ABSTRACT
Studies in animal models suggest a linkage between inflammatory response to injury and subsequent nephron loss during acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Failure of normal repair during CKD transition correlates with de novo expression of vascular cell adhesion protein-1 (VCAM-1) by a subset of injured proximal tubule cells. This study identifies the role of VCAM-1 expression in promoting the failed repair state. Single-cell transcriptome analysis of patients with AKI and CKD, and whole kidney RNA and protein analyses of mouse models of CKD, confirmed a marked increase of VCAM-1 expression in the proximal tubules of injured kidneys. In immortalized mouse proximal tubular (MPT) cells and primary cultured renal cells (PCRCs), VCAM-1 expression was induced by proinflammatory cytokines including TNFα and IL-1ß. Analyses of bulk RNA sequencing of TNFα-treated PCRCs or pseudo-bulk RNA sequencing of biopsies from the Kidney Precision Medicine Project (KPMP) datasets indicated activation of NF-κB and an enrichment of inflammatory response and cell adhesion pathways in VCAM-1-positive cells. Pharmacologic inhibition of NF-κB signaling or genetic deletion of myeloid differentiation factor 88 (Myd88) and TIR-domain-containing adapter-inducing interferon-ß (Trif) suppressed TNFα- and IL-1ß-induced VCAM-1 expression in vitro. TNFα stimulation or overexpression of VCAM-1 significantly increased splenocyte adhesion to the MPT monolayer in culture. These results demonstrate that persistence of proinflammatory cytokines after AKI can induce NF-κB-dependent VCAM-1 expression by proximal tubule cells, mediating increased immune cell adhesion to the tubule and thus promoting further tubule injury and greater risk of progression from AKI to CKD.
ABSTRACT
Incomplete repair after acute kidney injury can lead to development of chronic kidney disease. To define the mechanism of this response, we compared mice subjected to identical unilateral ischemia-reperfusion kidney injury with either contralateral nephrectomy (where tubule repair predominates) or contralateral kidney intact (where tubule atrophy predominates). By day 14, the kidneys undergoing atrophy had more macrophages with higher expression of chemokines, correlating with a second wave of proinflammatory neutrophil and T cell recruitment accompanied by increased expression of tubular injury genes and a decreased proportion of differentiated tubules. Depletion of neutrophils and T cells after day 5 reduced tubular cell loss and associated kidney atrophy. In kidney biopsies from patients with acute kidney injury, T cell and neutrophil numbers negatively correlated with recovery of estimated glomerular filtration rate. Together, our findings demonstrate that macrophage persistence after injury promotes a T cell- and neutrophil-mediated proinflammatory milieu and progressive tubule damage.
Subject(s)
Acute Kidney Injury , Renal Insufficiency, Chronic , Reperfusion Injury , Acute Kidney Injury/pathology , Animals , Atrophy/pathology , Kidney/metabolism , Kidney Tubules/pathology , Mice , Renal Insufficiency, Chronic/pathology , Reperfusion Injury/pathologyABSTRACT
BACKGROUND: After kidney injury, macrophages transition from initial proinflammatory activation to a proreparative phenotype characterized by expression of arginase-1 (Arg1), mannose receptor 1 (Mrc1), and macrophage scavenger receptor 1 (Msr1). The mechanism by which these alternatively activated macrophages promote repair is unknown. METHODS: We characterized the macrophage and renal responses after ischemia-reperfusion injury with contralateral nephrectomy in LysM-Cre;Arg1fl/fl mice and littermate controls and used in vitro coculture of macrophages and tubular cells to determine how macrophage-expressed arginase-1 promotes kidney repair. RESULTS: After ischemia-reperfusion injury with contralateral nephrectomy, Arg1-expressing macrophages were almost exclusively located in the outer stripe of the medulla adjacent to injured S3 tubule segments containing luminal debris or casts. Macrophage