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.
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 (snRNA-seq) on cryopreserved human diabetic kidney samples to generate 23,980 single-nucleus transcriptomes from 3 control and 3 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 thick ascending limb, 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.
The integration of single cell transcriptome and chromatin accessibility datasets enables a deeper understanding of cell heterogeneity. We performed single nucleus ATAC (snATAC-seq) and RNA ...(snRNA-seq) sequencing to generate paired, cell-type-specific chromatin accessibility and transcriptional profiles of the adult human kidney. We demonstrate that snATAC-seq is comparable to snRNA-seq in the assignment of cell identity and can further refine our understanding of functional heterogeneity in the nephron. The majority of differentially accessible chromatin regions are localized to promoters and a significant proportion are closely associated with differentially expressed genes. Cell-type-specific enrichment of transcription factor binding motifs implicates the activation of NF-κB that promotes VCAM1 expression and drives transition between a subpopulation of proximal tubule epithelial cells. Our multi-omics approach improves the ability to detect unique cell states within the kidney and redefines cellular heterogeneity in the proximal tubule and thick ascending limb.
Acute kidney injury (AKI) is a serious complication, commonly occurring in the critically ill population, with devastating short- and long-term consequences. Despite standardization of the definition ...and staging of AKI, early recognition remains challenging given that serum creatinine level is a marker, albeit imperfect, of kidney function and not kidney injury. Furthermore, the delay in increase in serum creatinine level after loss of glomerular filtration also prevents timely detection of decreased kidney function in patients with AKI. During the past decade, numerous clinical investigations have evaluated the utility of several biomarkers in the early diagnosis and risk stratification of AKI. In 2014, the US Food and Drug Administration approved the marketing of a test based on the combination of urine concentrations of tissue inhibitor of metalloproteinase 2 and insulin-like growth factor binding protein 7 (TIMP-2 × IGFBP7) to determine whether certain critically ill patients are at risk for developing moderate to severe AKI. The optimal role of this biomarker in the diagnosis, management, and prognosis of AKI in different clinical settings requires further clarification. In this perspective, we summarize the biological actions of these 2 cell-cycle arrest biomarkers and present important considerations regarding the clinical application, interpretation, and limitations of this novel test for the early detection of AKI.
Maximizing the potential of human kidney organoids for drug testing and regenerative medicine and to model development and disease requires addressing cell immaturity, the lack of a mature collecting ...system, and off-target cell types. By independently generating two kidney progenitor cell populations—metanephric mesenchyme and ureteric bud (UB)-like cells—we could generate kidney organoids with a collecting system. We also identify the hormones aldosterone and arginine vasopressin (AVP) as critical to promote differentiation of collecting duct cell types including both principal cells (PCs) and intercalated cells (ICs). The resulting PCs express aquaporin-2 (AQP2) protein, which undergoes translocation to the apical membrane after vasopressin or forskolin stimulation. By single-cell RNA sequencing (scRNA-seq), we demonstrate improved proximal tubule maturation and reduced off-target cell populations. We also show appropriate downregulation of progenitor cell types, improved modeling of tubular injury, the presence of urothelium (Uro), and the ability of Notch pathway modulation to regulate PC:IC ratios during organoid development.
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•Combining differentiated progenitors leads to kidney organoids with collecting duct•Aldosterone and vasopressin drive principal and intercalated cell differentiation•Organoids show improved maturation and model tubular injury•Notch regulates principal:intercalated cell ratios
Uchimura et al. combine independently differentiated metanephric mesenchyme-like and ureteric bud-like progenitors to generate human kidney organoids with a collecting system. Hormones aldosterone and arginine vasopressin drive principal and intercalated cell maturation, and Notch signaling could regulate cell ratios. Organoids also showed improved maturation and injury modeling.
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.
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.
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.
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.
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.
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.
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•Two human kidney organoid protocols were compared by single-cell transcriptomics•Both protocols generated 10%–20% non-renal cells•Kidney organoid cells are immature compared with fetal and adult human kidney•Inhibiting BDNF-NTRK2 signaling reduces off-target cell types by 90%
A strong characterization of cell types, lineages, and differentiation states present in human PSC-derived kidney organoids is critical to improve differentiation protocols. Wu et al. use single-cell transcriptomics to reveal non-renal cell types, describe lineage-specific expression of regulatory genes, and report a broadly applicable strategy to reduce off-target cell populations.
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