Search

Significance Statement: Understanding the mechanisms underlying adaptive and maladaptive renal repair after AKI and their long-term consequences is critical to kidney health. The authors used lineage tracing of cycling cells and single-nucleus multiomics (profiling transcriptome and chromatin accessibility) after AKI. They demonstrated that AKI triggers a cell-cycle response in most epithelial and nonepithelial kidney cell types. They also showed that maladaptive proinflammatory proximal tubule cells (PTCs) persist until 6 months post-AKI, although they decreased in abundance over time, in part, through cell death. Single-nucleus multiomics of lineage-traced cells revealed regulatory features of adaptive and maladaptive repair. These included activation of cell state-specific transcription factors and cis-regulatory elements, and effects in PTCs even after adaptive repair, weeks after the injury event., Background: AKI triggers a proliferative response as part of an intrinsic cellular repair program, which can lead to adaptive renal repair, restoring kidney structure and function, or maladaptive repair with the persistence of injured proximal tubule cells (PTCs) and an altered kidney structure. However, the cellular and molecular understanding of these repair programs is limited., Methods: To examine chromatin and transcriptional responses in the same cell upon ischemia-reperfusion injury (IRI), we combined genetic fate mapping of cycling ( Ki67+ ) cells labeled early after IRI with single-nucleus multiomics-profiling transcriptome and chromatin accessibility in the same nucleus-and generated a dataset of 83,315 nuclei., Results: AKI triggered a broad cell cycle response preceded by cell type-specific and global transcriptional changes in the nephron, the collecting and vascular systems, and stromal and immune cell types. We observed a heterogeneous population of maladaptive PTCs throughout proximal tubule segments 6 months post-AKI, with a marked loss of maladaptive cells from 4 weeks to 6 months. Gene expression and chromatin accessibility profiling in the same nuclei highlighted differences between adaptive and maladaptive PTCs in the activity of cis-regulatory elements and transcription factors, accompanied by corresponding changes in target gene expression. Adaptive repair was associated with reduced expression of genes encoding transmembrane transport proteins essential to kidney function., Conclusions: Analysis of genome organization and gene activity with single-cell resolution using lineage tracing and single-nucleus multiomics offers new insight into the regulation of renal injury repair. Weeks to months after mild-to-moderate IRI, maladaptive PTCs persist with an aberrant epigenetic landscape, and PTCs exhibit an altered transcriptional profile even following adaptive repair., (Copyright © 2023 by the American Society of Nephrology.)

Background: Gdf15 encodes a TGF- β superfamily member that is rapidly activated in response to stress in multiple organ systems, including the kidney. However, there has been a lack of information about Gdf15 activity and effects in normal kidney and in AKI., Methods: We used genome editing to generate a Gdf15 nuGFP-CE mouse line, removing Gdf15 at the targeted allele, and enabling direct visualization and genetic modification of Gdf15 -expressing cells. We extensively mapped Gdf15 expression in the normal kidney and following bilateral ischemia-reperfusion injury, and quantified and compared renal responses to ischemia-reperfusion injury in the presence and absence of GDF15. In addition, we analyzed single nucleotide polymorphism association data for GDF15 for associations with patient kidney transplant outcomes., Results: Gdf15 is normally expressed within aquaporin 1-positive cells of the S3 segment of the proximal tubule, aquaporin 1-negative cells of the thin descending limb of the loop of Henle, and principal cells of the collecting system. Gdf15 is rapidly upregulated within a few hours of bilateral ischemia-reperfusion injury at these sites and new sites of proximal tubule injury. Deficiency of Gdf15 exacerbated acute tubular injury and enhanced inflammatory responses. Analysis of clinical transplantation data linked low circulating levels of GDF15 to an increased incidence of biopsy-proven acute rejection., Conclusions: Gdf15 contributes to an early acting, renoprotective injury response, modifying immune cell actions. The data support further investigation in clinical model systems of the potential benefit from GDF15 administration in situations in which some level of tubular injury is inevitable, such as following a kidney transplant., (Copyright © 2020 by the American Society of Nephrology.)

Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2 + nephron progenitor cells (NPCs) and Foxd1 + interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1 , were readily detected within SIX2 + NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2 + NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2 , are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs., (Copyright © 2018 by the American Society of Nephrology.)

The nephron is the functional unit of the kidney, but the mechanism of nephron formation during human development is unclear. We conducted a detailed analysis of nephron development in humans and mice by immunolabeling, and we compared human and mouse nephron patterning to describe conserved and divergent features. We created protein localization maps that highlight the emerging patterns along the proximal-distal axis of the developing nephron and benchmark expectations for localization of functionally important transcription factors, which revealed unanticipated cellular diversity. Moreover, we identified a novel nephron subdomain marked by Wnt4 expression that we fate-mapped to the proximal mature nephron. Significant conservation was observed between human and mouse patterning. We also determined the time at which markers for mature nephron cell types first emerge-critical data for the renal organoid field. These findings have conceptual implications for the evolutionary processes driving the diversity of mammalian organ systems. Furthermore, these findings provide practical insights beyond those gained with mouse and rat models that will guide in vitro efforts to harness the developmental programs necessary to build human kidney structures., (Copyright © 2018 by the American Society of Nephrology.)

Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species., (Copyright © 2018 by the American Society of Nephrology.)

(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., (Copyright © 2017 by the American Society of Nephrology.)

We previously described a mesenchymal stem cell (MSC)-like population within the adult mouse kidney that displays long-term colony-forming efficiency, clonogenicity, immunosuppression, and panmesodermal potential. Although phenotypically similar to bone marrow (BM)-MSCs, kidney MSC-like cells display a distinct expression profile. FACS sorting from Hoxb7/enhanced green fluorescent protein (GFP) mice identified the collecting duct as a source of kidney MSC-like cells, with these cells undergoing an epithelial-to-mesenchymal transition to form clonogenic, long-term, self-renewing MSC-like cells. Notably, after extensive passage, kidney MSC-like cells selectively integrated into the aquaporin 2-positive medullary collecting duct when microinjected into the kidneys of neonatal mice. No epithelial integration was observed after injection of BM-MSCs. Indeed, kidney MSC-like cells retained a capacity to form epithelial structures in vitro and in vivo, and conditioned media from these cells supported epithelial repair in vitro. To investigate the origin of kidney MSC-like cells, we further examined Hoxb7(+) fractions within the kidney across postnatal development, identifying a neonatal interstitial GFP(lo) (Hoxb7(lo)) population displaying an expression profile intermediate between epithelium and interstitium. Temporal analyses with Wnt4(GCE/+):R26(tdTomato/+) mice revealed evidence for the intercalation of a Wnt4-expressing interstitial population into the neonatal collecting duct, suggesting that such intercalation may represent a normal developmental mechanism giving rise to a distinct collecting duct subpopulation. These results extend previous observations of papillary stem cell activity and collecting duct plasticity and imply a role for such cells in collecting duct formation and, possibly, repair., (Copyright © 2015 by the American Society of Nephrology.)

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 collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-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., (Copyright © 2014 by the American Society of Nephrology.)

