Organelle crosstalk between the ER and endolysosomes regulates TLR9–NF-κB inflammatory signaling in cisplatin-induced acute kidney injury

Inter-organelle communication is increasingly recognized as a critical determinant of cellular homeostasis, particularly under stress conditions. In the kidney, the contribution of membrane contact sites (MCSs) to inflammatory responses during remains largely unexplored. Endolysosomes serve as essential hubs for Toll-like receptor 9 (TLR9) activation and downstream NF-κB–mediated cytokine release in cisplatin-induced acute kidney injury (AKI). We hypothesized that endoplasmic reticulum (ER)–endolysosome contact sites, a key form of organelle crosstalk, support TLR9 signaling activation and contributes to AKI.

We investigated the functional significance of ER–endolysosome communication using Pdzd8 knockout (KO) mice and PDZD8-silenced HK-2 cells. Cisplatin-induced AKI was evaluated by histology and serum creatinine levels. NF-κB activation and inflammatory cytokine expression were assessed by immunoblotting and qPCR. In HK-2 cells, endolysosomal function was examined using LysoTracker, Magic Red, and cathepsin B assays, while TLR9 trafficking was analyzed via immunofluorescence and endolysosomal fraction immune blotting. Mitochondrial integrity and cytosolic mtDNA levels were also measured to check the severity of cellular damage. Bafilomycin was used to disrupt endolysosomal maturation pharmacologically.

Disruption of ER–endolysosome crosstalk through genetic loss of Pdzd8 led to reduced tubular injury and lower serum creatinine in cisplatin-treated mice, along with suppressed NF-κB activation and proinflammatory cytokine production. In HK-2 cells, loss of PDZD8 improved viability and reduced inflammation without affecting mitochondrial integrity or cytosolic mtDNA release, suggesting that cellular damage was not the primary driver of reduced inflammation. Instead, organelle crosstalk disruption impaired endolysosomal acidification and cathepsin B activity, preventing efficient TLR9 recruitment to LAMP1-positive endolysosome and attenuating MyD88-mediated downstream signaling. Bafilomycin treatment phenocopied these effects, supporting the link between endolysosomal maturation and TLR9–NF-κB activation.

This study highlights organelle crosstalk between the ER and endolysosomes as a key regulator of inflammation in cisplatin-AKI. By maintaining endolysosomal homeostasis, these contact sites enable effective TLR9–NF-κB signaling in response to injury. Disruption of this axis decouples tubular damage from inflammatory responses, offering a novel therapeutic target for limiting kidney inflammation without compromising epithelial cell survival.