Arg1 expression was reduced by more than 90% in injured LysM-Cre;Arg1fl/fl mice, resulting in decreased mouse survival, decreased renal tubular cell proliferation and decreased renal repair compared with littermate controls. In vitro studies demonstrate that tubular cells exposed apically to dead cell debris secrete high levels of GM-CSF and induce reparative macrophage activation, with those macrophages in turn secreting Arg1-dependent factor(s) that directly stimulate tubular cell proliferation. CONCLUSIONS: GM-CSF-induced, proreparative macrophages express arginase-1, which is required for the S3 tubular cell proliferative response that promotes renal repair after ischemia-reperfusion injury.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , Reperfusion Injury , Animals , Arginase/genetics , Arginase/metabolism , Macrophages/physiology , Mice , Mice, Inbred C57BL , Regeneration , Reperfusion Injury/metabolismABSTRACT
Renal tubular casts originating from detached epithelial cells after ischemia-reperfusion injury (IRI) can obstruct tubules and negatively impact glomerular filtration rate. Using multiphoton imaging of 400-µm-thick kidney sections, the distribution of casts and morphometric measurement of tubules was performed along the entire nephron for the first time. Tubular nuclei are shed before cell detachment, and visually occlusive casts (grade 3) appeared at 12 h after IRI at the S3/thin descending limb (tDL) junction. Grade 3 casts peaked at 24 h after injury [present in 99% of S3, 78% of tDL, 76% of thin ascending limb (tAL), 60% of medullary thick ascending limb (mTAL), and 10% of connecting tubule segments]. Cast formation in the S3 correlated with selective loss of cell numbers from this tubule segment. By day 3, most mTALs and connecting tubules were cast free, whereas 72% of S3 tubules and 58% of tDLs still contained grade 3 casts. Although bulk phagocytosis of cast material by surviving tubular cells was not observed, mass spectrometry identified large numbers of tryptic peptides in the outer medulla, and trypsin levels were significantly increased in the kidney and urine 24 h after IRI. Administration of either antipain or camostat to inhibit trypsin extended cast burden to the S2, led to sustained accumulation of S3 casts after IRI, but did not affect cast burden in the mTAL or renal function. Our data provide detailed and dynamic mapping of tubular cast formation and resolution after IRI that can inform future interventions to accelerate cast clearance and renal recovery.NEW & NOTEWORTHY This detailed characterization of the dynamic distribution of dead cell debris in ischemically injured kidney tubules reveals which cells in the kidney are most severely injured, when and where tubular casts form, and when (and to a lesser extent, how) they are cleared.
Subject(s)
Nephrons , Reperfusion Injury , Glomerular Filtration Rate , Humans , Kidney , Kidney TubulesABSTRACT
Biomedical research often requires primary cultures of specific cell types, which are challenging to obtain at high purity in a reproducible manner. Here we engineered the murine Rosa26 locus by introducing the diphtheria toxin receptor flanked by loxP sites. The resultant strain was nicknamed the Terminator mouse. This approach results in diphtheria toxin-receptor expression in all non-Cre expressing cell types, making these cells susceptible to diphtheria toxin exposure. In primary cultures of kidney cells derived from the Terminator mouse, over 99.99% of cells were dead within 72 h of diphtheria toxin treatment. After crossing the Terminator with the podocin-Cre (podocyte specific) mouse or the Ggt-Cre (proximal tubule specific) mouse, diphtheria toxin treatment killed non-Cre expressing cells but spared podocytes and proximal tubule cells, respectively, enriching the primary cultures to over 99% purity, based on both western blotting and immunostaining of marker proteins. Thus, the Terminator mouse can be a useful tool to selectively and reproducibly obtain even low-abundant cell types at high quantity and purity.
