Human Kidney Biopsy Single Cell Protocols and Analysis

Key Personnel

Project Description

RELATED DATA

Our partnership project aims to establish conditions for the transportation and storage of human kidney biopsies. These protocols will be optimized to ensure maximal cell viability and preservation of mRNA integrity. In the second phase of this project, we will compare single cell RNA sequencing technologies: FACS-seq and Drop-seq. Our goal is to determine which approach will be best adapted to the large-scale investigation of human kidney disease in large populations.

Publications

1. Abstract

   Translational profiles of medullary myofibroblasts during kidney fibrosis.

   Grgic, Ivica; Krautzberger, A. Michaela; Hofmeister, Andreas; Lalli, Matthew; DiRocco, Derek P.; Fleig, Susanne V.; Liu, Jing; Duffield, Jeremy S.; McMahon, Andrew P.; Aronow, Bruce; Humphreys, Benjamin D.. J Am Soc Nephrol . 25(9):1979–1990. September 2014.

   Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit protein under control of the collagen1alpha1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1alpha1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies.

2. Abstract

   Cell-specific translational profiling in acute kidney injury.

   Liu, Jing; Krautzberger, A. Michaela; Sui, Shannan H.; Hofmann, Oliver M.; Chen, Ying; Baetscher, Manfred; Grgic, Ivica; Kumar, Sanjeev; Humphreys, Benjamin D.; Hide, Winston A.; McMahon, Andrew P.. J Clin Invest . 124(3):1242–1254. March 2014.

   Acute kidney injury (AKI) promotes an abrupt loss of kidney function that results in substantial morbidity and mortality. Considerable effort has gone toward identification of diagnostic biomarkers and analysis of AKI-associated molecular events; however, most studies have adopted organ-wide approaches and have not elucidated the interplay among different cell types involved in AKI pathophysiology. To better characterize AKI-associated molecular and cellular events, we developed a mouse line that enables the identification of translational profiles in specific cell types. This strategy relies on CRE recombinase-dependent activation of an EGFP-tagged L10a ribosomal protein subunit, which allows translating ribosome affinity purification (TRAP) of mRNA populations in CRE-expressing cells. Combining this mouse line with cell type-specific CRE-driver lines, we identified distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Twenty-four hours following IRI, distinct translational signatures were identified in the nephron, kidney interstitial cell populations, vascular endothelium, and macrophages/monocytes. Furthermore, TRAP captured known IRI-associated markers, validating this approach. Biological function annotation, canonical pathway analysis, and in situ analysis of identified response genes provided insight into cell-specific injury signatures. Our study provides a deep, cell-based view of early injury-associated molecular events in AKI and documents a versatile, genetic tool to monitor cell-specific and temporal-specific biological processes in disease modeling.

3. Abstract

   Defining kidney biology to understand renal disease

   Little, MH; Brown, D.; Humphreys, BD; McMahon, AP; Miner, JH; Sands, JM; Weisz, OA; Mullins, C; Hoshizaki, D; Kidney Research National Dialogue, (KRND). Clin J Am Soc Nephrol . 9(4):809–11. April 2014.

   The Kidney Research National Dialogue represents a novel effort by the National Institute of Diabetes and Digestive and Kidney Diseases to solicit and prioritize research objectives from the renal research and clinical communities. The present commentary highlights selected scientific opportunities specific to the study of renal development, physiology, and cell biology. Describing such fundamental kidney biology serves as a necessary foundation for translational and clinical studies that will advance disease care and prevention. It is intended that these objectives foster and focus scientific efforts in these areas in the coming decade and beyond.

4. Lineage Tracing and Single-Nucleus Multiomics Reveal Novel Features of Adaptive and Maladaptive Repair after Acute Kidney Injury

   Gerhardt, Louisa M.S.; Koppitch, Kari; van Gestel, Jordi; Guo, Jinjin; Cho, Sam; Wu, Haojia; Kirita, Yuhei; Humphreys, Benjamin D.; McMahon, Andrew P.. Journal of the American Society of Nephrology . Publish Ahead of Print. January 2023.
5. Abstract

   Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing

   Li, Haikuo; Humphreys, Benjamin D.. STAR Protocols . 3(4):101904. 2022.

