Mesenchymal stem cells (MSCs) reside in the perivascular niche of many organs, including kidney, lung, liver, and heart, although their roles in these tissues are poorly understood. Here, we demonstrate that Gli1 marks perivascular MSC-like cells that substantially contribute to organ fibrosis. In vitro, Gli1(+) cells express typical MSC markers, exhibit trilineage differentiation capacity, and possess colony-forming activity, despite constituting a small fraction of the platelet-derived growth factor-β (PDGFRβ)(+) cell population. Genetic lineage tracing analysis demonstrates that tissue-resident, but not circulating, Gli1(+) cells proliferate after kidney, lung, liver, or heart injury to generate myofibroblasts. Genetic ablation of these cells substantially ameliorates kidney and heart fibrosis and preserves ejection fraction in a model of induced heart failure. These findings implicate perivascular Gli1(+) MSC-like cells as a major cellular origin of organ fibrosis and demonstrate that these cells may be a relevant therapeutic target to prevent solid organ dysfunction after injury.
The evolutionary conserved Wnt signaling transduction pathway plays essential roles in a wide array of biologic processes including embryonic development, branching morphogenesis, proliferation and carcinogenesis. Over the past ten years it has become increasingly clear that Wnt signaling also regulates the response of adult organs to disease processes, including kidney disease. This review will focus on the growing literature implicating important roles for Wnt signaling during disease in two separate kidney compartments: the tubular epithelium and the interstitium.
BACKGROUND: World Kidney Day 2013 focused on raising awareness of the impact and consequences of acute kidney injury (AKI). Although many studies have examined rates of AKI in hospitalized patients, we were interested in the impact of AKI on the workload of nephrologists. STUDY DESIGN: Cross-sectional forced-choice internet-based survey. SETTING & PARTICIPANTS: 598 survey respondents who were US-based nephrologist members of the American Society of Nephrology. OUTCOMES: Numbers of inpatients and outpatients seen on World Kidney Day 2013 for the management of AKI or other conditions (and specifically in-hospital renal replacement therapies [RRTs]), based on self-report of number/percentage of patients seen on World Kidney Day and in the prior year. RESULTS: Of 598 physician respondents (response rate, 12%), 310 saw patients in the hospital on World Kidney Day. Of 3,285 patients seen by respondents, 1,500 were seen for AKI (46%); 1,233, for end-stage renal disease (37%); and 552, for non-AKI/end-stage renal disease-related problems (17%). Of patients with AKI, 688 (46%) were in the intensive care unit and 415 (28%) received RRT. Intermittent hemodialysis was performed in 315 patients (76%) who received RRT. Delivered dialysis dose was quantified in only 48 (15%) of those receiving intermittent hemodialysis. 260 respondents saw 2,380 patients in the ambulatory setting, of whom 207 (9%) were seen for follow-up of AKI. LIMITATIONS: There was a low response rate to the survey. Numbers of patients were self-reported. CONCLUSIONS: This is the first physician survey examining the care of patients and impact of AKI on current in-hospital and ambulatory nephrology practices. In our sample, AKI was the most common reason for in-hospital nephrology consultation. Furthermore, our findings point to significant areas in which improvement is needed, including inadequate quantification of dialysis delivered dose. Finally, our survey highlights that AKI is a major public health issue.
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.
AKI predicts the future development of CKD, and one proposed mechanism for this epidemiologic link is loss of peritubular capillaries triggering chronic hypoxia. A precise definition of changes in peritubular perfusion would help test this hypothesis by more accurately correlating these changes with future loss of kidney function. Here, we have adapted and validated a fluorescence microangiography approach for use with mice to visualize, analyze, and quantitate peritubular capillary dynamics after AKI. A novel software-based approach enabled rapid and automated quantitation of capillary number, individual area, and perimeter. After validating perfusion in mice with genetically labeled endothelia, we compared peritubular capillary number and size after moderate AKI, characterized by complete renal recovery, and after severe AKI, characterized by development of interstitial fibrosis and CKD. Eight weeks after severe AKI, we measured a 40%±7.4% reduction in peritubular capillary number (P<0.05) and a 36%±4% decrease in individual capillary cross-sectional area (P<0.001) for a 62%±2.2% reduction in total peritubular perfusion (P<0.01). Whereas total peritubular perfusion and number of capillaries did not change, we detected a significant change of single capillary size following moderate AKI. The loss of peritubular capillary density and caliber at week 8 closely correlated with severity of kidney injury at day 1, suggesting irreparable microvascular damage. These findings emphasize a direct link between severity of acute injury and future loss of peritubular perfusion, demonstrate that reduced capillary caliber is an unappreciated long-term consequence of AKI, and offer a new quantitative imaging tool for understanding how AKI leads to future CKD in mouse models.