Subject(s)
Cell Lineage , Intercellular Signaling Peptides and Proteins/metabolism , Kidney Tubules, Proximal/metabolism , Podocytes/metabolism , Animals , Biomarkers/metabolism , Blotting, Western , Cell Separation , Cell Survival , Diphtheria Toxin/pharmacology , Genotype , Heparin-binding EGF-like Growth Factor , Immunohistochemistry , Integrases/genetics , Intercellular Signaling Peptides and Proteins/agonists , Intercellular Signaling Peptides and Proteins/genetics , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , Podocytes/drug effects , Podocytes/pathology , Primary Cell Culture , RNA, Untranslated/genetics , Time FactorsABSTRACT
Developing and adult ureters express the epigenetic regulator Brg1, but the role of Brg1 in ureter development is not well understood. We conditionally ablated Brg1 in the developing ureter using Hoxb7-Cre and found that Brg1 expression is upstream of p63, Pparγ, and sonic hedgehog (Shh) expression in the ureteral epithelium. In addition, epithelial stratification in the basal cells required Brg1-dependent p63 expression, whereas terminal differentiation of the umbrella cells required Brg1-dependent Pparγ expression. Furthermore, the loss of ureteric Brg1 resulted in failure of Shh expression, which correlated with reduced smooth muscle cell development and hydroureter. Taken together, we conclude that Brg1 expression unifies three aspects of ureter development: maintenance of the basal cell population, guidance for terminal differentiation of urothelial cells, and proper investment of ureteral smooth muscle cells.
Subject(s)
DNA Helicases/metabolism , Hedgehog Proteins/metabolism , Nuclear Proteins/metabolism , PPAR gamma/metabolism , Phosphoproteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Ureter/growth & development , Urothelium/metabolism , Animals , DNA Helicases/genetics , Mice , Mice, Knockout , Nuclear Proteins/genetics , Phosphoproteins/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Ureter/metabolism , Urothelium/cytologyABSTRACT
Although enhanced activation of the EGF receptor (EGFR) associates with the development and progression of renal fibrosis, the mechanisms linking these observations are not completely understood. Here, after unilateral ureteral obstruction (UUO), wild-type mice exhibited sustained EGFR phosphorylation in the kidney and developed renal fibrosis that was more severe than the renal fibrosis observed in waved-2 mice, which have reduced EGFR tyrosine kinase activity. Waved-2 mice also showed fewer renal tubular cells arrested at G2/M, reduced expression of α-smooth muscle actin (α-SMA), downregulation of multiple genes encoding profibrogenic cytokines, including TGF-ß1, and dephosphorylation of Smad3, STAT3, and ERK1/2. Administration of the specific EGFR inhibitor gefitinib recapitulated this phenotype in wild-type mice after UUO. Furthermore, inactivation of either EGFR or STAT3 reduced UUO-induced expression of lipocalin-2, a molecule associated with the pathogenesis of CKD. In cultured renal interstitial fibroblasts, inhibition of EGFR also abrogated TGF-ß1- or serum-induced phosphorylation of EGFR, STAT3, ERK1/2, and Smad3 as well as expression of α-SMA and extracelluar matrix proteins. Taken together, these data suggest that EGFR may mediate renal fibrogenesis by promoting transition of renal epithelial cells to a profibrotic phenotype, increased production of inflammatory factors, and activation of renal interstitial fibroblasts. Inhibition of EGFR may have therapeutic potential for fibrotic kidney disease.
Subject(s)
ErbB Receptors/antagonists & inhibitors , Kidney/pathology , Animals , Cell Cycle Checkpoints , Cytokines/biosynthesis , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibrosis , Lipocalins/analysis , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Phosphorylation , Smad3 Protein/physiology , Transforming Growth Factor beta1/physiology , Ureteral Obstruction/metabolismABSTRACT
Renal tubular atrophy accompanies many proteinuric renal diseases, suggesting that glomerular proteinuria injures the tubules. However, local or systemic inflammation and filtration of abnormal proteins known to directly injure tubules are also present in many of these diseases and animal models; therefore, whether glomerular proteinuria directly causes tubular injury is unknown. Here, we examined the renal response to proteinuria induced by selective podocyte loss. We generated mice that express the diphtheria toxin receptor exclusively in podocytes, allowing reproducible dose-dependent, specific ablation of podocytes by administering diphtheria toxin. Ablation of <20% of podocytes resulted in profound albuminuria that resolved over 1-2 weeks after the re-establishment of normal podocyte morphology. Immediately after the onset of albuminuria, proximal tubule cells underwent a transient burst of proliferation without evidence of tubular damage or increased apoptosis, resulting in an increase in total tubular cell numbers. The proliferative response coincided with detection of the growth factor Gas6 in the urine and phosphorylation of the Gas6 receptor Axl in the apical membrane of renal tubular cells. In contrast, ablation of >40% of podocytes led to progressive glomerulosclerosis, profound tubular injury, and renal failure. These data suggest that glomerular proteinuria in the absence of severe structural glomerular injury activates tubular proliferation, potentially as an adaptive response to minimize the loss of filtered proteins.