   Summary Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).1

6. Abstract

   Mapping the single-cell transcriptomic response of murine diabetic kidney disease to therapies

   Wu, Haojia; Villalobos, Romer Gonzalez; Yao, Xiang; Reilly, Dermot; Chen, Tao; Rankin, Matthew; Myshkin, Eugene; Breyer, Matthew D; Humphreys, Benjamin D. Cell Metabolism . 34(7):1064–1078.e6. July 2022.

   Diabetic kidney disease (DKD) occurs in ∼40% of patients with diabetes and causes kidney failure, cardiovascular disease, and premature death. We analyzed the response of a murine DKD model to five treatment regimens using single-cell RNA sequencing (scRNA-seq). Our atlas of ∼1 million cells revealed a heterogeneous response of all kidney cell types both to DKD and its treatment. Both monotherapy and combination therapies targeted differing cell types and induced distinct and non-overlapping transcriptional changes. The early effects of sodium-glucose cotransporter-2 inhibitors (SGLT2i) on the S1 segment of the proximal tubule suggest that this drug class induces fasting mimicry and hypoxia responses. Diabetes downregulated the spliceosome regulator serine/arginine-rich splicing factor 7 (Srsf7) in proximal tubule that was specifically rescued by SGLT2i. In vitro proximal tubule knockdown of Srsf7 induced a pro-inflammatory phenotype, implicating alternative splicing as a driver of DKD and suggesting SGLT2i regulation of proximal tubule alternative splicing as a potential mechanism of action for this drug class.

7. Abstract

   Kidney repair and regeneration: perspectives of the NIDDK (Re)Building a Kidney consortium

   Naved, Bilal A.; Bonventre, Joseph V.; Hubbell, Jeffrey A.; Hukriede, Neil A.; Humphreys, Benjamin D.; Kesselman, Carl; Valerius, M. Todd; McMahon, Andrew P.; Shankland, Stuart J.; Wertheim, Jason A.; White, Michael J.V.; de Caestecker, Mark P.; Drummond, Iain A. Kidney International . March 2022.

   Acute kidney injury impacts ∼13.3 million individuals and causes ∼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK’s open access data hub for accessing, downloading, and further analyzing data sets.

8. Abstract

   Regrow or Repair: An Update on Potential Regenerative Therapies for the Kidney

   Little, Melissa H.; Humphreys, Benjamin D. Journal of the American Society of Nephrology: JASN . 33(1):15–32. January 2022.

   Fifteen years ago, this journal published a review outlining future options for regenerating the kidney. At that time, stem cell populations were being identified in multiple tissues, the concept of stem cell recruitment to a site of injury was of great interest, and the possibility of postnatal renal stem cells was growing in momentum. Since that time, we have seen the advent of human induced pluripotent stem cells, substantial advances in our capacity to both sequence and edit the genome, global and spatial transcriptional analysis down to the single-cell level, and a pandemic that has challenged our delivery of health care to all. This article will look back over this period of time to see how our view of kidney development, disease, repair, and regeneration has changed and envision a future for kidney regeneration and repair over the next 15 years.

9. Abstract

   Spatially Resolved Transcriptomic Analysis of Acute Kidney Injury in a Female Murine Model

   Dixon, Eryn; Wu, Haojia; Muto, Yoshiharu; Wilson, Parker; Humphreys, Benjamin. Journal of the American Society of Nephrology . December 2021.

   Background Single cell sequencing technologies have advanced our understanding of kidney biology and disease but the loss of spatial information in these datasets hinders our interpretation of intercellular communication networks and regional gene expression patterns. New spatial transcriptomic sequencing platforms make it possible to measure the topography of gene expression at genome depth. Methods We optimized and validated a female bilateral ischemia reperfusion injury model. Using the 10X Genomics Visium Spatial Gene Expression solution, we generated spatial maps of gene expression across the injury and repair time course, and applied two open-source computational tools, Giotto and SPOTlight, to increase resolution and measure cell-cell interaction dynamics. Results An ischemia time of 34 minutes in a female murine model resulted in comparable injury to 22 minutes for males. We report a total of 16,856 unique genes mapped across injury and repair time course. Giotto, a computational toolbox for spatial data analysis, enabled increased resolution mapping of genes and cell types. Using a seeded non-negative matrix regression (SPOTlight) to deconvolute the dynamic landscape of cell-cell interactions, we find that injured proximal tubule cells are characterized by increasing macrophage and lymphocyte interactions even at 6 weeks after injury, potentially reflecting the AKI to CKD transition. Conclusions In this transcriptomic atlas, we defined region-specific and injury-induced loss of differentiation markers and their re-expression during repair, as well as region-specific injury and repair transcriptional responses. Lastly, we created a data visualization web application for the scientific community to explore these results (http://humphreyslab.com/SingleCell/; login: humphreyslab_visium password: irivisium).

