An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

Acute kidney injury results in long-term alterations of kidney lymphatics in mice.

The long-term effects of a single episode of acute kidney injury (AKI) induced by bilateral ischemia-reperfusion injury (BIRI) on kidney lymphatic dynamics are not known. The purpose of this study was to determine if alterations in kidney lymphatics are sustained in the long term and how they relate to inflammation and injury. Mice underwent BIRI as a model of AKI and were followed up to 9 mo. Although kidney function markers normalized following initial injury, histological analysis revealed sustained tissue damage and inflammation for up to 9 mo. Transcriptional analysis showed both acute and late-stage lymphangiogenesis, supported by increased expression of lymphatic markers, with unique signatures at each phase. Expression of Ccl21a was distinctly upregulated during late-stage lymphangiogenesis. Three-dimensional tissue cytometry confirmed increased lymphatic vessel abundance, particularly in the renal cortex, at early and late timepoints postinjury. In addition, the study identified the formation of tertiary lymphoid structures composed of CCR7+ lymphocytes and observed changes in immune cell composition over time, suggesting a complex and dynamic response to AKI involving tissue remodeling and immune cell involvement. This study provides new insights into the role of lymphatics in the progression of AKI to chronic kidney disease.NEW & NOTEWORTHY Here, we perform the first, comprehensive study of long-term lymphatic dynamics following a single acute kidney injury (AKI) event. Using improved three-dimensional image analysis and an expanded panel of transcriptional markers, we identify multiple stages of lymphatic responses with distinct transcriptional signatures, associations with the immune microenvironment, and collagen deposition. This research advances kidney lymphatic biology, emphasizing the significance of longitudinal studies in understanding AKI and the transition to chronic kidney disease.

Integrated Cytometry With Machine Learning Applied to High-Content Imaging of Human Kidney Tissue for In Situ Cell Classification and Neighborhood Analysis.

The human kidney is a complex organ with various cell types that are intricately organized to perform key physiological functions and maintain homeostasis. New imaging modalities, such as mesoscale and highly multiplexed fluorescence microscopy, are increasingly being applied to human kidney tissue to create single-cell resolution data sets that are both spatially large and multidimensional. These single-cell resolution high-content imaging data sets have great potential to uncover the complex spatial organization and cellular makeup of the human kidney. Tissue cytometry is a novel approach used for the quantitative analysis of imaging data; however, the scale and complexity of such data sets pose unique challenges for processing and analysis. We have developed the Volumetric Tissue Exploration and Analysis (VTEA) software, a unique tool that integrates image processing, segmentation, and interactive cytometry analysis into a single framework on desktop computers. Supported by an extensible and open-source framework, VTEA's integrated pipeline now includes enhanced analytical tools, such as machine learning, data visualization, and neighborhood analyses, for hyperdimensional large-scale imaging data sets. These novel capabilities enable the analysis of mesoscale 2- and 3-dimensional multiplexed human kidney imaging data sets (such as co-detection by indexing and 3-dimensional confocal multiplexed fluorescence imaging). We demonstrate the utility of this approach in identifying cell subtypes in the kidney on the basis of labels, spatial association, and their microenvironment or neighborhood membership. VTEA provides an integrated and intuitive approach to decipher the cellular and spatial complexity of the human kidney and complements other transcriptomics and epigenetic efforts to define the landscape of kidney cell types.

VEGFR3 tyrosine kinase inhibition aggravates cisplatin nephrotoxicity.

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity.NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

Large-scale, three-dimensional tissue cytometry of the human kidney: a complete and accessible pipeline.

The advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell's physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ.

In Situ Classification of Cell Types in Human Kidney Tissue Using 3D Nuclear Staining.

