Local microenvironment orchestrated by glomerular parietal epithelial cells gates kidney injury

Acute kidney injury (AKI) is a complex clinical syndrome with high morbidity and mortality and often progresses to chronic kidney disease (CKD) or end-stage renal disease (ESRD). Although injury to renal tubular epithelial cells is widely regarded as a key driver of AKI, the role of glomerular components remains poorly understood.

Recent evidence indicates that parietal epithelial cells (PECs), which line Bowman's capsule, are not merely passive structural cells but actively respond to various renal insults, including ischemia, inflammation, and nephrotoxic injury. This suggests that PECs may play a role in AKI induced by ischemia–reperfusion.

In this study, we aimed to characterize the phenotype and functional changes of PECs after IRI-AKI, explore their potential roles and underlying mechanisms in acute renal injury, and ultimately provide a foundation for PEC-targeted therapeutic strategies for AKI.

1. Unilateral and bilateral ischemia-reperfusion injury (uIRIbIRI) was performed to establish the AKI model in transgenic mice. 

2. Generation of PEC-specific Spp1 conditional knockout mice (Spp1-cKO).

3. Recombinant osteopontin (OPN) protein was administered via intraperitoneal injection to evaluate its functional role and therapeutic potential during acute renal injury.

Using a unilateral ischemia–reperfusion model (uIRI, 45 min), we generated a single-cell RNA-seq atlas of 78,250 cells grouped into 38 clusters. Sampling across multiple time points enabled us to trace glomerular cell dynamics during AKI and CKD transition. Among eight glomerular cell types, parietal epithelial cells (PECs) showed the most striking transcriptional and temporal changes after ischemia. UMAP analysis revealed an early and distinct PEC cluster shift at day 1, indicating rapid activation and phenotypic transition. Histology confirmed these findings: PEC numbers markedly declined on day 1, followed by partial recovery and crescent-like hyperplasia in later phases. TUNEL staining showed extensive apoptosis in PECs at day 1, suggesting that PEC loss resulted from direct ischemic injury rather than secondary effects.

Differential expression analysis identified Spp1 (Osteopontin, OPN) as the top secreted DEG. Immunostaining demonstrated that PEC-derived OPN peaked on day 1, implying a stress-induced compensatory or protective response. To define its function, we generated PEC-specific Spp1 conditional knockout mice (Spp1-cKO). After bilateral IRI, Spp1-cKO mice displayed higher serum creatinine and BUN, along with aggravated histological damage, compared with wild-type controls. Molecular assays showed stronger inflammatory activation in Spp1-cKO kidneys, indicating that PEC-derived OPN mitigates acute renal injury.

GSVA highlighted enrichment of vascular contraction/relaxation and renin–angiotensin system (RAS) pathways, suggesting a hemodynamic role. Consistently, super-resolution ultrasound imaging (SRUS) revealed markedly reduced renal perfusion in Spp1-cKO mice on day 1 post-AKI, confirming impaired microvascular flow. Given the link between RAS and renal perfusion, we next examined RAS components. ACE2, a central enzyme in the non-classical RAS axis, was significantly decreased in cKO kidneys, while classical components (ACE, AT1R) remained relatively unchanged. Immunostaining localized this reduction to PECs, indicating that OPN loss suppresses local ACE2 expression.

Recombinant OPN (rmOPN) administration to wild-type mice reversed these changes: ACE2 levels increased at both whole-kidney and PEC levels,  and Ang(1–7) increased at whole-kidney level, confirming that OPN promotes ACE2 expression and activates the ACE2–Ang(1–7)–Mas pathway. Together, these findings suggest that PEC-derived OPN preserves renal perfusion and limits AKI severity through modulation of ACE2-dependent vascular regulation.

In summary, our multi-dimensional findings support a mechanistic framework in which PEC-derived OPN functions as both a paracrine and autocrine regulator of renal injury responses. Under ischemic stress, PECs promptly release OPN, which in turn upregulates ACE2 expression and activates the ACE2–Ang(1–7)–Mas axis. This signaling promotes local vasodilation, improves microvascular perfusion, and dampens inflammatory activity within the glomerulus. When PEC-derived OPN is absent or impaired, this protective circuit is disrupted—ACE2 expression declines, renal blood flow decreases, and inflammatory injury intensifies, collectively worsening AKI outcomes. This study underscores the importance of PECs as both targets and regulators of ischemic renal injury.