10. Abstract

    Multi-omics integration in the age of million single-cell data

    Miao, Zhen; Humphreys, Benjamin D.; McMahon, Andrew P.; Kim, Junhyong. Nature Reviews Nephrology . 17(11):710–724. November 2021.

    An explosion in single-cell technologies has revealed a previously underappreciated heterogeneity of cell types and novel cell-state associations with sex, disease, development and other processes. Starting with transcriptome analyses, single-cell techniques have extended to multi-omics approaches and now enable the simultaneous measurement of data modalities and spatial cellular context. Data are now available for millions of cells, for whole-genome measurements and for multiple modalities. Although analyses of such multimodal datasets have the potential to provide new insights into biological processes that cannot be inferred with a single mode of assay, the integration of very large, complex, multimodal data into biological models and mechanisms represents a considerable challenge. An understanding of the principles of data integration and visualization methods is required to determine what methods are best applied to a particular single-cell dataset. Each class of method has advantages and pitfalls in terms of its ability to achieve various biological goals, including cell-type classification, regulatory network modelling and biological process inference. In choosing a data integration strategy, consideration must be given to whether the multi-omics data are matched (that is, measured on the same cell) or unmatched (that is, measured on different cells) and, more importantly, the overall modelling and visualization goals of the integrated analysis.

11. Abstract

    Single Cell Technologies: Beyond Microfluidics

    Li, Haikuo; Humphreys, Benjamin D. Kidney360 . July 2021.

    Single cell RNA-sequencing (scRNA-seq) has been widely adopted in recent years due to standardized protocols and automation, reliability and standardized bioinformatic pipelines. The most widely adopted platform is the 10X Genomics solution. While powerful, this system is also limited by its high cost, moderate throughput and the inability to customize due to fixed kit components. This review will cover new approaches that do not rely on microfluidics and thus have low entry costs, are highly customizable and are within the reach of any lab possessing molecular biology expertise.

12. Abstract

    Proximal Tubule Translational Profiling during Kidney Fibrosis Reveals Proinflammatory and Long Noncoding RNA Expression Patterns with Sexual Dimorphism

    Wu, Haojia; Lai, Chun-Fu; Chang-Panesso, Monica; Humphreys, Benjamin D. Journal of the American Society of Nephrology . September 2019.

    Having a comprehensive transcriptional profile of the proximal tubule in health and fibrosis would likely enhance understanding of fibrosis and perhaps help explain why CKD progresses more quickly in males versus females. To obtain a more complete picture of gene expression in the proximal tubule, the authors performed deep translational profiling of this segment in a mouse model of kidney fibrosis. Their findings demonstrate substantial sex differences in transcripts expressed in proximal tubule cells of males versus females, and indicate that the proximal tubule drives fibrosis through inflammatory and profibrotic paracrine signaling. The study also identified 439 long noncoding RNAs expressed in the proximal tubule, 143 of which undergo differential regulation in fibrosis, suggesting that this type of RNA has unanticipated regulatory roles kidney fibrosis.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.

13. Abstract

    Recent Insights into Kidney Injury and Repair from Transcriptomic Analyses.

    Kirita, Yuhei; Chang-Panesso, Monica; Humphreys, Benjamin D. Nephron . 21:1–4. May 2019.

    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.

14. Abstract

    Single Cell Transcriptomics and Solid Organ Transplantation.

    Malone, Andrew F.; Humphreys, Benjamin D. Transplantation . April 2019.

    Single cell RNA-sequencing (scRNA-seq) allows the measurement of transcriptomes from individual cells providing new insights into complex biological systems. scRNA-seq has enabled the identification of rare cell types, new cell states and intercellular communication networks that may be masked by traditional bulk transcriptional profiling. Researchers are increasingly using scRNA-seq to comprehensively characterize complex organs in health and disease. The diversity of immune cell types, some present at low frequency, in a transplanted organ undergoing rejection makes scRNA-seq ideally suited to characterize transplant pathologies because it can quantify subtle transcriptional differences between rare cell types. In this review we discuss single cell sequencing methods and their application in transplantation to date, current challenges and future directions. We believe that the remarkably rapid pace of technological development in this field makes it likely that single cell technologies such as scRNA-seq will have an impact in clinical transplantation within a decade.

