Parietal epithelial cells maintain the epithelial cell continuum forming Bowman's space in focal segmental glomerulosclerosis

In the glomerulus, Bowman's space is formed by a continuum of glomerular epithelial cells. In focal segmental glomerulosclerosis (FSGS), glomeruli show segmental scarring, a result of activated PECs invading the glomerular tuft. The segmental scars interrupt the epithelial continuum. However, non-sclerotic segments seem to be preserved even in glomeruli with advanced lesions. We studied the histology of the segmental pattern in Munich Wistar Frömter (MWF) rats, a model for secondary FSGS. Our results showed that matrix layers lined with PECs cover the sclerotic lesions. These PECs formed contacts with podocytes of the uninvolved tuft segments, restoring the epithelial continuum. Formed Bowman's spaces were still connected to the tubular system. Furthermore, in biopsies of patients with secondary FSGS we also detected matrix layers formed by PECs, separating the uninvolved from the sclerotic glomerular segments. While PECs have a major role in the formation of glomerulosclerosis, we showed that in FSGS, PECs also restore the glomerular epithelial cell continuum that surrounds Bowman's space. This process may be beneficial and indispensable for glomerular filtration in the uninvolved segments of sclerotic glomeruli.

Role of IRE1α in podocyte proteostasis and mitochondrial health

Glomerular epithelial cell (GEC)/podocyte proteostasis is dysregulated in glomerular diseases. The unfolded protein response (UPR) is an adaptive pathway in the endoplasmic reticulum (ER) that upregulates proteostasis resources. This study characterizes mechanisms by which inositol requiring enzyme-1α (IRE1α), a UPR transducer, regulates proteostasis in GECs. Mice with podocyte-specific deletion of IRE1α (IRE1α KO) were produced and nephrosis was induced with adriamycin. Compared with control, IRE1α KO mice had greater albuminuria. Adriamycin increased glomerular ER chaperones in control mice, but this upregulation was impaired in IRE1α KO mice. Likewise, autophagy was blunted in adriamycin-treated IRE1α KO animals, evidenced by reduced LC3-II and increased p62. Mitochondrial ultrastructure was markedly disrupted in podocytes of adriamycin-treated IRE1α KO mice. To pursue mechanistic studies, GECs were cultured from glomeruli of IRE1α flox/flox mice and IRE1α was deleted by Cre–lox recombination. In GECs incubated with tunicamycin, deletion of IRE1α attenuated upregulation of ER chaperones, LC3 lipidation, and LC3 transcription, compared with control GECs. Deletion of IRE1α decreased maximal and ATP-linked oxygen consumption, as well as mitochondrial membrane potential. In summary, stress-induced chaperone production, autophagy, and mitochondrial health are compromised by deletion of IRE1α. The IRE1α pathway is cytoprotective in glomerular disease associated with podocyte injury and ER stress.

Signatures of co-deregulated genes and their transcriptional regulators in colorectal cancer

The deregulated genes in colorectal cancer (CRC) vary significantly across different studies. Thus, a systems biology approach is needed to identify the co-deregulated genes (co-DEGs), explore their molecular networks, and spot the major hub proteins within these networks. We reanalyzed 19 GEO gene expression profiles to identify and annotate CRC versus normal signatures, single-gene perturbation, and single-drug perturbation signatures. We identified the co-DEGs across different studies, their upstream regulating kinases and transcription factors (TFs). Connectivity Map was used to identify likely repurposing drugs against CRC within each group. The functional changes of the co-upregulated genes in the first category were mainly associated with negative regulation of transforming growth factor β production and glomerular epithelial cell differentiation; whereas the co-downregulated genes were enriched in cotranslational protein targeting to the membrane. We identified 17 hub proteins across the co-upregulated genes and 18 hub proteins across the co-downregulated genes, composed of well-known TFs (MYC, TCF3, PML) and kinases (CSNK2A1, CDK1/4, MAPK14), and validated most of them using GEPIA2 and HPA, but also through two signature gene lists composed of the co-up and co-downregulated genes. We further identified a list of repurposing drugs that can potentially target the co-DEGs in CRC, including camptothecin, neostigmine bromide, emetine, remoxipride, cephaeline, thioridazine, and omeprazole. Similar analyses were performed in the co-DEG signatures in single-gene or drug perturbation experiments in CRC. MYC, PML, CDKs, CSNK2A1, and MAPKs were common hub proteins among all studies. Overall, we identified the critical genes in CRC and we propose repurposing drugs that could be used against them.

Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

BackgroundDNA variants in APOL1 associate with kidney disease, but the pathophysiologic mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types.MethodsWe performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-γ, or the combination of IFN-γ and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared with isogenic G0 controls.ResultsBoth G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-γ. scRNA-seq revealed cell type–specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids.ConclusionsSingle-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease.

Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

ABSTRACTBackgroundDNA variants in APOL1 associate with kidney disease, but the pathophysiological mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types.MethodsWe performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-γ, or the combination of IFN-γ and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared to isogenic G0 controls.ResultsBoth G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-γ. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids.ConclusionsSingle-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease.SIGNIFICANCE STATEMENTGaps persist in our mechanistic understanding of APOL1-mediated kidney disease. The authors apply genome-edited human kidney organoids, combined with single-cell transcriptomics, to profile APOL1 risk variants at the native genomic locus in different cell types. This approach captures interferon-mediated induction of APOL1 gene expression and reveals cellular dedifferentiation after a secondary insult of endoplasmic reticulum stress. This system provides a human cellular platform to interrogate complex mechanisms and human-specific regulators underlying APOL1-mediated kidney disease.

A small molecule screening to detect potential therapeutic targets in human podocytes

Widmeier E, Tan W, Airik M, Hildebrandt F. A small molecule screening to detect potential therapeutic targets in human podocytes. Am J Physiol Renal Physiol 312: F157–F171, 2017. First published October 19, 2016; doi:10.1152/ajprenal.00386.2016. Steroid-resistant nephrotic syndrome (SRNS) inevitably progresses to end-stage kidney disease, requiring dialysis or transplantation for survival. However, treatment modalities and drug discovery remain limited. Mutations in over 30 genes have been discovered as monogenic causes of SRNS. Most of these genes are predominantly expressed in the glomerular epithelial cell, the podocyte, placing it at the center of the pathogenesis of SRNS. Podocyte migration rate (PMR) represents a relevant intermediate phenotype of disease in monogenic causes of SRNS. We therefore adapted PMR in a high-throughput manner to screen small molecules as potential therapeutic targets for SRNS. We performed a high-throughput drug screening of a National Institutes of Health Clinical Collection (NCC) library ( n = 725 compounds) measuring PMR by videomicroscopy. We used the Woundmaker to perform individual 96-well scratch wounds and screened compounds using a quantitative kinetic live cell imaging migration assay using IncuCyte ZOOM technology. Using a normal distribution for the average PMR in wild-type podocytes with a vehicle control (DMSO), we applied a 90% confidence interval to define “distinct” compounds (5% faster/slower PMR) and found that 12 of 725 compounds (at 10 μM) reduced PMR. Clusters of drugs that alter PMR included actin/tubulin modulators such as the azole class of antifungals and antineoplastic vinca-alkaloids. We hereby identify compounds that alter PMR. The PMR assay provides a new avenue to test therapeutics for nephrotic syndrome. Positive results may reveal novel pathways in the study of glomerular diseases such as SRNS.

Role of the deubiquitinating enzyme ubiquitin-specific protease-14 in proteostasis in renal cells

Kidney cell injury may be associated with protein misfolding and induction of endoplasmic reticulum (ER) stress. Examples include complement-induced glomerular epithelial cell (GEC)/podocyte injury in membranous nephropathy and ischemia-reperfusion injury. Renal cell injury can also result from mutations in integral proteins, which lead to their misfolding and accumulation. Certain nephrin missense mutants misfold, accumulate in the ER, and induce ER stress. We examined if enhancement of ubiquitin-proteasome system function may facilitate proteostasis and confer protection against injury. Ubiquitin-specific protease 14 (Usp14) is reported to retard proteasomal protein degradation. Thus inhibition of Usp14 may enhance degradation of misfolded proteins and attenuate cell injury. In GEC, the reporter proteins GFPu (a “misfolded” protein) and CD3δ (an ER-associated degradation substrate) undergo time-dependent proteasomal degradation. Complement did not affect degradation of CD3δ-yellow fluorescent protein (YFP), but accelerated degradation of GFPu, and the Usp14-directed inhibitor IU1 further accelerated this degradation. Conversely, overexpression of Usp14 reduced degradation of GFPu and CD3δ-YFP. In 293T cells, IU1 did not enhance degradation of disease-associated nephrin missense mutants I171N and S724C, whereas overexpression of Usp14 reduced degradation. IU1 was cytoprotective after injury induced by the ER stressor tunicamycin and in vitro ischemia-reperfusion, but did not affect complement-induced cytotoxicity. In conclusion, Usp14 controls proteasomal degradation of some misfolded proteins. In addition, a Usp14-directed inhibitor reduces cytotoxicity in the context of global protein misfolding during certain types of renal cell injury.