To understand the physiology and pathology of disease, capturing the heterogeneity of cell types within their tissue environment is fundamental. In such an endeavor, the human kidney presents a formidable challenge because its complex organizational structure is tightly linked to key physiological functions. Advances in imaging-based cell classification may be limited by the need to incorporate specific markers that can link classification to function. Multiplex imaging can mitigate these limitations, but requires cumulative incorporation of markers, which may lead to tissue exhaustion. Furthermore, the application of such strategies in large scale 3-dimensional (3D) imaging is challenging. Here, we propose that 3D nuclear signatures from a DNA stain, DAPI, which could be incorporated in most experimental imaging, can be used for classifying cells in intact human kidney tissue. We developed an unsupervised approach that uses 3D tissue cytometry to generate a large training dataset of nuclei images (NephNuc), where each nucleus is associated with a cell type label. We then devised various supervised machine learning approaches for kidney cell classification and demonstrated that a deep learning approach outperforms classical machine learning or shape-based classifiers. Specifically, a custom 3D convolutional neural network (NephNet3D) trained on nuclei image volumes achieved a balanced accuracy of 80.26%. Importantly, integrating NephNet3D classification with tissue cytometry allowed in situ visualization of cell type classifications in kidney tissue. In conclusion, we present a tissue cytometry and deep learning approach for in situ classification of cell types in human kidney tissue using only a DNA stain. This methodology is generalizable to other tissues and has potential advantages on tissue economy and non-exhaustive classification of different cell types.

Clinical, histopathologic and molecular features of idiopathic and diabetic nodular mesangial sclerosis in humans.

BACKGROUND: Idiopathic nodular mesangial sclerosis, also called idiopathic nodular glomerulosclerosis (ING), is a rare clinical entity with an unclear pathogenesis. The hallmark of this disease is the presence of nodular mesangial sclerosis on histology without clinical evidence of diabetes mellitus or other predisposing diagnoses. To achieve insights into its pathogenesis, we queried the clinical, histopathologic and transcriptomic features of ING and nodular diabetic nephropathy (DN). METHODS: All renal biopsy reports accessioned at Indiana University Health from 2001 to 2016 were reviewed to identify 48 ING cases. Clinical and histopathologic features were compared between individuals with ING and DN (n = 751). Glomeruli of ING (n = 5), DN (n = 18) and reference (REF) nephrectomy (n = 9) samples were isolated by laser microdissection and RNA was sequenced. Immunohistochemistry of proline-rich 36 (PRR36) protein was performed. RESULTS: ING subjects were frequently hypertensive (95.8%) with a smoking history (66.7%). ING subjects were older, had lower proteinuria and had less hyaline arteriolosclerosis than DN subjects. Butanoate metabolism was an enriched pathway in ING samples compared with either REF or DN samples. The top differentially expressed gene, PRR36, had increased expression in glomeruli 248-fold [false discovery rate (FDR) P = 5.93 × 10-6] compared with the REF and increased 109-fold (FDR P = 1.85 × 10-6) compared with DN samples. Immunohistochemistry revealed a reduced proportion of cells with perinuclear reaction in ING samples as compared to DN. CONCLUSIONS: Despite similar clinical and histopathologic characteristics in ING and DN, the uncovered transcriptomic signature suggests that ING has distinct molecular features from nodular DN. Further study is warranted to understand these relationships.

Cigarette Smoking Impairs Adipose Stromal Cell Vasculogenic Activity and Abrogates Potency to Ameliorate Ischemia.