Identifying new biomarkers and therapeutic targets for podocytopathies such as focal segmental glomerulosclerosis (FSGS) requires a detailed analysis of transcriptional changes in podocytes over the course of disease. Here we used translating ribosome affinity purification (TRAP) to isolate and profile podocyte-specific mRNA in two different models of FSGS. We expressed enhanced green fluorescent protein-tagged to ribosomal protein L10a in podocytes under the control of the collagen-1α1 promoter, enabling one-step podocyte-specific mRNA isolation over the course of disease. This TRAP protocol robustly enriched known podocyte-specific mRNAs. We crossed Col1α1-eGFP-L10a mice with the Actn4(-/-) and Actn4(+/K256E) models of FSGS and analyzed podocyte transcriptional profiles at 2, 6, and 44 weeks of age. Two upregulated podocyte genes in murine FSGS (CXCL1 and DMPK) were found to be upregulated at the protein level in biopsies from patients with FSGS, validating this approach. There was no dilution of podocyte-specific transcripts during disease. These are the first podocyte-specific RNA expression data sets during aging and in two models of FSGS. This approach identified new podocyte proteins that are upregulated in FSGS and defines novel biomarkers and therapeutic targets for human glomerular disease.Kidney International advance online publication, 18 June 2014; doi:10.1038/ki.2014.204.
Fibrinogen (Fg) has been implicated in the pathogenesis of several fibrotic disorders by acting as profibrotic ligand for a variety of cellular surface receptors and by modulating the provisional fibrin matrix formed after injury. We demonstrate increased renal Fg expression following unilateral ureteral obstruction and folic acid (FA) nephropathy in mice respectively. Urinary Fg excretion was also increased in the FA nephropathy. Using in vitro and in vivo approaches, our results suggest that IL-6 mediates STAT3 activation in kidney fibrosis and phosphorylated STAT3 binds to Fgα, Fgβ and Fgγ promoters in the kidney to regulate their transcription. Genetically modified Fg heterozygote mice (~75% of normal plasma Fg levels) exhibited only 3% kidney interstitial fibrosis and tubular atrophy following FA nephropathy compared to 24% for wild type. Fibrinogenolysis through Ancrod following FA reduced interstitial fibrosis more than 3-fold compared to vehicle treated controls. Mechanistically, Fg seems to act synergistically with TGF-β1 to induce fibroblast proliferation and activates TGF-β1/pSMAD2 signaling. This study offers increased understanding of Fg expression and molecular interaction with TGF-β1 in progression to kidney fibrosis and, importantly, indicates that fibrinogenolytics like Ancrod present a treatment opportunity for a yet intractable disease.
Renal pericytes have been neglected for many years, but recently they have become an intensively studied cell population in renal biology and pathophysiology. Pericytes are stromal cells that support vasculature, and a subset of pericytes are mesenchymal stem cells. In kidney, pericytes have been reported to play critical roles in angiogenesis, regulation of renal medullary and cortical blood flow, and serve as progenitors of interstitial myofibroblasts in renal fibrogenesis. They interact with endothelial cells through distinct signaling pathways and their activation and detachment from capillaries after acute or chronic kidney injury may be critical for driving chronic kidney disease progression. By contrast, during kidney homeostasis it is likely that pericytes serve as a local stem cell population that replenishes differentiated interstitial and vascular cells lost during aging. This review describes both the regenerative properties of pericytes as well as involvement in pathophysiologic conditions such as fibrogenesis.
In recent years it has become clear that most organs and tissues, including kidney, contain resident stem/progenitor cells. Stem cells are undifferentiated, long-lived cells that are unique in their ability to produce differentiated daughter cells and to retain their stem cell identity by self-renewal. A primary goal of this meeting was to review the current understanding of kidney stem cells and mechanisms of kidney regeneration in both lower vertebrates and mammals. Presenters covered a broad range of topics including stem cell quiescence, epigenetics, transcriptional control circuits, dedifferentiation, pluripotent stem cells, renal progenitors, and novel imaging approaches in kidney regeneration. By the end of this highly interactive conference it was clear we are entering into very exciting times for regenerative medicine and the kidney.