15. Abstract

    Single-cell genomics and gene editing: implications for nephrology

    Wilson, Parker C.; Humphreys, Benjamin D. Nature Reviews Nephrology . 15(2):63–64. February 2019.

    Discoveries in 2018 using single-cell sequencing and gene-editing technologies have revealed their transformative potential for the investigation of kidney physiology and disease. Their promise is matched by the speed of their evolution.

16. Abstract

    Advantages of Single-Nucleus over Single-Cell RNA Sequencing of Adult Kidney: Rare Cell Types and Novel Cell States Revealed in Fibrosis.

    Wu, Haojia; Kirita, Yuhei; Donnelly, Erinn L.; Humphreys, Benjamin D. J Am Soc Nephrol . 30(1):23–32. January 2019.

    BACKGROUND: A challenge for single-cell genomic studies in kidney and other solid tissues is generating a high-quality single-cell suspension that contains rare or difficult-to-dissociate cell types and is free of both RNA degradation and artifactual transcriptional stress responses. METHODS: We compared single-cell RNA sequencing (scRNA-seq) using the DropSeq platform with single-nucleus RNA sequencing (snRNA-seq) using sNuc-DropSeq, DroNc-seq, and 10X Chromium platforms on adult mouse kidney. We validated snRNA-seq on fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery. RESULTS: A total of 11,391 transcriptomes were generated in the comparison phase. We identified ten clusters in the scRNA-seq dataset, but glomerular cell types were absent, and one cluster consisted primarily of artifactual dissociation-induced stress response genes. By contrast, snRNA-seq from all three platforms captured a diversity of kidney cell types that were not represented in the scRNA-seq dataset, including glomerular podocytes, mesangial cells, and endothelial cells. No stress response genes were detected. Our snRNA-seq protocol yielded 20-fold more podocytes compared with published scRNA-seq datasets (2.4% versus 0.12%, respectively). Unexpectedly, single-cell and single-nucleus platforms had equivalent gene detection sensitivity. For validation, analysis of frozen day 14 UUO kidney revealed rare juxtaglomerular cells, novel activated proximal tubule and fibroblast cell states, and previously unidentified tubulointerstitial signaling pathways. CONCLUSIONS: snRNA-seq achieves comparable gene detection to scRNA-seq in adult kidney, and it also has substantial advantages, including reduced dissociation bias, compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and successful performance on inflamed fibrotic kidney.

17. Abstract

    Kidney and organoid single-cell transcriptomics: the end of the beginning.

    Wilson, Parker C.; Humphreys, Benjamin D. Pediatr Nephrol . January 2019.

    Single-cell RNA sequencing (scRNA-seq) technologies are increasingly being applied to reveal cellular heterogeneity in kidney development and disease. In just the last year, multiple scRNA-seq datasets have been generated from kidney organoids, developing mouse and human kidney, adult kidney, and kidney cancer. The data generated enables a much deeper understanding of biological processes within and between cells. It has also elucidated unforeseen cell lineage relationships, defined the presence of off-target cell types in kidney organoids, and revealed a diverse inflammatory response in a human kidney allograft undergoing rejection. This review summarizes the recent rapid progress in scRNA-seq of the kidney and outlines future directions for single-cell technologies as applied to the kidney.

18. Abstract

    The Single Cell Transcriptomic Landscape of Early Human Diabetic Nephropathy

    Wilson, Parker C.; Wu, Haojia; Kirita, Yuhei; Uchimura, Kohei; Rennke, Helmut G.; Welling, Paul A.; Waikar, Sushrut S.; Humphreys, Benjamin D. bioRxiv . 2019.

    Diabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed unbiased single nucleus RNA sequencing (snRNAseq) on cryopreserved human diabetic kidney samples to generate 23,980 single nucleus transcriptomes from three control and three early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side by side comparison demonstrated cell-type-specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic loop of Henle, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na-K+-ATPase, WNK1, mineralocorticoid receptor and NEDD4L expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy.Significance Statement Single nucleus RNA sequencing revealed gene expression changes in early diabetic nephropathy that promote urinary potassium secretion and decreased calcium and magnesium reabsorption. Multiple cell types exhibited angiogenic signatures, which may represent early signs of aberrant angiogenesis. These alterations may help to identify biomarkers for disease progression or signaling pathways amenable to early intervention.

19. Abstract

    Comparative Analysis and Refinement of Human PSC-Derived Kidney Organoid Differentiation with Single-Cell Transcriptomics

    Wu, H; Uchimura, K; Donnelly, E.L.; Kirita, Y; Morris, S.A.; Humphreys, B.D. Cell Stem Cell . November 2018.

    Kidney organoids derived from human pluripotent stem cells have great utility for investigating organogenesis and disease mechanisms and, potentially, as a replacement tissue source, but how closely organoids derived from current protocols replicate adult human kidney is undefined. We compared two directed differentiation protocols by single-cell transcriptomics of 83,130 cells from 65 organoids with single-cell transcriptomes of fetal and adult kidney cells. Both protocols generate a diverse range of kidney cells with differing ratios, but organoid-derived cell types are immature, and 10%?20% of cells are non-renal. Reconstructing lineage relationships by pseudotemporal ordering identified ligands, receptors, and transcription factor networks associated with fate decisions. Brain-derived neurotrophic factor (BDNF) and its cognate receptor NTRK2 were expressed in the neuronal lineage during organoid differentiation. Inhibiting this pathway improved organoid formation by reducing neurons by 90% without affecting kidney differentiation, highlighting the power of single-cell technologies to characterize and improve organoid differentiation.

20. Abstract

    A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition

    Ó hAinmhire, E; Wu, H; Muto, Y; Donnelly, EL; Machado, FG; Fan, LX; Chang-Panesso, M; Humphreys, BD. Am. J. Physiol. Renal Physiol. . October 2018.

    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 (KG1) 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 TGFb. Co-culture of KG1 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 which potently inhibited TGFb-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 KG1 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.

21. Abstract

    FoxM1 drives proximal tubule proliferation during repair from acute kidney injury

    Chang-Panesso, Monica; Kadyrov, Farid F.; Lalli, Matthew; Wu, Haojia; Ikeda, Shiyo; Kobayashi, Akio; Humphreys, Benjamin D. bioRxiv . 2018.

    The proximal tubule has a remarkable capacity for repair after acute injury but the cellular lineage and molecular mechanisms underlying this repair response have been poorly characterized. Here, we developed a Kim-1-GFPCreERt2 knockin mouse line (Kim-1-GCE), performed genetic lineage analysis after injury and measured the cellular transcriptome of proximal tubule during repair. Acutely injured genetically labeled clones co-expressed Kim-1, Vimentin, Sox9 and Ki67, indicating a dedifferentiated and proliferative state. Clonal analysis revealed clonal expansion of Kim-1+ cells, indicating that acutely injured, dedifferentiated proximal tubule cells account for repair rather than a fixed tubular progenitor. 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, and was dependent on epidermal growth factor receptor (EGFR) stimulation. In conclusion, dedifferentiated proximal tubule cells effect proximal tubule repair and we reveal a novel EGFR-FoxM1-dependent signaling pathway that drives proliferative repair after injury.

22. Abstract

    Single-Cell Transcriptomics of a Human Kidney Allograft Biopsy Specimen Defines a Diverse Inflammatory Response

    Wu, H; Malone, AF; Donnelly, EL; Kirita, Y; Uchimura, K; Ramakrishnan, SM; Gaut, JP; Humphreys, BD. JASN . 29(8):2069–2080. May 2018.

    Background Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen. Methods We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection. Results Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16− group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database. Conclusions We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community.

23. Abstract

    Bringing Renal Biopsy Interpretation Into the Molecular Age With Single-Cell RNA Sequencing

    Malone, AF; Wu, H; Humphreys, BD. Semin Nephrol . January 2018.

