Nephrotic syndrome (NS) is a common clinical glomerular disease, and massive proteinuria is its main clinical indication. Central to NS is immune system dysfunction, particularly the dysregulation of podocytes and inflammation factors that damage the glomerular filtration barrier (GFB), mainly composed of three layers, namely, glomerular endothelial cells, basement membrane, and podocytes, respectively, from inside to outside (Vivarelli et al., 2023). Release of proteinuria or NS are symptoms of podocytopathies, glomerular disease caused by direct or indirect podocyte injury, which link to immune system (Myette et al., 2025). Nephrin, the major podocyte antigen, contributes to renal injury through the production of autoantibodies, and prevents macromolecular proteins carrying the same charge properties from leaking out of the filtration barrier (Al-Aubodah et al., 2025; Duan et al., 2025; Qu and Jiao, 2023; Colucci et al., 2022; Meng et al., 2025).

As the last barrier to proteinuria, podocytes are the key target cells in the whole process of occurrence and development of NS (Vivarelli et al., 2023; Meliambro et al., 2024). Structural podocyte abnormalities, such as abnormalities in actin cytoskeleton or slit diagram (SD), can cause foot processes (FPs) fusion, and affect the integrity of the GFB, indicating that podocyte injury and proteinuria, which account for up to 90% of cases of worsening kidney function worldwide (Meliambro et al., 2024; Loreth et al., 2025). Nevertheless, podocyte foot process morphology has diagnostic value in differentiating diabetic nephropathy (DN) and minimal change disease (MCD) (Li et al., 2025).

Moreover, podocytes are postmitotic cells and have a very limited capacity for self-renewal (Loreth et al., 2025; Haydak and Azeloglu, 2024). Podocyte loss, whether due to detachment or cell death, results in irreversible damage and scarring of the renal filtration units (Meliambro et al., 2024; Haydak and Azeloglu, 2024). Podocyte injury has been reported to be associated with intracellular calcium (Ca²⁺) overload (Ilatovskaya et al., 2025). Transient receptor potential ion channel 6 (TRPC6) has been recognized as a novel SD protein involved in maintaining the structural stability of the podocyte skeleton and regulating Ca²⁺ homeostasis (Lu et al., 2019). It has been confirmed by TRPC6-specific inhibitor through attenuating the degradation of podocyte structural proteins, inhibiting fluorescence intensity of intracellular Ca²⁺, and podocyte apoptosis, resulted in podocyte injury and recovery in vitro (Feng et al., 2022). It was also shown that TRPC6 gene variation in glomerular human glomerular diseases, including MCD, FSGS, and immune complex associated glomerulonephritis (Sun et al., 2021), and TRPC6 overexpression in podocytes correlate with decreased calpastatin expression, autophagy blockade, and podocyte injury in DN (Salemkour et al., 2023). Therefore, TRPC6-directed therapy is therefore currently being targeted for treatment for podocytopathies.

Corticosteroids are the cornerstone of the treatment of NS. However, 5%–15% children who do not respond to a cycle of oral steroids, and 55%–60% have frequent relapses and require repeated or ongoing use of glucocorticoids, therefore, most of them require steroid-sparing immunosuppressive agents, including calcineurin inhibitors, rituximab (RTX) (Vivarelli et al., 2023). Aside from depleting CD20 B cells, RTX binds to podocyte SMPDL3b and has non-immunological effect on podocytes by reducing podocyte injury and apoptosis, increasing cell adhesion, and stabilizing actin cytoskeleton, contributing to its effectiveness in reducing proteinuria (Jeruschke et al., 2022; Aslam and Koirala, 2023). Furthermore, the depletion of antigen-presenting B cells by RTX may target B-cell survival signaling through the BAFF/APRIL pathway, restore the balance between autoreactive T cells and regulatory T cells, and suppress interleukin (IL)-13 secretion by Th2 cells in autoimmune diseases (Lin et al., 2024).

Clinical studies have demonstrated that combining RTX treatment with methylprednisolone (MP) pulse therapy might be more effective in reducing proteinuria and relapse rates in patients suffering from NS than RTX alone, however, the precise pharmacological mechanism of RTX and MP are not well understood yet (Chan and Tullus, 2021; Chan et al., 2023; Yokota et al., 2024; Nozu et al., 2024). In vivo and vitro, TRPC6-targeted dexamethasone (Dex) nanobubles could alleviate podocyte apoptosis and inflammation, suggesting that TRPC6 might be an ideal guiding target for glucocorticoids-based renal therapy (Wu et al., 2025). Puromycin aminonucleoside (PAN) is used mainly in vitro and vivo, not in clinic, and there is no standard for the safe doses at human level for its potential toxicity. Furthermore, PAN treatment could significantly disrupt the cytoskeletal architecture of cultured mouse podocytes, and reduce the formation of focal adhesions and stress fibers. Interdigiting intercellular junctions were replaced by dot-like structures with accumulated filamentous actin (Huang et al., 2025). In this study, we utilized a podocyte injury model with PAN treatment to explore how MP and RTX impact TRPC6’s expression and localization, and assess MP’s and RTX’s effects on podocyte injury and recovery.

