Description
Despite the recent advances in our understanding of the role of lipids, metabolites and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving human kidney damage (KD). The limited availability of kidney biopsies from living donors with kidney disease has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study kidney disease in humans and characterized these kidneys using imaging and multi-omics approaches. We demonstrated that changes in kidney injury and inflammatory markers following KD were consistent with the changes in pre-donation renal function in donors. Neighborhood and correlation analyses of imaging mass cytometry data showed that a subset of renal cells (e.g., fibroblasts) are associated with the expression profile of renal immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that renal arachidonic acid metabolism and seven other metabolic pathways were upregulated following KD. To validate the therapeutic potential of targeting the arachidonic acid pathway, we demonstrated increased levels of cytosolic phospholipase A2 (cPLA2) protein and related lipid mediators (e.g., prostaglandin E2) in the injured kidneys. The inhibition of cPLA2 reduced injury and inflammation in human renal proximal tubular epithelial cells (RPTEC) in vitro. This study identifies cell types and metabolic pathways that may be critical for controlling inflammation associated with KD in humans.
Overall Design
Frozen kidney cortex samples were put in RNA later stabilization solution (Thermo Fisher Scientific, UK) and stored at -80C until use. Samples were moved into Agencourt RNAdvance Tissue kit (Beckman Coulter) lysis buffer and homogenized at 1000 rpm for 2min with 5mm Stainless Steel Bead (Qiagen) using 1600 MiniG Tissue Homogenizer (SPEX SamplePrep). Next, total RNA was extracted using Agencourt RNAdvance Tissue kit including DNase treatment for 15 min at 37 C (Ambion DNaseI, ThermoFisher) on an automated Biomek i7 Hybrid system (Beckman Coulter). RNA quantification and quality control was performed on 48 capillary Fragment Analyzer 5300 system (Agilent). All samples showed fragment distribution values (% DV200) above 74 and were subjected to a total RNA-seq library preparation using KAPA RNA HyperPrep Kit with RiboErase (Roche) with 450ng of input RNA, 5min fragmentation at 95 C, and 9 PCR cycles performed on a Tecan Fluent 1080 system (Tecan). Resulting libraries were quantified on 48 capillary Fragment Analyzer 5300 system showing 295bp average peak size, and pooled equimolarly to a final concentration of 1.9nM. 51bp paired-end sequencing was performed on a v1.5 S1 NovaSeq 6000 kit (Illumina) including 1% of 1.9nM PhiX v3 control (Illumina).
Curator
yq_pan