#4359 LOSS OF GASDERMIN D LEADS TO AN EXACERBATION OF KIDNEY DAMAGE IN NEPHROCALCINOSIS-RELATED CHRONIC KIDNEY DISEASE
Authors: Kusunoki Yoshihiro , Steiger Stefanie , Linkermann Andreas , Anders Hans-Joachim 
Published: June 2023

Abstract

Background and Aims
Gasdermin D (GSDMD) is a membrane-targeting and pore-forming protein that is primarily expressed in immune cells. In the proinflammatory cell death process known as pyroptosis, cleaved GSDMD plays a crucial role through cell membrane pore formation, leading to the release of proinflammatory cytokines and membrane rapture. Despite this, the majority of GSDMD's other physiological functions remain to be elucidated. Previous reports have implicated GSDMD in the pathogenesis of kidney disease, utilizing some acute kidney injury (AKI) animal models and a few chronic kidney disease (CKD) animal models. However, it remains uncertain if GSDMD contributes to the pathogenesis of nephrocalcinosis-related CKD. Therefore, this study aimed to investigate the role of GSDMD in in this context.

Method
In our study, 8-week-old GSDMD-knockout (KO) mice and wildtype (WT) mice received an oxalate-rich diet (50 μmol/g sodium oxalate) for three weeks. All mice were sacrificed on day 21 and kidneys were harvested after the sacrifice. Plasma and urine samples were collected at different time points before the sacrifice. The glomerular filtration rate (GFR) was measured on day 0 and day 20 to assess kidney excretion function. Histological analysis was performed using Periodic Acid-Schiff (PAS) staining to assess renal injury, Sirius Red staining to check renal fibrosis, Pizzolato staining to evaluate calcium crystal deposition, F4/80 staining to evaluate the infiltration of macrophages, TUNEL staining to check for cell death, RIPK3 and phosphorylated MLKL (pMLKL) staining to detecting necroptosis. And GSDMD staining was also performed to examine the expression of GSDMD in the kidney.

Results
Pizzolato staining showed no differences in calcium crystal deposition between WT mice and KO mice. GFR was decreased in both WT mice and KO mice after the oxalate-rich diet (day 0 WT: 199.5±40.1 μl/min vs KO 175.0±44.8 μl/min, day 20 WT: 156.5±37.9 μl/min vs KO: 73.0±48.1 μl/min), and ΔGFR was much greater in KO mice (WT: −22.4±26.5% vs KO: −59.4±19.3%). The level of blood urea nitrogen was significantly higher in KO mice (WT: 41.9±3.90 mg/dl vs KO: 58.7±22.5 mg/dl) in accordance with the result of the GFR. PAS staining revealed more severe kidney injury in KO mice, and Sirius Red staining indicated a higher degree of renal fibrosis in KO mice. Furthermore, F4/80 staining showed greater infiltration of macrophages in KO mice. Although TUNEL staining showed no significant differences in positive cells between WT and KO, KO mice showed greater positive areas implicating more necroptosis in RIPK3 and pMLKL staining. GSDMD staining revealed that injured tubular cells during the chronic phase express GSDMD (Fig. 1). In in vitro experiments, we evaluated the functionality of macrophages such as phagocytic ability and migratory ability using bone marrow-derived macrophages (BMDMs), but we didn't find any significant disparities between BMDMs from WT mice and those from KO mice.

Conclusion
Genetic deletion of GSDMD resulted in more severe kidney injury, macrophage infiltration and fibrosis in mice with nephrocalcinosis-related CKD. Our results suggest a potential role for pyroptosis, especially GSDMD, in contributing to renal tubular cell necroptosis, a process downstream of calcium-oxalate crystal formation and deposition, in the progression of nephrocalcinosis-related CKD, similar to previous reports in mouse models of AKI.