METABOLIC ALTERATIONS IN GLYCOLIPIDS DURING ACUTE KIDNEY INJURY DRIVE RENAL INFLAMMATION

Acute kidney injury (AKI) can progress to chronic kidney disease (CKD), via a mechanism that is still largely unknown. We previously reported that levels of hexosylceramide (HexCer) increase after AKI, and that this lipid species is likely glucosylceramide (GlcCer). Furthermore, GlcCer activates macrophages via the innate immune receptor macrophage-inducible C-type lectin (Mincle) in vitro, suggesting that the GlcCer–Mincle pathway may contribute to the AKI-to-CKD transition. However, it remains unclear whether the HexCer species elevated after AKI is really GlcCer and how it changes dynamically, whether GlcCer functions as a damage-associated molecular pattern (DAMP) in vivo, and how its accumulation is regulated in the injured kidney.

Male mice aged 8-12 weeks were subjected to ischemia-reperfusion injury (IRI) and observed until Day 14. HexCer species were identified by LC-MS/MS in hydrophilic interaction liquid chromatography (HILIC) mode in the harvested injured kidneys, and spatial distribution in the kidneys was analyzed using MALDI mass spectrometry imaging. Gene expression of glycosyltransferases at day 3 after AKI was comprehensively assessed by microarray analysis. and temporal changes up to day 14 were evaluated by Quantitative real-time PCR. Gene expression changes were also examined in cisplatin and folic acid models. Protein levels of glycosyltransferase genes after IRI were observed by Western blotting until Day 14. In vitro, mouse proximal tubular cells (mProx24) were treated with menadione or cisplatin to induce oxidative stress, with N-acetylcysteine (NAC) rescue experiments performed to assess reversibility. siRNA-mediated knockdown of candidate glycosyltransferase genes was conducted to evaluate their effects on GlcCer accumulation. Furthermore, a GlcCer–cholesterol emulsion was injected under the renal capsule, and inflammatory responses were compared between wild-type and Mincle-deficient mice.

LC-MS/MS in HILIC mode revealed that the HexCer elevated after AKI consisted of GlcCer. MALDI mass spectrometry imaging demonstrated selective accumulation of GlcCer in injured proximal tubules. Microarray analysis indicated that, among glycosyltransferase genes associated with increased GlcCer, β-1,4-galactosyltransferase 5 (B4galt5) was decreased. In the IRI model, both mRNA and protein levels of B4galt5 remained reduced up to day 14, and its mRNA expression was also decreased in cisplatin- and folic acid-induced AKI models. Menadione and cisplatin suppressed B4galt5 expression in proximal tubular cells, and this suppression was reversed by NAC. Knockdown of glycosyltransferase genes increased GlcCer accumulation in vitro. Subcapsular injection of GlcCer with cholesterol induced a robust inflammatory response in wild-type mice, whereas this response was almost completely abolished in Mincle-deficient mice.

Our findings demonstrate that the increase in HexCer during AKI results primarily from GlcCer accumulation, driven by oxidative stress–mediated downregulation of B4galt5 in renal proximal tubules. We also show that GlcCer, in combination with free cholesterol, induces Mincle-mediated inflammatory responses in the kidneys in vivo. These findings identify that B4galt5 as a potential therapeutic target for preventing the AKI-to-CKD transition.