to be reduced in DKD mice, was further weakened upon PIK3CD knockdown (Figure 2D). Additionally, HE staining (Figure 2E) and picrosirius red staining (Figure 2F) revealed significant tubular atrophy and interstitial fibrosis in the kidneys of DKD mice. However, these symptoms were ameliorated upon PIK3CD knockdown in mice. Immunofluorescence staining demonstrated a decrease in the staining of AQP1, a water channel protein, in the kidney sections of DKD mice, which was restored following PIK3CD knockdown (Figure 2G). ELISA results showed that the PIK3CD‐KD decreased the urinary NGAL level which was significantly elevated in DKD mice (Figure 2H). Additionally, DKD mice presented an increased β‐NAG/urinary creatinine ratio, which was decreased by PIK3CD knockdown as well (Figure 2I). Additionally, the WT‐1 transcription factor, a reliable marker for podocytes, was found to be reduced in the kidney of DKD mice but restored following PIK3CD knockdown (Figure 2J). This indicates that PIK3CD reduction can also protect podocytes from injury in DKD.

FIGURE 2.

PIK3CD knockdown mitigates kidney injury in DKD mice. DKD mice were administered PIK3CD‐KD for artificial PIK3CD silencing. (A), PIK3CD protein level in the mouse kidney examined by WB analysis; (B, C), blood glucose levels (B) and body weight (C) of mice in each group before animal sacrifice; (D), insulin sensitivity of mice in each group; (E), pathological injury in mouse kidney examined by haematoxylin and eosin staining; (F), interstitial fibrosis in the mouse kidney examined by picrosirius red staining; (G), AQP1 expression in the kidney tissue sections determined by immunofluorescence staining; (H), urinary NGAL level in mice examined using ELISA; (I), β‐NAG/creatinine ratio of mice examined using colorimetric assay kits; (J), expression of the podocyte marker WT‐1 in the mouse kidney examined by IHC. Each group contained five mice. Differences were analysed by the unpaired t‐test (B, C) or one‐way anova. *p < .05, **p < .01, ***p < .001, ****p < .0001; ns denotes to no significance. DKD, diabetic kidney disease; IHC, immunohistochemistry; PIK3CD, phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta.

This evidence demonstrates that PIK3CD knockdown has an ameliorating role in DKD symptoms in mice.

3.3. SC79, an Akt activator, enhances kidney injury in DKD mice in the presence of PIK3CD knockdown

The PIK3CD gene encodes PI3Kdelta (p110 delta), a catalytic subunit isoform of PI3K participating in signalling transduction of the PI3K/Akt/mTOR pathway predominantly in B lymphocytes. ⁷ Not surprisingly, we found that the phosphorylation of Akt and mTOR in the mouse kidney was significantly blocked by the PIK3CD knockdown, which was, however, substantially restored by the Akt‐specific activator SC79 (Figure 3A). Additionally, the SC79 treatment significantly enhanced UACR, BUN level and β‐NAG/creatinine ratio in the DKD mice (Figure 3B). The tubular injury and interstitial fibrosis in the mouse kidney were aggravated by SC79 treatment as well (Figure 3C,D), along with a reduction in the immunofluorescence staining intensity of AQP1 (Figure 3E). The urinary NGAL level in mice was enhanced by SC79 as well (Figure 3F). Moreover, the SC79 treatment reduced the positive staining of WT‐1 in the mouse kidney which was increased upon PIK3CD knockdown (Figure 3G). However, in the presence of SC79 treatment, the blood glucose levels in mice were reduced (Figure 3H), yet the body weight of mice was decreased as well (Figure 3I).

FIGURE 3.

The Akt activator SC79 enhances kidney injury in DKD mice alleviated by PIK3CD knockdown. DKD mice administered PIK3CD‐KD were additionally treated with SC79 or DMSO. (A), phosphorylation of Akt and mTOR in the mouse kidney determined by WB analysis; (B), UACR, BUN level and β‐NAG/creatinine ratio in mice examined using colorimetric assay kits; (C), pathological injury in mouse kidney examined by haematoxylin and eosin staining; (D), interstitial fibrosis in the mouse kidney examined by picrosirius red staining; (E), AQP1 expression in the kidney tissue sections determined by immunofluorescence staining; (F), urinary NGAL level in mice examined using ELISA; (G), expression of the podocyte marker WT‐1 in the mouse kidney examined by IHC; (H, I), blood glucose levels (H) and body weight (I) of mice in each group before animal sacrifice. Each group contained five mice. Differences were analysed by the one‐way (B–I) or two‐way (A) anova. *p < .05, **p < .01, ***p < .001, ****p < .0001; ns denotes to no significance. DKD, diabetic kidney disease; IHC, immunohistochemistry; mTOR, mechanistic target of rapamycin; PIK3CD, phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta.