    The renal biopsy provides critical diagnostic and prognostic information to clinicians including cases of acute kidney injury, chronic kidney disease, and allograft dysfunction. Today, biopsy specimens are read using a combination of light microscopy, electron microscopy, and indirect immunofluorescence, with a limited number of antibodies. These techniques all were perfected decades ago with only incremental changes since then. By contrast, recent advances in single-cell genomics are transforming scientists’ ability to characterize cells. Rather than measure the expression of several genes at a time by immunofluorescence, it now is possible to measure the expression of thousands of genes in thousands of single cells simultaneously. Here, we argue that the development of single-cell RNA sequencing offers an opportunity to describe human kidney disease comprehensively at a cellular level. It is particularly well suited for the analysis of immune cells, which are characterized by multiple subtypes and changing functions depending on their environment. In this review, we summarize the development of single-cell RNA sequencing methodologies. We discuss how these approaches are being applied in other organs, and the potential for this powerful technology to transform our understanding of kidney disease once applied to the renal biopsy.

24. Abstract

    The promise of single-cell RNA sequencing for kidney disease investigation

    Wu, H; Humphreys, Benjamin D. Kidney International . 92(6):1334–1342. December 2017.

    Recent techniques for single-cell RNA sequencing (scRNA-seq) at high throughput are leading to profound new discoveries in biology. The ability to generate vast amounts of transcriptomic data at cellular resolution represents a transformative advance, allowing the identification of novel cell types, states, and dynamics. In this review, we summarize the development of scRNA-seq methodologies and highlight their advantages and drawbacks. We discuss available software tools for analyzing scRNA-Seq data and summarize current computational challenges. Finally, we outline ways in which this powerful technology might be applied to discovery research in kidney development and disease.

25. Abstract

    (Re)Building a Kidney.

    Oxburgh, L; Carroll, TJ; Cleaver, O; Gossett, DR; Hoshizaki, DK; Hubbell, JA; Humphreys, BD; Jain, S; Jensen, J; Kaplan, DL; Kesselman, C; Ketchum, CJ; Little, MH; McMahon, AP; Shankland, SJ; Spence, JR; Valerius, MT; Wertheim, JA; Wessely, O; Zheng, Y; Drummond, IA. J Am Soc Nephrol . 28(5):1370–1378. May 2017.

    (Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.

26. Abstract

    Gli1+ Pericyte Loss Induces Capillary Rarefaction and Proximal Tubular Injury.

    Kramann, Rafael; Wongboonsin, Janewit; Chang-Panesso, Monica; Machado, Flavia G; Humphreys, Benjamin D. J Am Soc Nephrol . 2017.

    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-CreERt2; R26tdTomato reporter mice, we observed increased distance between Gli1+ pericytes and endothelial cells after AKI (mean6 SEM: 3.360.1 mm before injury versus 12.560.2 mm 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 (mean6SEM capillaries/high-power field: 67.664.7 in control versus 44.164.8 at 56 days; P,0.05) and increased cross-sectional area (mean6 SEM: 21.960.4 mm2 in control versus 24.160.6 mm2 at 10 days; P,0.01 and 24.66 0.6 mm2 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.

27. Abstract

    Comparative analysis of kidney organoid and adult human kidney single cell and single nucleus transcriptomes

    Wu, Haojia; Uchimura, Kohei; Donnelly, Erinn; Kirita, Yuhei; Morris, Samantha A; Humphreys, Benjamin D. bioRxiv . January 2017.

    Kidney organoids differentiated from human pluripotent stem cells hold great promise for understanding organogenesis, modeling disease and ultimately as a source of replacement tissue. Realizing the full potential of this technology will require better differentiation strategies based upon knowledge of the cellular diversity and differentiation state of all cells within these organoids. Here we analyze single cell gene expression in 45,227 cells isolated from 23 organoids differentiated using two different protocols. Both generate kidney organoids that contain a diverse range of kidney cells at differing ratios as well as non-renal cell types. We quantified the differentiation state of major organoid kidney cell types by comparing them against a 4,259 single nucleus RNA-seq dataset generated from adult human kidney, revealing immaturity of all kidney organoid cell types. We reconstructed lineage relationships during organoid differentiation through pseudotemporal ordering, and identified transcription factor networks associated with fate decisions. These results define impressive kidney organoid cell diversity, identify incomplete differentiation as a major roadblock for current directed differentiation protocols and provide a human adult kidney snRNA-seq dataset against which to benchmark future progress.

28. A Plumbing Solution for Stem Cell-Derived Kidneys

    Ó\hAinmhire, Eoghainín; Humphreys, Benjamin D. Transplantation . 100(1):3–4. January 2016.