Subject(s)
Albuminuria/physiopathology , Cell Proliferation , Kidney Glomerulus/physiopathology , Kidney Tubules, Proximal/pathology , Podocytes/pathology , Proteinuria/physiopathology , Albuminuria/metabolism , Albuminuria/pathology , Animals , Disease Models, Animal , Female , Heparin-binding EGF-like Growth Factor , Integrases/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Male , Membrane Proteins/genetics , Mice , Mice, Transgenic , Proteinuria/metabolism , Proteinuria/pathology , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Axl Receptor Tyrosine KinaseABSTRACT
The mammalian kidney is a highly complex organ that requires the precise structural arrangement of multiple cell types for effective function. The need to filter large volumes of plasma at the glomerulus followed by active reabsorption of nearly 99% of that filtrate by the tubules creates vulnerability in both compartments for cell injury. Thus maintenance of cell viability and replacement of those cells that are lost are essential for functional stability of the kidney. This review addresses our current understanding of how cells from the glomerular, tubular, and interstitial compartments arise during development and the manner in which they may be regenerated in the adult organ. In addition, we discuss the data regarding the role of organ-specific and bone marrow-derived stem and progenitor cells in the replacement/repair process, as well as the potential for ex vivo programming of stem cells toward a renal lineage.
Subject(s)
Kidney Diseases/pathology , Kidney/physiology , Regeneration/physiology , Animals , Bone Marrow Cells/physiology , Epithelial Cells/physiology , Glomerular Mesangium/cytology , Glomerular Mesangium/metabolism , Glomerular Mesangium/physiology , Humans , Kidney/growth & development , Kidney/metabolism , Kidney/pathology , Kidney Tubules/metabolism , Kidney Tubules/pathology , Pluripotent Stem Cells/physiology , Podocytes/physiology , Stem Cells/physiologyABSTRACT
Recent studies have demonstrated that erythropoietin (EPO) receptors are expressed on tubular epithelial cells and that EPO can protect tubular cells from injury in vitro and in vivo. Separate studies have demonstrated that marrow stromal cells (MSCs) exert a renoprotective effect in ischemia-reperfusion and cisplatin tubular injury via the secretion of factors that reduce apoptosis and increase proliferation of tubular epithelial cells. In the present study we demonstrate that MSCs express EPO receptors and that EPO can protect MSCs from serum deprivation-induced cell death and can stimulate MSC proliferation in vitro. The administration of EPO to mice resulted in the expansion of CD45-Flk1-CD105+ MSCs in the bone marrow and in the spleen and mobilized these cells as well as CD45-Flk1+ endothelial progenitor cells into the peripheral circulation. Consistent with previous reports, the administration of EPO diminished the decline in renal function associated with cisplatin administration. This effect was partially reproduced by intraperitoneal injection of cultured EPO-mobilized cells in cisplatin-treated mice. Thus the in vivo expansion and/or activation of these cells may contribute to the renoprotective effects of EPO to protect tubular cells from toxic injury.