Injury to the adult kidney induces a number of developmental genes thought to regulate repair, including Wnt4. During kidney development, early nephron precursors and medullary stroma both express Wnt4, where it regulates epithelialization and controls smooth muscle fate, respectively. Expression patterns and roles for Wnt4 in the adult kidney, however, remain unclear. In this study, we used reporters, lineage analysis, and conditional knockout or activation of the Wnt/β-catenin pathway to investigate Wnt4 in the adult kidney. Proliferating, medullary, interstitial myofibroblasts strongly expressed Wnt4 during renal fibrosis, whereas tubule epithelia, except for the collecting duct, did not. Exogenous Wnt4 drove myofibroblast differentiation of a pericyte-like cell line, suggesting that Wnt4 might regulate pericyte-to-myofibroblast transition through autocrine signaling. However, conditional deletion of Wnt4 in interstitial cells did not reduce myofibroblast proliferation, cell number, or myofibroblast gene expression during fibrosis. Because the injured kidney expresses multiple Wnt ligands that might compensate for the absence of Wnt4, we generated a mouse model with constitutive activation of canonical Wnt/β-catenin signaling in interstitial pericytes and fibroblasts. Kidneys from these mice exhibited spontaneous myofibroblast differentiation in the absence of injury. Taken together, Wnt4 expression in renal fibrosis defines a population of proliferating medullary myofibroblasts. Although Wnt4 may be dispensable for myofibroblast transformation, canonical Wnt signaling through β-catenin stabilization is sufficient to drive spontaneous myofibroblast differentiation in interstitial pericytes and fibroblasts, emphasizing the importance of this pathway in renal fibrosis.

Formation of a functional renal network requires the interconnection of two epithelial tubes: the nephron, which arises from kidney mesenchyme, and the collecting system, which originates from the branching ureteric epithelium. How this connection occurs, however, is incompletely understood. Here, we used high-resolution image analysis in conjunction with genetic labeling of epithelia to visualize and characterize this process. Although the focal absence of basal lamina from renal vesicle stages ensures that both epithelial networks are closely apposed, we found that a patent luminal interconnection is not established until S-shaped body stages. Precursor cells of the distal nephron in the interconnection zone exhibit a characteristic morphology consisting of ill-defined epithelial junctional complexes but without expression of mesenchymal markers such as vimentin and Snai2. Live-cell imaging revealed that before luminal interconnection, distal cells break into the lumen of the collecting duct epithelium, suggesting that an invasive behavior is a key step in the interconnection process. Furthermore, loss of distal cell identity, which we induced by activating the Notch pathway, prevented luminal interconnection. Taken together, these data support a model in which establishing the distal identity of nephron precursor cells closest to the nascent collecting duct epithelium leads to an active cell invasion, which in turn contributes to a patent tubular interconnection between the nephron and collecting duct epithelia.

In late 2004, an International Consortium of research groups were charged with the task of producing a high-quality molecular anatomy of the developing mammalian urogenital tract (UGT). Given the importance of these organ systems for human health and reproduction, the need for a systematic molecular and cellular description of their developmental programs was deemed a high priority. The information obtained through this initiative is anticipated to enable the highest level of basic and clinical research grounded on a 21st-century view of the developing anatomy. There are three components to the Genitourinary Developmental Molecular Anatomy Project GUDMAP; all of these are intended to provide resources that support research on the kidney and UGT. The first provides ontology of the cell types during UGT development and the molecular hallmarks of those cells as discerned by a variety of procedures, including in situ hybridization, transcriptional profiling, and immunostaining. The second generates novel mouse strains. In these strains, cell types of particular interest within an organ are labeled through the introduction of a specific marker into the context of a gene that exhibits appropriate cell type or structure-specific expression. In addition, the targeting construct enables genetic manipulation within the cell of interest in many of the strains. Finally, the information is annotated, collated, and promptly released at regular intervals, before publication, through a database that is accessed through a Web portal. Presented here is a brief overview of the Genitourinary Developmental Molecular Anatomy Project effort.

Nearly 50 yr. ago, Clifford Grobstein made the observation that the ureteric bud induced the nephrogenic mesenchyme to undergo tubulogenesis. Since that discovery, scientists have attempted to characterize the molecular nature of the inducer. To date, no single molecule that is both necessary and sufficient for nephric induction has been identified. Because of recent insights regarding the role of several secreted molecules in tubulogenesis, it has become necessary to revise the classic model of metanephric induction. The studies of the classic ureteric inducer performed to date have most likely been characterizations of a mesenchyme-specific inducer, Wnt-4, and its role in tubulogenesis. Ureteric induction most likely involves a series of distinct events that provide proliferative, survival, and condensation signals to the mesenchyme, integrating the growth of the ureteric system with tubulogenesis.