3.4. POU2F2 is a positive regulator of PIK3CD

To explore the possible cause for aberrant PIK3CD upregulation in DKD, we predicted transcription factors targeting PIK3CD using the hTFtarget system and had the candidates cross‐referenced with the DEGs identified from the GSE3012, GSE30528 and GSE142025 datasets (adjusted p <.05), resulting in 11 intersecting genes (Figure 4A). PML, KMT2A and POU2F2 were three of the factors whose functions in DKD remain uninvestigated. Data from the GEPIA system suggest that all PML, KMT2A and POU2F2 show a significant positive correlation with PIK3CD (Figure 4B). However, existing evidence demonstrates that PML ¹⁵ and KMT2A ¹⁶ exert suppressive effects of Akt activity, while POU2F2 has been demonstrated as a positive regulator of the PI3K/Akt/mTOR signalling in B cell lymphoma cells through PDPK1. ¹³ We therefore selected POU2F2 for further investigation.

FIGURE 4.

POU2F2 is a positive regulator of PIK3CD. (A), intersections of transcription factors targeting PIK3CD predicted using the hTFtarget system and DEGs identified using GSE30529, GSE30528 and GSE142025 datasets; (B), correlations between PML, KMT2A and POU2F2 and PIK3CD predicted using the GEPIA system; (C), direct binding relationship between POU2F2 and the PIK3CD promoter in B lymphocytes examined by the ChIP‐qPCR assay. B lymphocytes were administered NC‐KD + NC‐OE, POU2F2‐KD + NC‐OE, or POU2F2‐KD + PIK3CD‐OE. (D), protein levels of POU2F2, PIK3CD, p‐Akt and p‐mTOR determined by WB analysis; (E), the regulatory role of POU2F2 on PIK3CD promoter examined by dual luciferase reporter gene assay. For cellular experiments, three biological replicates were performed. Differences were compared by the unpaired t‐test (C and E) or two‐way anova (D). **p < .01, ***p < .001, ****p < .0001; ns denotes to no significance. mTOR, mechanistic target of rapamycin; PIK3CD, phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta; POU2F2, POU class 2 homeobox 2.

Importantly, we identified a high enrichment of anti‐POU2F2 near the PIK3CD promoter region in B cells using the ChIP‐qPCR assay (Figure 4C). Subsequently, we induced POU2F2‐KD in the B cells. This led to a significant decrease in the protein levels of POU2F2 and PIK3CD as well as the phosphorylation of Akt and mTOR (Figure 4D). As anticipated, additional artificial overexpression of PIK3CD in these cells restored the activity of the Akt/mTOR signalling pathway (Figure 4D). Moreover, the dual luciferase reporter gene assay revealed that the transcription activity of the PIK3CD in B cells was substantially decreased following POU2F2‐KD (Figure 4E).

3.5. POU2F2 and PIK3CD affect B cell function

The functions of POU2F2 and PIK3CD in the proliferation and apoptosis of B lymphocytes in vitro were first investigated. MTT assay (Figure 5A) and flow cytometry (Figure 5B) revealed that the POU2F2‐KD in B cells significantly reduced cell proliferation while promoting cell apoptosis. However, these trends were reversed by the additional PIK3CD overexpression in cells. Additionally, we induced POU2F2‐KD and PIK3CD overexpression in DKD mice. Flow cytometric analysis showed that the population of plasmablasts (CD138⁺CD44⁺), a B cell subset, in the kidney of mice was significantly decreased by POU2F2 downregulation but restored following PIK3CD overexpression (Figure 5C). In addition, by performing immunofluorescence staining using fluorescence‐conjugated anti‐mouse IgG, we identified IgG deposition in the mouse kidney. Importantly, this deposition was decreased by POU2F2 downregulation but restored following PIK3CD overexpression (Figure 5D). Interestingly, the POU2F2 silencing was found to reduce the populations of CD3⁺ T cells as well as pro‐inflammatory CD68⁺ F4/80⁺ CD11c⁺ macrophages in the single‐cell suspensions of mouse kidney tissues. However, these cell populations were found to be restored following the additional PIK3CD overexpression (Figure 5E,F). Moreover, the POU2F2‐KD administration significantly decreased the IHC of POU2F2 and PIK3CD (Figure 5G) and increased immunofluorescence staining of AQP1 (Figure 5H) in the kidney tissues, decreased the urinary NGAL level (Figure 5I), decreased UACR, BUN level and β‐NAG/creatinine ratio (Figure 5J), mitigated tubular injury and interstitial fibrosis in the kidney (Figure 5K,L) and enhanced positive staining of WT‐1 in the kidney tissues (Figure 5M). These changes were reversed upon the additional PIK3CD overexpression (Figure 5G–M). Furthermore, the POU2F2‐KD was found to increase the blood glucose levels in mice (Figure 5N) while improving the body weight (Figure 5O), which was negated by the PIK3CD upregulation as well (Figure 5N,O).