Subject(s)
Acute Kidney Injury/drug therapy , Erythropoietin/pharmacology , Kidney Tubules/cytology , Stromal Cells/cytology , Stromal Cells/drug effects , Acute Kidney Injury/chemically induced , Acute Kidney Injury/pathology , Adipocytes/cytology , Adipocytes/drug effects , Animals , Antineoplastic Agents/toxicity , Cell Division/drug effects , Cell Survival/drug effects , Cells, Cultured , Cisplatin/toxicity , Drug Interactions , Female , Kidney Tubules/drug effects , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mice , Mice, Inbred C57BL , Receptors, Erythropoietin/metabolismABSTRACT
Green fluorescent protein (GFP) transgenic animals are widely used in biomedical research. We observed that the commonly used beta-actin-GFP transgenic mouse has renal defects with proteinuria starting as early as 5 weeks of age. Histological analysis reveals a widespread increase in glomerular extracellular matrix, occasional mesangiolysis, and secondary tubulointerstitial injury. Electron microscopic (EM) analysis reveals dramatic thickening of the glomerular basement membrane (GBM). Several other transgenic strains with GFP on ubiquitous promoters including beta-actin (with insertion in a different location) and ubiquitin C show no renal abnormalities. Western blot analysis on crude glomerular preparations from several GFP transgenic strains revealed that higher levels of GFP expression might be responsible for the observed pathogenesis. Mapping of the transgene insertion site by inverse PCR indicates that the beta-actin GFP transgene does not cause insertional mutagenesis nor does it modify the transcription level of adjacent genes. Taken together, this strain of beta-actin-GFP transgenic mouse may be used to study the mechanism of GBM expansion. Moreover, experiments using this strain of GFP mouse should be hereafter carefully planned because its renal pathology may interfere with data interpretation.
Subject(s)
Actins/genetics , Glomerulosclerosis, Focal Segmental/genetics , Green Fluorescent Proteins/genetics , Animals , Glomerulosclerosis, Focal Segmental/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis, InsertionalABSTRACT
Pax2 is a transcription factor with important functions during kidney development . Ectopic expression of Pax2 in podocytes has been reported in various glomerular diseases , but the functional relevance remains unknown. We developed an inducible mouse model that allows activation of Pax2 specifically in podocytes. Persistent expression of Pax2 did not interfere with the initial differentiation of podocytes, but mice ectopically expressing PAX2 developed end-stage renal failure soon after birth. Similarly, activation of PAX2 in healthy adult animals resulted in renal disease within 3 weeks after podocyte-specific induction of a deleter Cre. PAX2 activation caused repression of the podocyte key regulator molecule Wt1 and consequently a dramatic reduction of nephrin expression. Recruitment of the groucho-related protein TLE4 may be involved in converting Pax2 into a transcriptional repressor of Wt1. Finally, treatment of mice with an angiotensin-converting enzyme (ACE) inhibitor normalized renal function and induced upregulation of the important structural molecule nephrin via a Wt1-independent pathway. Our data demonstrate the functional significance of PAX2 reexpression in mature podocytes for the development of glomerular diseases and suggest that reactivation of PAX genes in terminally differentiated cells leads to a more dedifferentiated phenotype.