Whether kidney proximal tubule harbors a scattered population of epithelial stem cells is a major unsolved question. Lineage-tracing studies, histologic characterization, and ex vivo functional analysis results conflict. To address this controversy, we analyzed the lineage and clonal behavior of fully differentiated proximal tubule epithelial cells after injury. A CreER(T2) cassette was knocked into the sodium-dependent inorganic phosphate transporter SLC34a1 locus, which is expressed only in differentiated proximal tubule. Tamoxifen-dependent recombination was absolutely specific to proximal tubule. Clonal analysis after injury and repair showed that the bulk of labeled cells proliferate after injury with increased clone size after severe compared with mild injury. Injury to labeled proximal tubule epithelia induced expression of CD24, CD133, vimentin, and kidney-injury molecule-1, markers of putative epithelial stem cells in the human kidney. Similar results were observed in cultured proximal tubules, in which labeled clones proliferated and expressed dedifferentiation and injury markers. When mice with completely labeled kidneys were subject to injury and repair there was no dilution of fate marker despite substantial proliferation, indicating that unlabeled progenitors do not contribute to kidney repair. During nephrogenesis and early kidney growth, single proximal tubule clones expanded, suggesting that differentiated cells also contribute to tubule elongation. These findings provide no evidence for an intratubular stem-cell population, but rather indicate that terminally differentiated epithelia reexpress apparent stem-cell markers during injury-induced dedifferentiation and repair.
PURPOSE OF REVIEW: The review summarizes the most recent advances in stem cell and regenerative approaches to treat kidney injury, and highlights areas of active controversy. Over the past year, a number of findings have been reported that have brought this field much closer to clinical translation. RECENT FINDINGS: Recent progress in regenerative nephrology includes the directed differentiation of embryonic stem cells to kidney fates, understanding the proliferative capacity of tubules after injury, the use of mesenchymal stem cells for kidney disease and tissue engineering approaches to renal replacement. Controversies persist, however, including whether adult epithelial stem cells exist at all, the best therapeutic strategy for the treatment of kidney injury and how to use mesenchymal stem cells optimally for the prevention of acute kidney injury. SUMMARY: Although recent progress in kidney regeneration is very encouraging, current controversies must be resolved before clinical breakthroughs can occur.
Generation of differentiated kidney cell types from pluripotent stem cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. In three recent reports, mature kidney cell types and three-dimensional nephron-like structures were generated from pluripotent cells rapidly and efficiently. A detailed understanding of the signals that drive nephrogenesis proved critical for these achievements.
Acute kidney injury (AKI) is common and urgently requires new preventative therapies. Expression of a cyclin-dependent kinase (CDK) inhibitor transgene protects against AKI, suggesting that manipulating the tubular epithelial cell cycle may be a viable therapeutic strategy. Broad spectrum small molecule CDK inhibitors are protective in some kidney injury models, but these have toxicities and epithelial proliferation is eventually required for renal repair. Here, we tested a well-tolerated, novel and specific small molecule inhibitor of CDK4 and CDK6, PD 0332991, to investigate the effects of transient cell cycle inhibition on epithelial survival in vitro and kidney injury in vivo. We report that CDK4/6 inhibition induced G0/G1 cycle arrest in cultured human renal proximal tubule cells (hRPTC) at baseline and after injury. Induction of transient G0/G1 cycle arrest through CDK4/6 inhibition protected hRPTC from DNA damage and caspase 3/7 activation following exposure to the nephrotoxins cisplatin, etoposide, and antimycin A. In vivo, mice treated with PD 0332991 before ischemia-reperfusion injury (IRI) exhibited dramatically reduced epithelial progression through S phase 24 h after IRI. Despite reduced epithelial proliferation, PD 0332991 ameliorated kidney injury as reflected by improved serum creatinine and blood urea nitrogen levels 24 h after injury. Inflammatory markers and macrophage infiltration were significantly decreased in injured kidneys 3 days following IRI. These results indicate that induction of proximal tubule cell cycle arrest with specific CDK4/6 inhibitors, or "pharmacological quiescence," represents a novel strategy to prevent AKI.
The kidney possesses profound regenerative potential and in some cases can recover completely 'restitutio at integrum' following an acute kidney injury (AKI). Emerging evidence strongly suggests that sometimes repair is incomplete, however, and, in this situation, an episode of AKI leads to future chronic kidney disease (CKD). Understanding the tubular response after AKI will shed light on the relationship between incomplete repair and future risk of CKD. The first repair phase after AKI is characterized by robust proliferation of epithelial cells in the proximal tubule. The exact source of these proliferating cells has been a source of controversy for the last decade. While nearly everyone now agrees that reparative cells arise within the proximal tubule, there is disagreement about whether all surviving cells possess an equivalent repair capacity through dedifferentiation, or alternatively whether a pre-existing intratubular stem cell population [so-called scattered tubular cells (STC)] is responsible for repair. This review will summarize the evidence on both sides of this issue and will discuss very recent genetic fate-tracing data that strongly points against the existence of intratubular stem cells but rather indicates that terminally differentiated proximal tubule epithelial cells undergo dedifferentiation upon injury to replace lost neighboring tubular epithelial cells through proliferative self-duplication. This new evidence includes data clearly indicating that STC are not committed tubular stem cells but instead represent individual dedifferentiated tubular epithelial cells that transiently express putative stem cell markers.
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.