FIGURE 5.

POU2F2 and PIK3CD affect B cell proliferation. B lymphocytes were administered NC‐KD + NC‐OE, POU2F2‐KD + NC‐OE or POU2F2‐KD + PIK3CD‐OE. (A), proliferation of B lymphocytes examined by the MTT assay; (B), apoptosis of B lymphocytes examined by flow cytometry. DKD mice were administered POU2F2‐KD or PIK3CD‐OE. (C), population of plasmablasts (CD138⁺CD44⁺) in the mouse kidney examined by flow cytometry; (D), IgG deposition in the mouse kidney determined by immunofluorescence staining; (E, F), populations of CD3⁺ T cells (C) and CD68⁺ F4/80⁺ CD11c⁺ macrophages (F) in the single‐cell suspensions of mouse kidney tissues determined using flow cytometry; (G), expression of POU2F2 and PIK3CD in the mouse kidney examined by IHC; (H), AQP1 expression in the kidney tissue sections determined by immunofluorescence staining; (I), urinary NGAL level in mice examined using ELISA; (J), UACR, BUN level and β‐NAG/creatinine ratio in mice examined using colorimetric assay kits; (K), pathological injury in mouse kidney examined by haematoxylin and eosin staining; (L), interstitial fibrosis in mouse kidney examined by picrosirius red staining; (M), WT‐1 expression in the mouse kidney examined by IHC; (N, O), blood glucose levels (N) and body weight (O) of mice in each group before animal sacrifice. For cellular experiments, three biological replicates were performed. For animal experiments, each group contained five mice. Differences were analysed by the one‐way (A–F and H–O) or two‐way (G) anova. *p < .05, **p < .01, ***p < .001, ****p < .0001; ns denotes to no significance. DKD, diabetic kidney disease; IHC, immunohistochemistry; PIK3CD, phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta; POU2F2, POU class 2 homeobox 2.

4. DISCUSSION

The surge in DKD prevalence mirrors the notable increase in global diabetes rates, ¹⁷ imposing significant financial burdens and profound impacts on daily living. Moreover, this scenario underscores the pressing need for researchers to unveil additional molecular drivers implicated in DKD advancement. Within this study, the authors illustrate how POU2F2 promotes the transcription of PIK3CD, consequently activating the subsequent PI3K/Akt/mTOR signalling pathway in B lymphocytes. This cascade, in turn, fosters kidney damage and contributes to the progression of DKD.

Following the bioinformatics predictions indicating aberrant expression of PIK3CD in diabetic kidneys and its association with disease progression, we validated this heightened expression pattern in the kidneys of diabetic NOD mice. Through activation of the PI3K/Akt signalling cascade, PIK3CD has been implicated in orchestrating epithelial‐to‐mesenchymal transition in renal tubular epithelial cells and promoting kidney fibrosis. ¹⁸ Activation of this signalling has also been correlated with the onset of glomerular lesions and the development of DKD. ¹⁹ , ²⁰ , ²¹ Importantly, our findings revealed that artificial knockdown of PIK3CD significantly improved the body weight of DKD mice, ameliorated tubular injury, kidney fibrosis, improved markers of kidney function (AQP1, urinary NGAL, UACR, BUN and β‐NAG/creatinine) and increased the expression of podocyte marker WT‐1. It is widely recognized that activated PI3Ks interact with PIP2, generating PIP3 at the plasma membrane, which in turn recruits proteins containing pleckstrin homology (PH) domains, such as Akt, to the cell membrane, leading to subsequent activation of various downstream targets, including mTOR. ²² Notably, our subsequent observations revealed that SC79, a small molecule that specifically binds to the PH domain of Akt and paradoxically activates Akt in the cytosol, ²³ counteracted the mitigating effects of PIK3CD on DKD in mice. This underscores the pivotal role of PIK3CD in DKD progression through the PI3K/Akt signalling pathway. However, we found that PIK3CD knockdown increased insulin resistance and blood glucose levels in mice. Indeed, the PI3K/Akt cascade is critically involved in glucose uptake and metabolism in various tissues through several mechanisms, such as promotes the translocation of glucose transporter 4 to the cell membrane, facilitating glucose uptake into cells. ²⁴ , ²⁵ Targeting PIK3CD has also been correlated with increased insulin resistance in hepatocytes. ²⁶ This evidence suggests that PIK3CD knockdown or PI3K/Akt signalling inhibition might protect DKD injury independent of regulating insulin resistance or glucose metabolism.

