#2903 Comprehensive lipidomic analysis reveals further insights into the molecular mechanism of podocyte lipotoxicity in Alport syndrome
Authors: Shabaka Amir , Requena Belen Fernandez , Gonzalez-Riano Carolina , Lanzon Borja , Martinez Sara , Gradillas Ana , Carrillo Isabel Galan , Bosch Teresa Bada , Sevillano Angel , Medina-Gomez Gema , Barbas Coral , Juarez Gema Maria Fernandez 
Published: May 2024

Abstract

Background and Aims
The accumulation of lipids in podocytes has been shown to be a mediator of kidney injury in diabetic kidney disease (DKD). Recent animal studies have shown evidence of podocyte lipotoxicity in Alport syndrome (AS). First, discoidin domain receptor 1 (DDR1) is overactivated by excessive accumulation of α1 chain type 1 collagen in AS resulting in CD36 activation, producing direct podocyte injury by increasing reuptake of free fatty acids (FFAs). Furthermore, local overexpression of glomerular tumor necrosis factor has been observed in AS, which produces suppression of ABCA1, which affects mitochondrial function and decreases cholesterol efflux from podocytes, causing an accumulation of cholesterol and podocyte lipotoxicity. This study aimed to compare serum and urine lipidome variation between patients with AS and patients with DKD compared to healthy controls to explore the underlying molecular mechanisms of podocyte lipotoxicity in AS.

Method
A total of 63 samples from AS patients (COL4A3/4, n = 50, COL4A5, n = 13) were employed for this study, and compared to 15 samples from DKD patients and 20 samples from healthy volunteers considered as controls. We performed an untargeted lipidomics analysis to cover the broader spectrum of plasma and urine lipidome. Samples were analyzed by using an Agilent 1290 Infinity II Ultra-High-Performance Liquid-Chromatography system coupled to an Agilent 6546 Quadrupole Time-of-Flight Mass Spectrometer equipped with dual Agilent Jet Stream Electrospray ion source. For plasma samples, raw data matrix underwent normalization for sequence intensity correction based on the QC samples by using the support vector regression. For urine samples, normalization was performed using the Probabilistic Quotient Normalization to account for unwanted variance due to sample preparation and the analytical run.

Results
We generated a heatmap employing hierarchical clustering to visually represent the distinctions in statistically significant lipids across AS COL4A3/4, AS COL4A5, and DKD groups compared to the healthy control group, categorized by classes.
Figure 1 summarizes the results of our study. In urine samples, a distinct decrease in acylcarnitines is evident in AS COL4A3/4 and AS COL4A5. Regarding the fatty acid (FA) lipid class, there is an increased general trend. FA 20:3 shows a meaningful increase in AS COL4A3/4 and DKD compared to the healthy control group, whereas in AS COL4A5, the trend of this lipid species is to decline when compared with COL4A3/4 and DKD levels. Concerning plasma samples, different trends were observed in AS COL4A3/4 and DKD depending on specific lipid species, while general reduced levels were reported in AS COL4A5.
The phosphatidylcholine (PC) lipid species, implicated in various biological functions, display a drastic increase in the DKD urine group compared to the healthy control. The same trend is followed by AS COL4A3/4 and AS COL4A5 urine groups, with relatively similar data.
Among the sphingolipids class, in urine, the overall trend of sphingomyelins (SM) for both AS COL4A3/4 and AS COL4A5 indicates a decrease in the levels of the statistically significant lipid species, with relatively similar levels between them. The neutral sphingolipids (HexCer) experience a general decrease in AS COL4A3/4, AS COL4A5 and DKD urine samples compared to the healthy control group.
Lastly, the ceramides (Cer) follow a decreasing trend, where the most remarkable differences could be observed in the DKD urine group compared to the healthy controls.

Conclusion
Overall, the present study provided new insights into the mechanism of podocyte lipotoxicity in patients with AS. Increased specific plasma Cer and SM that are responsible for correct COL4A1 and COL4A2 formation might be responsible for aberrant COL1 expression DDR1 activation. Furthermore, specific essential FFAs that are involved in collagen synthesis are increased in AS, and might be indicating aberrant CD36 levels increasing their levels and enhancing their accumulation. Increased ABCA1 efflux activity is evident in higher extracellular levels of PC observed in urine samples from patients with AS.