Cigarette smoking (CS) adversely affects the physiologic function of endothelial progenitor, hematopoietic stem and progenitor cells. However, the effect of CS on the ability of adipose stem/stromal cells (ASC) to promote vasculogenesis and rescue perfusion in the context of ischemia is unknown. To evaluate this, ASC from nonsmokers (nCS-ASC) and smokers (CS-ASC), and their activity to promote perfusion in hindlimb ischemia models, as well as endothelial cell (EC) survival and vascular morphogenesis in vitro were assessed. While nCS-ASC improved perfusion in ischemic limbs, CS-ASC completely lost this therapeutic effect. In vitro vasculogenesis assays revealed that human CS-ASC and ASC from CS-exposed mice showed compromised support of EC morphogenesis into vascular tubes, and the CS-ASC secretome was less potent in supporting EC survival/proliferation. Comparative secretome analysis revealed that CS-ASC produced lower amounts of hepatocyte growth factor (HGF) and stromal cell-derived growth factor 1 (SDF-1). Conversely, CS-ASC secreted the angiostatic/pro-inflammatory factor Activin A, which was not detected in nCS-ASC conditioned media (CM). Furthermore, higher Activin A levels were measured in EC/CS-ASC cocultures than in EC/nCS-ASC cocultures. CS-ASC also responded to inflammatory cytokines with 5.2-fold increase in Activin A secretion, whereas nCS-ASC showed minimal Activin A induction. Supplementation of EC/CS-ASC cocultures with nCS-ASC CM or with recombinant vascular endothelial growth factor, HGF, or SDF-1 did not rescue vasculogenesis, whereas inhibition of Activin A expression or activity improved network formation up to the level found in EC/nCS-ASC cocultures. In conclusion, ASC of CS individuals manifest compromised in vitro vasculogenic activity as well as in vivo therapeutic activity. Stem Cells 2018;36:856-867.

AMD3100 ameliorates cigarette smoke-induced emphysema-like manifestations in mice.

We have shown that cigarette smoke (CS)-induced pulmonary emphysema-like manifestations are preceded by marked suppression of the number and function of bone marrow hematopoietic progenitor cells (HPCs). To investigate whether a limited availability of HPCs may contribute to CS-induced lung injury, we used a Food and Drug Administration-approved antagonist of the interactions of stromal cell-derived factor 1 (SDF-1) with its chemokine receptor CXCR4 to promote intermittent HPC mobilization and tested its ability to limit emphysema-like injury following chronic CS. We administered AMD3100 (5mg/kg) to mice during a chronic CS exposure protocol of up to 24 wk. AMD3100 treatment did not affect either lung SDF-1 levels, which were reduced by CS, or lung inflammatory cell counts. However, AMD3100 markedly improved CS-induced bone marrow HPC suppression and significantly ameliorated emphysema-like end points, such as alveolar airspace size, lung volumes, and lung static compliance. These results suggest that antagonism of SDF-1 binding to CXCR4 is associated with protection of both bone marrow and lungs during chronic CS exposure, thus encouraging future studies of potential therapeutic benefit of AMD3100 in emphysema.

Human adipose-derived stem cells ameliorate cigarette smoke-induced murine myelosuppression via secretion of TSG-6.

OBJECTIVE: Bone marrow-derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose-derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. METHODS: C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA-transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony-forming unit assays. RESULTS: In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS-induced myelosuppression. This effect was specifically dependent on the anti-inflammatory factor TSG-6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. CONCLUSION: Our results suggest that systemic administration of hASC or TSG-6 may be novel approaches to reverse CS-induced myelosuppression.

Plasma proteomics of acute tubular injury.

The kidney tubules constitute two-thirds of the cells of the kidney and account for the majority of the organ's metabolic energy expenditure. Acute tubular injury (ATI) is observed across various types of kidney diseases and may significantly contribute to progression to kidney failure. Non-invasive biomarkers of ATI may allow for early detection and drug development. Using the SomaScan proteomics platform on 434 patients with biopsy-confirmed kidney disease, we here identify plasma biomarkers associated with ATI severity. We employ regional transcriptomics and proteomics, single-cell RNA sequencing, and pathway analysis to explore biomarker protein and gene expression and enriched biological pathways. Additionally, we examine ATI biomarker associations with acute kidney injury (AKI) in the Kidney Precision Medicine Project (KPMP) (n = 44), the Atherosclerosis Risk in Communities (ARIC) study (n = 4610), and the COVID-19 Host Response and Clinical Outcomes (CHROME) study (n = 268). Our findings indicate 156 plasma proteins significantly linked to ATI with osteopontin, macrophage mannose receptor 1, and tenascin C showing the strongest associations. Pathway analysis highlight immune regulation and organelle stress responses in ATI pathogenesis.