Subject(s)
Disease Models, Animal , Kidney Failure, Chronic/metabolism , PAX2 Transcription Factor/physiology , Podocytes/metabolism , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Enalapril/therapeutic use , Gene Expression Regulation , Kidney Failure, Chronic/drug therapy , Kidney Failure, Chronic/pathology , Male , Membrane Proteins/metabolism , Mice , PAX2 Transcription Factor/genetics , Podocytes/pathology , Proteinuria/drug therapy , WT1 Proteins/metabolismABSTRACT
Bone marrow (BM) transplantation has been shown to provide beneficial effects in injured organs, including heart, liver, and kidney. We explored the therapeutic potential of BM transplantation (BMT) in Wilms' tumor suppressor 1 (Wt1) heterozygous mice, which represent a model of mesangial sclerosis. After transplantation of wild-type BM, there is statistically significantly lower urinary albumin and increased survival in Wt1+/- recipients. Control BMT using Wt1+/- donors showed no significant beneficial effects. The long-term beneficial effect of BMT was dependent on the dose of irradiation applied to the recipients before BMT. At a lethal dose of 1,000 cGy, the decrease in albuminuria and prolongation of lifespan in Wt1+/- mice were transient, with maximal amelioration at 12 weeks and resumption of albuminuria by 24 weeks after BMT. This was, at least in part, due to irradiation and not Wt1 heterozygosity because wild-type recipients also developed albuminuria within 24 weeks of BMT with 1,000 cGy. In contrast, Wt1+/- mice transplanted after 400 cGy showed long-term improvement in albuminuria and lifespan. Approximately 0.4% of podocytes were marrow derived, a level that is unlikely to be responsible for the therapeutic effects. In addition, donor BM cells formed rings surrounding the glomeruli, and approximately one third of the cells in these rings were macrophages. In conclusion, transplantation of wild-type BM attenuates progression of mesangial sclerosis in the Wt1+/- model of renal disease, and the mechanism by which this occurs may involve engraftment of BM-derived cells in the renal parenchyma.
Subject(s)
Bone Marrow Transplantation , Glomerular Mesangium , Kidney Diseases/surgery , Sclerosis/surgery , Albuminuria/etiology , Animals , Cell Survival/radiation effects , Disease Progression , Dose-Response Relationship, Drug , Mice , Regeneration , Sclerosis/pathology , WT1 Proteins/geneticsABSTRACT
Prior to sex determination the gonadal anlage is formed as a bipotential primordium with the capacity to differentiate into either testes or ovaries depending on the presence or absence of the Sry gene. Knockout experiments have implicated five genes in the formation or survival of the gonadal primordium: Wt1, Sf1, Lim1, Lhx9 and Emx2. We are particularly interested in the Wilms tumour suppressor, WT1, which is characterized by complex posttranscriptional modifications. Here we will focus on published in vitro evidence suggesting distinct functions for the various isoforms and present our own results from in vivo experiments. Our data suggest that WT1 is an important regulator of the transcription or stability of the sex-determining gene Sry. One of the first genes expressed after the initial male sex-determining signal is the Sox9 gene. Human SOX9 has been implicated in male-to-female sex reversal. To analyse Sox9 function in mouse development we have performed transgenic experiments and ectopically expressed this gene in XX gonads. Our data indicate that Sox9 is sufficient to induce testis formation in mice. Here we will discuss our new data and present an updated model for Wt1 and Sox9 function in gonad formation and sex determination.
Subject(s)
High Mobility Group Proteins/genetics , Ovary/embryology , Sex Differentiation/genetics , Testis/embryology , Transcription Factors/genetics , WT1 Proteins/genetics , Animals , Female , Male , SOX9 Transcription Factor , Sex Determination ProcessesABSTRACT
Glomerular disease is one of the most common causes of end-stage renal failure. Increasing evidence suggests that these glomerulopathies are frequently caused by primary lesions in the renal podocytes. One of the major consequences of podocyte lesions is the accumulation of mesangial matrix in the glomerular basement membrane, a process called glomerulosclerosis. Mesangial sclerosis is one of the most consistent findings in Denys-Drash patients and can be caused by dominant mutations in the Wilms' tumor 1 gene (WT1). The underlying mechanism, however, is poorly understood. WT1 is expressed in the podocytes throughout life, but its function in this cell type is unknown. Combining Wt1-knockout and inducible yeast artificial chromosome transgenic mouse models, we demonstrate that reduced expression levels of WT1 result in either crescentic glomerulonephritis or mesangial sclerosis depending on the gene dosage. Strikingly, the two podocyte-specific genes nphs1 and podocalyxin are dramatically downregulated in mice with decreased levels of Wt1, suggesting that these two genes act downstream of Wt1. Taken together, our data provide genetic evidence that reduced levels of Wt1 are responsible for the pathogenesis of two distinct renal diseases and offer a molecular explanation for the increased occurrence of glomerulosclerosis in patients with WAGR syndrome.