HYPOXIA-INDUCED PODOCYTE INJURY IN PRIMARY NEPHROTIC SYNDROME VIA ITGB6

Primary nephrotic syndrome (PNS) is a glomerular disease characterized by massive proteinuria, hypoalbuminemia, edema and hyperlipidemia, which may progress to end-stage renal disease without appropriate intervention. Podocyte injury contributes to proteinuria and glomerulosclerosis. Emerging evidence suggests that ischemia and hypoxia are pivotal drivers of podocyte injury. However, the molecular mechanism remains unclear. This study aims to explore the underlying mechanism of hypoxia-induced podocyte injury and novel therapeutic approach.

HIF1α immunohistochemical staining was performed on renal tissues from patients with PNS and isolated hematuria individuals (for comparison) to verify the presence of hypoxia. Hypoxia-inducing kits were used to create hypoxia in differentiated conditionally immortalized human podocytes. Podocyte functional assessment included Transwell migration assays, fibronectin-based cell adhesion assays, flow cytometry for apoptosis detection via Annexin V-FITC/PI staining, and F-actin cytoskeletal visualization using phalloidin-Alexa Fluor 488 staining. Transcriptomic analysis was performed using RNA-seq, with differential gene expression analysis employing TopHat alignment and Cuffdiff analysis (screening criteria: |log₂FC|≥1, P<0.05). ITGB6 expression was validated through RT-qPCR using specific primers, Western blot analysis with anti-ITGB6 antibodies, and immunofluorescence microscopy. The functional significance of ITGB6 was assessed through siRNA-mediated knockdown experiments using specific sequences (5'-GCAUGUUCUGUCUUCAAUA-3') and Lipofectamine 2000 transfection. The protective effects of trehalose (50 mM) were evaluated using pretreatment and co-treatment protocols. In vivo validation employed male Sprague-Dawley rats (200-220 g) in three models: doxorubicin-induced nephrotic syndrome (NS; 6 mg/kg i.v.), unilateral renal vascular ligation-induced acute kidney injury (AKI), and a combined NS+AKI model. Renal function was assessed through 24-hour urine protein/creatinine ratios, serum creatinine, and albumin measurements. Renal ITGB6 expression was analyzed through Western blot analysis of kidney tissue homogenates.

Immunohistochemical staining for HIF1α in renal tissues from PNS patients revealed increased expression in podocytes, positively correlating with disease severity. This confirms an association between podocyte hypoxia and disease severity. Hypoxic exposure profoundly impaired podocyte function. Migration capacity was significantly reduced in hypoxic podocytes (58.77 ± 9.99 vs. 131.93 ± 18.1 migrated cells per field in controls, P < 0.05), accompanied by diminished adhesion ability (37.77 ± 4.3 vs. 46.07 ± 3.4 adherent cells per field in controls, P < 0.05). Apoptotic cells were significantly increased under hypoxic conditions (10.27 ± 3.27% vs. 4.22 ± 1.26% in controls, P < 0.05). Meanwhile, F-actin staining revealed severe cytoskeletal disorganization with loss of characteristic "ring-shaped" cortical structures and disrupted radial stress fiber arrangements. Comprehensive transcriptomic analysis identified 3,537 significantly differentially expressed genes in hypoxic podocytes, including 1,640 upregulated and 1,897 downregulated transcripts. Gene Ontology enrichment analysis revealed alterations in endoplasmic reticulum-to-nucleus signalling, apoptotic pathways, and cytoskeletal regulation, while KEGG pathway analysis highlighted protein processing in endoplasmic reticulum, TNF signalling, and TGF-β signalling pathways. Notably, ITGB6 emerged as a significantly downregulated gene, with validation studies confirming reduced mRNA and protein expression in hypoxic conditions by RT-qPCR, Western blotting, and immunofluorescence analyses. Functional validation via siRNA-mediated ITGB6 knockdown recapitulated the hypoxic phenotype, demonstrating reduced migration (51% decrease vs. control), impaired adhesion (36% decrease vs. control), increased apoptosis (265% increase vs. control), and comparable F-actin disorganization patterns. These findings established ITGB6 downregulation as a critical mediator of hypoxia-induced podocyte dysfunction. Trehalose pretreatment provided substantial protection against hypoxic injury, improving migration capacity (85% increase vs. hypoxia alone), enhancing adhesion (64% increase vs. hypoxia alone), and reducing apoptotic cell death (25% decrease vs. hypoxia alone). F-actin staining demonstrated preserved cytoskeletal organization following trehalose treatment. Protective effects were partially maintained in ITGB6-knockdown podocytes, suggesting both ITGB6-dependent and -independent mechanisms. In vivo validation across three rat disease models confirmed clinical relevance, with doxorubicin-induced NS, renal vascular ligation AKI, and combined NS+AKI models all demonstrating elevated urinary protein/creatinine ratios, increased serum creatinine levels, decreased serum albumin levels, and significantly reduced renal ITGB6 expression. The combined NS+AKI model exhibited the most severe renal dysfunction and ITGB6 downregulation, supporting the synergistic effects of hypoxic injury in pre-existing nephropathy.

Hypoxia induces podocyte dysfunction through ITGB6 downregulation. Trehalose emerges as a promising cytoprotective agent capable of mitigating hypoxia-induced podocyte injury.