The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. Comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measure dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We establish a spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we note distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3, KLF6, and KLF10 regulates the transition between health and injury, while in thick ascending limb cells this transition is regulated by NR2F1. Further, combined perturbation of ELF3, KLF6, and KLF10 distinguishes two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Molecular Signatures of Glomerular Neovascularization in a Patient with Diabetic Kidney Disease.

The Kidney Precision Medicine Project (KPMP) aims to create a kidney tissue atlas, define disease subgroups, and identify critical cells, pathways, and targets for novel therapies through molecular investigation of human kidney biopsies obtained from participants with acute kidney injury (AKI) or chronic kidney disease (CKD). We present the case of a 66-year-old woman with diabetic kidney disease who underwent a protocol KPMP kidney biopsy. Her clinical history included diabetes mellitus complicated by neuropathy and eye disease, increased insulin resistance, hypertension, albuminuria, and relatively preserved glomerular filtration rate (early CKD stage 3a). The patient's histopathology was consistent with diabetic nephropathy and arterial and arteriolar sclerosis. Three-dimensional, immunofluorescence imaging of the kidney biopsy specimen revealed extensive peri-glomerular neovascularization that was underestimated by standard histopathologic approaches. Spatial transcriptomics was performed to obtain gene expression signatures at discrete areas of the kidney biopsy. Gene expression in the areas of glomerular neovascularization revealed increased expression of genes involved in angiogenic signaling, proliferation and survival of endothelial cells, as well as new vessel maturation and stability. This molecular correlation provides additional insights into the development of kidney disease in patients with diabetes and spotlights how novel molecular techniques employed by the KPMP can supplement and enrich the histopathologic diagnosis obtained from a kidney biopsy.

A spatially anchored transcriptomic atlas of the human kidney papilla identifies significant immune injury in patients with stone disease.

Kidney stone disease causes significant morbidity and increases health care utilization. In this work, we decipher the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease and healthy subjects. In addition to identifying cell types important in papillary physiology, we characterize collecting duct cell subtypes and an undifferentiated epithelial cell type that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncover a global injury signature characterized by immune activation, oxidative stress and extracellular matrix remodeling. We also identify the association of MMP7 and MMP9 expression with stone disease and mineral deposition, respectively. MMP7 and MMP9 are significantly increased in the urine of patients with CaOx stone disease, and their levels correlate with disease activity. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla and identify associated urinary biomarkers.

The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. However, comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measured dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We established a comprehensive and spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we noted distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3 , KLF6 , and KLF10 regulated the transition between health and injury, while in thick ascending limb cells this transition was regulated by NR2F1 . Further, combined perturbation of ELF3 , KLF6 , and KLF10 distinguished two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Tissue Cytometry With Machine Learning in Kidney: From Small Specimens to Big Data.

Advances in cellular and molecular interrogation of kidney tissue have ushered a new era of understanding the pathogenesis of kidney disease and potentially identifying molecular targets for therapeutic intervention. Classifying cells in situ and identifying subtypes and states induced by injury is a foundational task in this context. High resolution Imaging-based approaches such as large-scale fluorescence 3D imaging offer significant advantages because they allow preservation of tissue architecture and provide a definition of the spatial context of each cell. We recently described the Volumetric Tissue Exploration and Analysis cytometry tool which enables an interactive analysis, quantitation and semiautomated classification of labeled cells in 3D image volumes. We also established and demonstrated an imaging-based classification using deep learning of cells in intact tissue using 3D nuclear staining with 4',6-diamidino-2-phenylindole (DAPI). In this mini-review, we will discuss recent advancements in analyzing 3D imaging of kidney tissue, and how combining machine learning with cytometry is a powerful approach to leverage the depth of content provided by high resolution imaging into a highly informative analytical output. Therefore, imaging a small tissue specimen will yield big scale data that will enable cell classification in a spatial context and provide novel insights on pathological changes induced by kidney disease.