Podocytes, are specialized cells within the GFB, which are crucial for maintaining glomerular structural integrity and convective ultrafiltration (Loreth et al., 2025). Podocyte dysfunction, resulting from oxidative stress, dysregulated prosurvival signaling, or structural damage, can drive the development of proteinuria and glomerulosclerosis (Loreth et al., 2025; Haydak and Azeloglu, 2024). Functionally, podocyte injury leads to actin cytoskeleton rearrangement and the merging and disappearance of FPs, and dysregulates SD protein expression, which could induce podocyte depletion and impair ultrafiltration (Meliambro et al., 2024). The mechanism of PAN associated podocyte injuries was that it could trigger the effacement of podocyte FPs, resulting in cytoskeletal disruption and atypical expression and allocation of podocyte molecules (Ding et al., 2021; Qiu et al., 2021; Huang et al., 2025). The current research constructed a model of podocyte damage utilizing PAN, and showed that the numbers of podocytes were markedly reduced in the PAN stimulation group following 48 h of exposure to PAN, and the apoptotic rates were notably elevated after 8, 24, and 48 h of PAN treatment, indicating that PAN can lead to podocyte depletion. Moreover, following treatment with MP, and RTX over a 48-h period, there was a notable rise in podocyte counts, and when applied for both 24 and 48 h, these agents markedly reduced the apoptosis rates, suggesting that MP, and RTX might be possible therapeutic implications in podocytes depletion.

The increased expression and activity of TRPC6 leads to aberrant cytoskeletal rearrangements in podocytes, podocyte FPs effacement, and eventually podocyte death (Ding et al., 2021; Qiu et al., 2021; Hart et al., 2023). A gain-of-function mutation in TRPC6 was identified as monogenic cause of FSGS, however, a small number of mutations with a loss-of-function TRPC6 phenotype have also been associated with FSGS (Riehle et al., 2016) and a novel heterozygous loss-of-function TRPC6 mutation was not associated with FSGS (Batool et al., 2023). TRPC6 expression was also increased in non-hereditary proteinuric kidney disorders (Salemkour et al., 2023; Möller et al., 2007), indicating that it can be targeted for treatment. Nevertheless, in the current study, elevations in levels of TRPC6 mRNA and protein following exposure to PAN for durations of 8, 24, and 48 h, surpassed those observed in the control set. These findings align with previously published studies (Tu et al., 2023; Ma et al., 2024). Concurrently, the data from our experiments further revealed that post-intervention with MP or RTX, there were diminished expressions of TRPC6 at corresponding time intervals at both the mRNA and protein levels, suggesting that MP’s or RTX’s ability to lessen the damage PAN causes to podocytes.

The primary therapies for many glomerular diseases are glucocorticoids, which exert their immunosuppressive and direct podocyte protective effects via the glucocorticoid receptor (Agrawal et al., 2021). Podocyte-targeted delivery of TRPC6 short-interfering RNA using an antibody delivery system reduced podocyte TRPC6 expression in rats, and TRPC6 short-interfering RNA prevented AngII-induced apoptosis and increased markers of autophagy in cultured mouse podocytes (Feng et al., 2022). Recently, in an adriamycin-induced mouse nephropathy model, TRPC6-targeted Dex-loaded nanobubles (Dex@NBs), administered at half the dosage of free Dex, markedly alleviated proteinuria, glomerular and tubular damage, renal apoptosis, inflammation, and fibrosis (Wu et al., 2025), which were aligned with our findings, enhancing MP’s organization within the cells and reducing both mRNA expression and protein dispersion during PAN-induced podocyte damage featuring elevated TRPC6 expression.

