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Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to kidney failure. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single-nucleus multimodal atlas of an orthologous mouse PKD model at early, mid, and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalog differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single-nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution., Competing Interests: Competing interests statement:B.D.H. is a consultant for Janssen Research & Development, LLC, Pfizer and Chinook Therapeutics. B.D.H holds equity in Chinook Therapeutics. B.D.H holds grant funding from Chinook Therapeutics and Janssen Research & Development, LLC. O.M.W has received grants from AstraZeneca unrelated to the current work.

Acute kidney injury (AKI) strongly upregulates the transcription factor Foxm1 in the proximal tubule in vivo, and Foxm1 drives epithelial proliferation in vitro. Here, we report that deletion of Foxm1 either with a nephron-specific Cre driver or by inducible global deletion reduced proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI to chronic kidney disease transition, with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report ERK mediated FOXM1 induction downstream of the EGFR in primary proximal tubule cells. We defined FOXM1 genomic binding sites by cleavage under targets and release using nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned data sets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identified 2 cis regulatory elements that bound FOXM1 and regulated CCNF expression, demonstrating that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK/FOXM1/CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to end-stage kidney disease. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single nucleus multimodal atlas of an orthologous mouse PKD model at early, mid and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalogue differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution., Competing Interests: Competing Interest Statement: B.D.H. is a consultant for Janssen Research & Development, LLC, Pfizer and Chinook Therapeutics, holds equity in Chinook Therapeutics and grant funding from Chinook Therapeutics and Janssen Research & Development, LLC. O.M.W has received grants from AstraZeneca unrelated to the current work. The remaining authors declare no competing interests.

Our aging population is growing and developing treatments for age-related diseases such as Alzheimer's and Parkinson's disease has taken on an increasing urgency and is accompanied by high public awareness. The already high and rising incidence of acute kidney injury (AKI) in the elderly, however, has received relatively little attention despite the potentially fatal outcomes associated with an episode of AKI in this age group. When discussing AKI and aging, one should consider two aspects: first, elderly patients have an increased susceptibility to an AKI episode, and second, they have decreased kidney repair after AKI given the high incidence of progression to chronic kidney disease (CKD). It is unclear if the same factors that drive the increased susceptibility to AKI could be playing a role in the decreased repair capacity or if they are totally different and unrelated. This review will examine current knowledge on the risk factors for the increased susceptibility to AKI in the elderly and will also explore potential aspects that might contribute to a decreased kidney repair response in this age group., (© 2021. IPNA.)

Background: Proximal tubule injury can initiate CKD, with progression rates that are approximately 50% faster in males versus females. The precise transcriptional changes in this nephron segment during fibrosis and potential differences between sexes remain undefined., Methods: We generated mice with proximal tubule-specific expression of an L10a ribosomal subunit protein fused with enhanced green fluorescent protein. We performed unilateral ureteral obstruction surgery on four male and three female mice to induce inflammation and fibrosis, collected proximal tubule-specific and bulk cortex mRNA at day 5 or 10, and sequenced samples to a depth of 30 million reads. We applied computational methods to identify sex-biased and shared molecular responses to fibrotic injury, including up- and downregulated long noncoding RNAs (lncRNAs) and transcriptional regulators, and used in situ hybridization to validate critical genes and pathways., Results: We identified >17,000 genes in each proximal tubule group, including 145 G-protein-coupled receptors. More than 700 transcripts were differentially expressed in the proximal tubule of males versus females. The >4000 genes displaying altered expression during fibrosis were enriched for proinflammatory and profibrotic pathways. Our identification of nearly 150 differentially expressed proximal tubule lncRNAs during fibrosis suggests they may have unanticipated regulatory roles. Network analysis prioritized proinflammatory and profibrotic transcription factors such as Irf1 , Nfkb1 , and Stat3 as drivers of fibrosis progression., Conclusions: This comprehensive transcriptomic map of the proximal tubule revealed sexually dimorphic gene expression that may reflect sex-related disparities in CKD, proinflammatory gene modules, and previously unappreciated proximal tubule-specific bidirectional lncRNA regulation., (Copyright © 2020 by the American Society of Nephrology.)

The proximal tubule has a remarkable capacity for repair after acute injury, but the cellular lineage and molecular mechanisms underlying this repair response are incompletely understood. Here, we developed a Kim1-GFPCreERt2 knockin mouse line (Kim1-GCE) in order to perform genetic lineage tracing of dedifferentiated cells while measuring the cellular transcriptome of proximal tubule during repair. Acutely injured genetically labeled clones coexpressed KIM1, VIMENTIN, SOX9, and KI67, indicating a dedifferentiated and proliferative state. Clonal analysis revealed clonal expansion of Kim1+ cells, indicating that acutely injured, dedifferentiated proximal tubule cells, rather than fixed tubular progenitor cells, account for repair. Translational profiling during injury and repair revealed signatures of both successful and unsuccessful maladaptive repair. The transcription factor Foxm1 was induced early in injury, was required for epithelial proliferation in vitro, and was dependent on epidermal growth factor receptor (EGFR) stimulation. In conclusion, dedifferentiated proximal tubule cells effect proximal tubule repair, and we reveal an EGFR/FOXM1-dependent signaling pathway that drives proliferative repair after injury.

Glioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-β (TGF-β). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-β-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.

