Long-chain ceramides as compartment-specific markers of renal injury in chronic kidney disease

Background: Ceramides are bioactive sphingolipids involved in cellular stress responses, inflammation, and apoptosis. Dysregulated ceramide metabolism has been associated with chronic kidney disease (CKD) in both clinical and experimental studies. Elevated ceramide levels have been reported in patients with reduced kidney function, supporting clinical interest in their role in disease progression. Among different ceramide species, long-chain ceramides have been implicated in cellular injury pathways and have been suggested to play a role in renal damage. However, the distribution of long-chain ceramides in human kidney tissue across different kidney diseases and renal compartments is not well defined. Clarifying these patterns may help identify markers of active injury and support the development of new therapeutic strategies. Methods: Formalin-fixed, paraffin-embedded human kidney biopsy samples with confirmed diagnoses were stained with a long-chain ceramide-selective antibody (Enzo Life Sciences, Cat# ALX-804-196). We further analyzed diabetic nephropathy (DN) and histologically normal controls from both sexes (male DN: 47±8 years, eGFR 53±11 mL/min/1.73m 2 ; female DN: 39±4 years, eGFR 73±10; male controls: 53±9 years, eGFR 74±7; female controls: 61±8 years, eGFR 77±15; n=4 per group). Female samples from lupus nephritis (LN) and focal segmental glomerulosclerosis (FSGS) were evaluated in two age groups (LN: 45±2 years, eGFR 66±3, and 26±2 years, eGFR 69±20; FSGS: 62±5 years, eGFR 33±5, and 26±2 years, eGFR 67±12; n=4 per group). Ceramide signal was evaluated in glomerular and tubulointerstitial compartments. Further, immunofluorescence staining was used to assess long-chain ceramide localization in podocytes and tubular epithelial cells. Staining intensity was quantified in regions of interest and analyzed using mean gray value measurements. Quantification was performed in a blinded manner and stratified by diagnosis, sex, and age. Results: Long-chain ceramide abundance showed distinct disease- and compartment-specific patterns. In control kidneys, males had higher baseline ceramide levels than females in glomeruli (23.5±2.1 vs 7.6±1.0; p< 0.01) and tubules (47.2±2.8 vs 27.3±2.7; p< 0.01), indicating overall sex differences in abundance. DN exhibited the highest accumulation, particularly in the tubulointerstitial region (64.2±5.7 in males and 43.0±3.8 in females; p< 0.01 vs control), and elevated glomerular levels (46.4±4.7 and 30.9±2.2; p< 0.01 vs control). LN demonstrated robust increases in glomerular ceramide abundance (35.5±3.2 and 34.2±3.1; p< 0.001 vs female control in both older and younger groups). FSGS showed moderate increases in glomerular ceramides (22.4±2.1 and 17.1±2.1; p< 0.01 vs female control in older and younger female groups) while tubular ceramide levels in FSGS did not differ significantly from controls. Conclusion: Across CKD groups, DN showed the highest levels of long-chain ceramide accumulation, followed by LN and FSGS. Sex-related differences were observed in controls and DN, and age-associated differences were present within LN and FSGS samples. These findings indicate that long-chain ceramide abundance differs across CKD types and renal compartments. The results support the potential value of long-chain ceramides as compartment-specific tissue markers of kidney injury and highlight the need for further studies to evaluate their diagnostic and therapeutic relevance in CKD. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.