Alterations in Protein Translation and Carboxylic Acid Catabolic Processes in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease despite decades of study. Alterations in the glomerulus and kidney tubules both contribute to the pathogenesis of DKD although the majority of investigative efforts have focused on the glomerulus. We sought to examine the differential expression signature of human DKD in the glomerulus and proximal tubule and corroborate our findings in the db/db mouse model of diabetes. A transcriptogram network analysis of RNAseq data from laser microdissected (LMD) human glomerulus and proximal tubule of DKD and reference nephrectomy samples revealed enriched pathways including rhodopsin-like receptors, olfactory signaling, and ribosome (protein translation) in the proximal tubule of human DKD biopsy samples. The translation pathway was also enriched in the glomerulus. Increased translation in diabetic kidneys was validated using polyribosomal profiling in the db/db mouse model of diabetes. Using single nuclear RNA sequencing (snRNAseq) of kidneys from db/db mice, we prioritized additional pathways identified in human DKD. The top overlapping pathway identified in the murine snRNAseq proximal tubule clusters and the human LMD proximal tubule compartment was carboxylic acid catabolism. Using ultra-performance liquid chromatography-mass spectrometry, the fatty acid catabolism pathway was also found to be dysregulated in the db/db mouse model. The Acetyl-CoA metabolite was down-regulated in db/db mice, aligning with the human differential expression of the genes ACOX1 and ACACB. In summary, our findings demonstrate that proximal tubular alterations in protein translation and carboxylic acid catabolism are key features in both human and murine DKD.

A reference tissue atlas for the human kidney.

Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

Integration of spatial and single-cell transcriptomics localizes epithelial cell-immune cross-talk in kidney injury.

Single-cell sequencing studies have characterized the transcriptomic signature of cell types within the kidney. However, the spatial distribution of acute kidney injury (AKI) is regional and affects cells heterogeneously. We first optimized coordination of spatial transcriptomics and single-nuclear sequencing data sets, mapping 30 dominant cell types to a human nephrectomy. The predicted cell-type spots corresponded with the underlying histopathology. To study the implications of AKI on transcript expression, we then characterized the spatial transcriptomic signature of 2 murine AKI models: ischemia/reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were associated with injury pathways. Using single-cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. Neutrophils infiltrated the renal medulla in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubules. In the CLP model, infiltrating macrophages dominated the outer cortical signature, and Mdk was identified as a corresponding chemotactic factor. The regional distribution of these immune cells was validated with multiplexed CO-Detection by indEXing (CODEX) immunofluorescence. Spatial transcriptomic sequencing complemented single-cell sequencing by uncovering mechanisms driving immune cell infiltration and detection of relevant cell subpopulations.

Molecular characterization of the human kidney interstitium in health and disease.

The gene expression signature of the human kidney interstitium is incompletely understood. The cortical interstitium (excluding tubules, glomeruli, and vessels) in reference nephrectomies (N = 9) and diabetic kidney biopsy specimens (N = 6) was laser microdissected (LMD) and sequenced. Samples underwent RNA sequencing. Gene signatures were deconvolved using single nuclear RNA sequencing (snRNAseq) data derived from overlapping specimens. Interstitial LMD transcriptomics uncovered previously unidentified markers including KISS1, validated with in situ hybridization. LMD transcriptomics and snRNAseq revealed strong correlation of gene expression within corresponding kidney regions. Relevant enriched interstitial pathways included G-protein coupled receptor. binding and collagen biosynthesis. The diabetic interstitium was enriched for extracellular matrix organization and small-molecule catabolism. Cell type markers with unchanged expression (NOTCH3, EGFR, and HEG1) and those down-regulated in diabetic nephropathy (MYH11, LUM, and CCDC3) were identified. LMD transcriptomics complements snRNAseq; together, they facilitate mapping of interstitial marker genes to aid interpretation of pathophysiology in precision medicine studies.