Injured tubular epithelium exhibits cellular plasticity in that it can dedifferentiate, reenter the cell cycle, and subsequently either redifferentiate or adopt a chronically injured phenotype. Although some nephrogenic genes are reexpressed during injury and repair, developmental pathways are only partially recapitulated and the process is more accurately viewed as an entirely new program intrinsic to the regenerative response to injury. Recent advances in our understanding of the molecular circuitry underpinning epithelial plasticity have come from bulk, cell-specific, and single-cell transcriptomic analyses. These results have begun to define the signaling pathways and gene regulatory networks governing the epithelial injury response. In this review, we highlight recent transcriptomic analyses in kidney injury, repair and fibrosis, and outline the ways that these studies are improving our understanding of kidney regeneration., (© 2019 S. Karger AG, Basel.)

The homeobox transcription factor Meis1 is required for mammalian development, and its overexpression plays a role in tumorigenesis, especially leukemia. Meis1 is known to be expressed in kidney stroma, but its function in kidney is undefined. We hypothesized that Meis1 may regulate stromal cell proliferation in kidney development and disease and tested the hypothesis using cell lineage tracing and cell-specific Meis1 deletion in development, aging, and fibrotic disease. We observed strong expression of Meis1 in platelet-derived growth factor receptor-β-positive pericytes and perivascular fibroblasts, both in adult mouse kidney and to a lesser degree in human kidney. Either bilateral ischemia-reperfusion injury or aging itself led to strong upregulation of Meis1 protein and mRNA in kidney myofibroblasts, and genetic lineage analysis confirmed that Meis1-positive cells proliferate as they differentiate into myofibroblasts after injury. Conditional deletion of Meis1 in all kidney stroma with two separate tamoxifen-inducible Cre recombinase drivers had no phenotype with the exception of consistent induction of the tubular injury marker kidney injury molecule-1 (Kim-1) only in Meis1 mutants. Further examination of Kim-1 expression revealed linkage disequilibrium of Kim-1 and Meis1, such that Meis1 mutants carried the longer BALB/c Kim-1 allele. Unexpectedly, we report that this Kim-1 allele is expressed at baseline in wild-type BALB/c mice, without any associated abnormalities, including long-term fibrosis, as predicted from the literature. We conclude that Meis1 is specifically expressed in stroma and myofibroblasts of mouse and human kidney, that it is not required for kidney development, disease, or aging, and that BALB/c mice unexpectedly express Kim-1 protein at baseline without other kidney abnormality.

Peritubular capillary rarefaction is hypothesized to contribute to the increased risk of future CKD after AKI. Here, we directly tested the role of Gli1 + kidney pericytes in the maintenance of peritubular capillary health, and the consequences of pericyte loss during injury. Using bigenic Gli1-CreER t2 ; R26tdTomato reporter mice, we observed increased distance between Gli1 + pericytes and endothelial cells after AKI (mean±SEM: 3.3±0.1 µ m before injury versus 12.5±0.2 µ m after injury; P <0.001). Using a genetic ablation model, we asked whether pericyte loss alone is sufficient for capillary destabilization. Ten days after pericyte ablation, we observed endothelial cell damage by electron microscopy. Furthermore, pericyte loss led to significantly reduced capillary number at later time points (mean±SEM capillaries/high-power field: 67.6±4.7 in control versus 44.1±4.8 at 56 days; P <0.05) and increased cross-sectional area (mean±SEM: 21.9±0.4 µ m 2 in control versus 24.1±0.6 µ m 2 at 10 days; P <0.01 and 24.6±0.6 µ m 2 at 56 days; P <0.001). Pericyte ablation also led to hypoxic focal and subclinical tubular injury, reflected by transient expression of Kim1 and vimentin in scattered proximal tubule segments. This analysis provides direct evidence that AKI causes pericyte detachment from capillaries, and that pericyte loss is sufficient to trigger transient tubular injury and permanent peritubular capillary rarefaction., (Copyright © 2017 by the American Society of Nephrology.)

Terminally differentiated cells can be reprogrammed to pluripotency or directly to another differentiated cell type in vitro, a capacity termed cellular plasticity. Plasticity is not limited to in vitro manipulations but rather represents an important aspect of the regenerative response to injury in organs. Differentiated adult cells retain the capacity to dedifferentiate, adopting a progenitor-like phenotype after injury or, alternatively, to transdifferentiate, directly converting to a different mature cell type. Emerging concepts on cellular plasticity have relevance to our understanding of repair after kidney injury, including epithelial regeneration. Here we discuss work published in the past 5 years on the cellular hierarchies and mechanisms underlying kidney injury and repair, with a particular focus on potential roles for cellular plasticity in this response.

Mesenchymal stem cell (MSC)-like cells reside in the vascular wall, but their role in vascular regeneration and disease is poorly understood. Here, we show that Gli1 + cells located in the arterial adventitia are progenitors of vascular smooth muscle cells and contribute to neointima formation and repair after acute injury to the femoral artery. Genetic fate tracing indicates that adventitial Gli1 + MSC-like cells migrate into the media and neointima during athero- and arteriosclerosis in ApoE -/- mice with chronic kidney disease. Our data indicate that Gli1 + cells are a major source of osteoblast-like cells during calcification in the media and intima. Genetic ablation of Gli1 + cells before induction of kidney injury dramatically reduced the severity of vascular calcification. These findings implicate Gli1 + cells as critical adventitial progenitors in vascular remodeling after acute and during chronic injury and suggest that they may be relevant therapeutic targets for mitigation of vascular calcification., (Copyright © 2016 Elsevier Inc. All rights reserved.)