Subsequent bioinformatics analyses focusing on upstream regulators of PIK3CD highlighted POU2F2 as an intriguing target. POU2F2, predominantly expressed in B lymphocytes, ²⁷ shares this characteristic with PIK3CD. ⁸ While the precise role of B cells in DKD pathogenesis remains unclear, existing evidence suggests a close interplay between the two. Plasmablasts have been identified as antigen‐presenting cells that facilitate the activation and proinflammatory response of CD4⁺ T cells, thereby contributing to T cell‐mediated beta cell destruction and insulin resistance. ²⁸ B cells have been demonstrated to be required for the development of diabetes in NOD mice. ²⁹ Additionally, the CD38⁺CD19⁺ B cell population has been reported to exhibit a positive correlation with urinary protein levels and a negative correlation with estimated glomerular filtration rate in DKD patients. ³⁰ Meanwhile, the CD38⁺CD19⁺ plasma cells have shown a positive association with serum IgG levels in these patients. ³⁰ This might be attributed to increased activation and differentiation of B cells upon antigen stimulation. ³¹ Collaborating with CD4⁺ helper T cells, activated B cells secrete inflammatory cytokines and form immune complexes with autoantibodies secreted by plasma cells. ³² These immune complexes can infiltrate the glomeruli, leading to mesangial expansion, thickening of the glomerular basement membrane, macrophage accumulation and inflammation overload. ³² Building on these insights, we explored the roles of POU2F2 and PIK3CD in B cell activity. Our results demonstrated that PIK3CD overexpression restored B cell proliferation and survival, and reversed the suppression of plasmablast populations and IgG deposition in mouse kidneys induced by POU2F2‐KD. In partial agreement with our findings, gain‐of‐function mutations in PIK3CD have been shown to over‐activate the PI3K/Akt/mTOR signalling pathway, influencing the biology of B and C lymphocytes and natural killer cells. ³³ Inhibition of PIK3CD has been found to reduce the chemotaxis of chronic lymphocytic leukaemia cells to stroma. ⁸ It is noteworthy that the POU2F2‐KD and PI3KCD overexpression in mice were achieved through tail vein injection of lentiviral vectors. In this scenario, the entry of lentiviral vectors to the kidney parenchyma is blocked by the intrinsic stringent filtering functions of the kidney, and the vectors are too large to pass slit diaphragms generated by podocytes within the glomerulus. ³⁴ Therefore, the detected POU2F2 and PIK3CD alteration in mouse kidney tissues following the tail vein injection might be due to infiltrating circulating lymphocytes, including B lymphocytes. The changes in the B cell population might be a direct effect of POU2F2 and PIK3CD manipulation rather than a secondary change due to alterations in the kidney. These findings underscore the significant role of the POU2F2/PIK3CD axis in modulating B cell activity during DKD progression. Additionally, we also observed that the populations of infiltrating CD3⁺ T cells and CD68⁺ F4/80⁺ CD11c⁺ macrophages in the mouse kidney were decreased following POU2F2‐KD but restored after PIK3CD overexpression. However, whether these changes in T cell and macrophage populations are directly influenced by POU2F2 or PIK3CD intervention, or indirectly affected by B cell‐mediated inflammatory responses or immune complexes, ³² or both, remain to be further investigated.

5. CONCLUSION

In summary, our study unveils a novel POU2F2/PIK3CD axis that drives the overactivation of the PI3K/Akt/mTOR signalling pathway in B lymphocytes, thereby fostering the deposition of immune complexes, tubular injury, kidney fibrosis and dysfunction (Figure 6). These findings underscore the potential of POU2F2 and PIK3CD as promising targets for mitigating B cell overactivation and curbing the severity and progression of DKD. However, many limitations should be acknowledged. First, the effects of POU2F2 and PIK3CD in other types of immune cells are not extensively investigated. In addition, the lack of clinical samples for the assessment of circulating or kidney POU2F2 and PIK3CD levels may weaken the translational value of this study. Furthermore, given the fact that the POU2F2/PIK3CD cascade favours glucose metabolism and reduces blood glucose levels, targeting these molecules alone may aggravate hyperglycemia in diabetic patients. Therefore, a combination of other glucose‐lowing regimens may be required. We would like to focus on these issues in future research endeavours.

FIGURE 6.

In the context of DKD, the POU2F2/PIK3CD axis drives the overactivation of the PI3K/Akt/mTOR signalling pathway in B lymphocytes, thereby promoting the deposition of immune complexes, tubular injury and kidney fibrosis and dysfunction. DKD, diabetic kidney disease; mTOR, mechanistic target of rapamycin; PI3K, phosphatidylinositol 3‐kinases; PIK3CD, phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta; POU2F2, POU class 2 homeobox 2.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Data S1. Supporting Information.