Single Cell Dissection of Epithelial-Immune Cellular Interplay in Acute Kidney Injury Microenvironment

Abstract

BackgroundUnderstanding the acute kidney injury (AKI) microenvironment changes and the complex cellular interaction is essential to elucidate the mechanisms and develop new targeted therapies for AKI.MethodsWe employed unbiased single-cell RNA sequencing to systematically resolve the cellular atlas of kidney tissue samples from mice at 1, 2 and 3 days after ischemia-reperfusion AKI and healthy control. The single-cell transcriptome findings were validated using multiplex immunostaining, western blotting, and functional experiments.ResultsWe constructed a systematic single-cell transcriptome atlas covering different AKI timepoints with immune cell infiltration increasing with AKI progression. Three new proximal tubule cells (PTCs) subtypes (PTC-S1-new/PTC-S2-new/PTC-S3-new) were identified, with upregulation of injury and repair-regulated signatures such as Sox9, Vcam1, Egr1, and Klf6 while with downregulation of metabolism. PTC-S1-new exhibited pro-inflammatory and pro-fibrotic signature compared to normal PTC, and trajectory analysis revealed that proliferating PTCs were the precursor cell of PTC-S1-new, and part of PTC-S1-new cells may turn into PTC-injured and then become fibrotic. Cellular interaction analysis revealed that PTC-S1-new and PTC-injured interacted closely with infiltrating immune cells through CXCL and TNF signaling pathways. Immunostaining validated that injured PTCs expressed a high level of TNFRSF1A and Kim-1, and functional experiments revealed that the exogenous addition of TNF-α promoted kidney inflammation, dramatic injury, and specific depletion of TNFRSF1A would abrogate the injury.ConclusionsThe single-cell profiling of AKI microenvironment provides new insight for the deep understanding of molecular changes of AKI, and elucidates the mechanisms and developing new targeted therapies for AKI.

Single-cell dissection of cellular and molecular features underlying human cervical squamous cell carcinoma initiation and progression

“…After washing with phosphate-buffered saline (PBS), the samples were enzymatically digested to obtain single-cell suspensions. Following similar procedures previously described ( 57 , 58 ), the samples were minced into <1 mm 3 pieces and digested with 5 ml of digestion buffer containing collagenase IV (2 mg/ml; Sigma-Aldrich) and deoxyribonuclease I (1 mg/ml; Sigma-Aldrich) for 30 min at 37°C. Then, the resulting suspension was mixed with 5 ml of 2% fetal bovine serum (FBS)/PBS and centrifuged at 300 g for 5 min at 4°C to get the cell pellet.…”

“…Following similar procedures previously described ( 57 , 58 ), the Single-Cell 3′ Library Kit v3 (10x Genomics) was used for single-cell transcriptome amplification and library preparation according to the manufacturer’s instructions. The sorted single-cell suspension was loaded onto a microfluidic chip from 10x Genomics to generate the cDNA library.…”

“…The data were processed as previously described ( 57 , 58 ). Using the cellranger count function of CellRanger (10x Genomics, v4), raw sequencing FASTQ files were aligned to the GRCh38 reference genome to produce a gene expression library via the STAR algorithm.…”

“…As previously described ( 58 ), we analyzed the differentially expressed TFs with a standard pipeline implemented in R using the SCENIC R package ( https://github.com/aertslab/SCENIC ) to explore the single-cell gene regulatory network (GRN) among different cell subclusters. Briefly, we estimated the gene expression matrices of the cell subclusters using GENIE3 to build the initial coexpression GRNs.…”

AFM negatively regulates the infiltration of monocytes to mediate sepsis-associated acute kidney injury

“…Sepsis is defined as end-organ dysfunction due to the host’s inflammatory response to infection, in which the damaged kidney, a major source of inflammatory chemokines, may have local and remote deleterious effects on the body and increase the risk of mortality in patients with sepsis ( 3 ). Several studies have shown that the occurrence and development of SA-AKI are closely related to the infiltration of various immune cells ( 31 ). AKI is characterized by inflammatory infiltration within the kidney that induces apoptosis and promotes tubular epithelial cell loss ( 32 ).…”