Analysis of gene expression and use of connectivity mapping to identify drugs for treatment of human glomerulopathies

Abstract

BackgroundHuman glomerulonephritis (GN)—membranous nephropathy (MN), focal segmental glomerulosclerosis (FSGS) and IgA nephropathy (IgAN), as well as diabetic nephropathy (DN) are leading causes of chronic kidney disease. In these glomerulopathies, distinct stimuli disrupt metabolic pathways in glomerular cells. Other pathways, including the endoplasmic reticulum (ER) unfolded protein response (UPR) and autophagy, are activated in parallel to attenuate cell injury or promote repair.MethodsWe used publicly available datasets to examine gene transcriptional pathways in glomeruli of human GN and DN and to identify drugs.ResultsWe demonstrate that there are many common genes upregulated in MN, FSGS, IgAN, and DN. Furthermore, these glomerulopathies were associated with increased expression of ER/UPR and autophagy genes, a significant number of which were shared. Several candidate drugs for treatment of glomerulopathies were identified by relating gene expression signatures of distinct drugs in cell culture with the ER/UPR and autophagy genes upregulated in the glomerulopathies (“connectivity mapping”). Using a glomerular cell culture assay that correlates with glomerular damage in vivo, we showed that one candidate drug – neratinib (an epidermal growth factor receptor inhibitor) is cytoprotective.ConclusionThe UPR and autophagy are activated in multiple types of glomerular injury. Connectivity mapping identified candidate drugs that shared common signatures with ER/UPR and autophagy genes upregulated in glomerulopathies, and one of these drugs attenuated injury of glomerular cells. The present study opens the possibility for modulating the UPR or autophagy pharmacologically as therapy for GN.

Genetic Deletion of Calcium-independent Phospholipase A2γ Protects Mice from Diabetic Nephropathy

“…co-culture of cell lines, may be required to address mechanistic questions [46]. Evaluation of iPLA2γ metabolic effects and autophagy in human diabetic kidneys is difficult, although autophagy genes and gene ontology pathways are upregulated in glomeruli in human diabetic nephropathy [47].…”

Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice

“…The publicly accessible Nephroseq dataset “JuCKD-Glom” (GSE47183) was used for the expression analysis of glomerular genes, including genes associated with the ER/UPR and autophagy 16 , 31 , 32 . Nephroseq contains microarray gene expression data (mRNAs) of microdissected whole glomeruli from human kidney biopsies and the data are presented as the fold-increase of gene expression in disease above healthy control.…”

“…P-values for ER/UPR and autophagy genes were corrected for false discovery according to the Benjamini–Hochberg method. Pathway overrepresentation and gene ontology (GO) enrichment analyses were performed using the ConsensusPathDB interaction database 16 , 33 . This database provides the adjusted P-values (hypergeometric test, corrected for multiple comparisons) that we present in this manuscript.…”

“…There is evidence for ER stress in human glomerulopathies—ER stress markers are increased in the glomeruli of kidney biopsies from patients with glomerulonephritis and diabetic nephropathy 4 , 13 , 14 . Analysis of a human glomerular gene expression dataset (Nephroseq) supports activation of a UPR-IRE1α axis in patients with various glomerulopathies 15 , 16 . We demonstrated the functional importance of the IRE1α-UPR axis using mice with podocyte-specific deletion of IRE1α (IRE1α knockout; KO mice) 17 .…”

Role of the Ste20‐like kinase SLK in podocyte adhesion

“…Alternatively, GECs were fixed with ice-cold methanol. Cells were stained with antibodies against FAC proteins for 24 h (4°C) followed by secondary antibodies and Hoechst H33342 (to stain nuclei), as described previously (Chung et al, 2019(Chung et al, , 2023Kachurina et al, 2016). Z-stack images were acquired on a Zeiss Axio Observer inverted fluorescence microscope with visual output connected to an AxioCam MRm monochrome camera (Carl Zeiss AG; Toronto, ON).…”