The kidney is an important organ for the maintenance of Ca²⁺ homeostasis in the body (Semenikhina et al., 2023). Enhanced Ca²⁺ entry stimulates the development of actin-myosin contractility along with stress fibers in the cellular structure, which, when activated improperly, can induce architectural disarray of FPs and damage or even kill podocytes, cause the onset of various renal disorders (Hart et al., 2023; Tu et al., 2023). During the progression of kidney disease, Ca²⁺ signaling plays a key role in various cell activities such as necrosis, apoptosis, eryptosis, and autophay (Zhang et al., 2021; Semenikhina et al., 2023). In vivo and vitro, inhibiting TRPC6 expression could alleviate Ca²⁺ influx and the degradation of podocyte structural proteins, and reduce podocyte injury and proteinuria excretion ['t Hart et al., 2021; Ding et al., 2021; Feng et al., 2022]. Podocytes express large conductance Ca²⁺-activated K⁺ channel (BK channels) increasing Ca²⁺ influx via TRPC6 channels and KCa1.1 subunits interacting directly with TRPC6 channels in PAN-induced podocytes damage (Kim et al., 2024). Furthermore, our research confirmed that TRPC6 overexpression can activate Ca²⁺ influx in PAN-induced podocyte injury, and MP could decrease Ca²⁺ influx for 8, 24, and 48 h, whereas RTX decreased Ca²⁺ influx for 24 h. Accordingly, we propose that MP and RTX can reduce Ca²⁺ influx by inhibiting TRPC6 expression, stabilizing the number of podocytes, further protecting podocytes, and decreasing proteinuria excretion (Ning et al., 2021). Therefore, altering Ca²⁺ signaling pathways may serve as a viable therapeutic approach for diseases linked to podocytes.

This study provides evidence that following PAN stimulation, the concentrations of IL-1β and IL-18 were notably elevated compared to the normal control group at different time intervals. When compared to the PAN treated cohort, the group receiving MP intervention exhibited substantially reduced quantities of IL-1β and IL-18 at the same time point, while the RTX intervention led to significant reductions at both 24 and 48 h. Levels of IL-1β at 24 and 48 h and level of IL-18 at 24 h were lower in the group treated with RTX and MP compared to the group subjected to PAN stimulation. In addition, the maturation and secretion of pro-inflammatory cytokines IL-1β and Il-18 were triggered by Nod-like receptor protein 3 (NLRP3) inflammasome activation, which was induced by the increase of intracellular calcium in vitro and in vivo studies (Yang et al., 2019; Werner and Wagner, 2023). Moreover, in vitro, knockout of TRPC6 could decrease NLRP3 expression and intracellular Ca²⁺ concention and suppress the release of IL-1β and Il-18 in macrophages (Chen et al., 2025). TRPC6 knockout in type 2 diabetes mellitus induced hepatic inflammation and fibrosis, inhibited calcium overload, and suppressed the calcineurin/nuclear factor of activated T cells 2/NLRP3 signaling pathway in mice (Liu et al., 2025). These studies showed that NLRP3 inflammasome could be activated via the TRPC6/Ca²⁺/NLRP3 pathway, contributing to inflammation, concurring with our findings that the expression of TRPC6 and its channel could promote calcium influx in podocytes, stimulate inflammatory agents, cause podocyte injury, and release IL-1β and Il-18.

This in vitro study also confirmed that a common distribution existed between MP and RTX ligand on TRPC6; thus, we inferred that MP and RTX might interact with TRPC6. Moreover, in the current study, MP and RTX treatment decreased the expressions of TRPC6 mRNA and protein at 24 and 48 h, respectively, but increased Ca²⁺ influx at 24 h, suggesting that the Ca²⁺ signal network may participate in the regulation of podocyte injury, and TRPC6 might mediate extracellular Ca²⁺ influx. Following a period of 48 h, levels of TRPC6 mRNA and protein were found to be diminished in combined RTX and MP intervention as compared to those observed in the MP and the RTX intervention. Nonetheless, at intervals of 8 and 24 h, there was a noticeably increased intake of Ca²⁺, leading us to surmise that different channels or regulatory molecules could be involved in the damage to podocytes. Additionally, there are at least two other TRPC channels, such as TRPC3 and TRPC5, expressed in podocytes. The TRPC3 channels cannot be activated by application of ATP in the absence of TRPC6 (Staruschenko et al., 2023). Untill now, TRPC5 expression couldn’t compared with TRPC6 in human renal biopsies, moreover, TRPC5 plays a role redundant to that of TRPC6 in podocytes (Staruschenko et al., 2023; Polat et al., 2023). Concurrently, it is imperative to conduct more extensive research into the precise molecular processes and to corroborate these findings through further examinations employing TRPC6 inhibitors, as indicated by our research or subsequent studies utilizing pertinent animal models (Hart et al., 2023; Tu et al., 2023; Batool et al., 2023).

In conclusion, our study indicates that both MP and RTX have the potential to diminish apoptotic rates and maintain podocyte counts, achieved through suppression of excessive TRPC6 expression, enhancement of TRPC6 arrangement within podocytes, reduction of calcium entry, and mitigation of PAN’s detrimental impact on these cells. These could offer foundational rationales for the therapeutic employment of MP, and RTX in renal pathologies. Collectively, the findings imply a contributory factor of TRPC6 in the harm to podocytes via the disruption of the calcium signaling cascade.