Gli1+ Pericyte Loss Induces Capillary Rarefaction and Proximal Tubular Injury.
ABSTRACT: Peritubular capillary rarefaction is hypothesized to contribute to the increased risk of future CKD after AKI. Here, we directly tested the role of Gli1+ kidney pericytes in the maintenance of peritubular capillary health, and the consequences of pericyte loss during injury. Using bigenic Gli1-CreERt2; R26tdTomato reporter mice, we observed increased distance between Gli1+ pericytes and endothelial cells after AKI (mean±SEM: 3.3±0.1 µm before injury versus 12.5±0.2 µm after injury; P<0.001). Using a genetic ablation model, we asked whether pericyte loss alone is sufficient for capillary destabilization. Ten days after pericyte ablation, we observed endothelial cell damage by electron microscopy. Furthermore, pericyte loss led to significantly reduced capillary number at later time points (mean±SEM capillaries/high-power field: 67.6±4.7 in control versus 44.1±4.8 at 56 days; P<0.05) and increased cross-sectional area (mean±SEM: 21.9±0.4 µm2 in control versus 24.1±0.6 µm2 at 10 days; P<0.01 and 24.6±0.6 µm2 at 56 days; P<0.001). Pericyte ablation also led to hypoxic focal and subclinical tubular injury, reflected by transient expression of Kim1 and vimentin in scattered proximal tubule segments. This analysis provides direct evidence that AKI causes pericyte detachment from capillaries, and that pericyte loss is sufficient to trigger transient tubular injury and permanent peritubular capillary rarefaction.
Project description: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.
Project description:Damage to endothelial cells contributes to acute kidney injury (AKI) by causing impaired perfusion, while the permanent loss of the capillary network following AKI has been suggested to promote chronic kidney disease. Therefore, strategies to protect renal vasculature may impact both short-term recovery and long-term functional preservation post-AKI. Human adipose stromal cells (hASCs) possess pro-angiogenic and anti-inflammatory properties and therefore have been tested as a therapeutic agent to treat ischaemic conditions. This study evaluated hASC potential to facilitate recovery from AKI with specific attention to capillary preservation and inflammation. Male Sprague Dawley rats were subjected to bilateral ischaemia/reperfusion and allowed to recover for either two or seven days. At the time of reperfusion, hASCs or vehicle was injected into the suprarenal abdominal aorta. hASC-treated rats had significantly greater survival compared to vehicle-treated rats (88.7% versus 69.3%). hASC treatment showed hastened recovery as demonstrated by lower creatinine levels at 48 hrs, while tubular damage was significantly reduced at 48 hrs. hASC treatment resulted in a significant decrease in total T cell and Th17 cell infiltration into injured kidneys at 2 days post-AKI, but an increase in accumulation of regulatory T cells. By day 7, hASC-treated rats showed significantly attenuated capillary rarefaction in the cortex (15% versus 5%) and outer medulla (36% versus 18%) compared to vehicle-treated rats as well as reduced accumulation of interstitial alpha-smooth muscle actin-positive myofibroblasts. These results suggest for the first time that hASCs improve recovery from I/R-induced injury by mechanisms that contribute to decrease in inflammation and preservation of peritubular capillaries.
Project description:BACKGROUND:Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury (AKI), which is associated with high morbidity and mortality. AKI is a serious and costly medical condition. Effective therapy for AKI is an unmet clinical need, and molecular mechanisms underlying the interactions between an injured kidney and distant organs remain unclear. Therefore, novel therapeutic strategies should be developed. METHODS:We directed the differentiation of human induced pluripotent stem (iPS) cells into endothelial progenitor cells (iEPCs), which were then applied for treating mouse AKI. The mouse model of AKI was induced by I/R injury. RESULTS:We discovered that intravenously infused iEPCs were recruited to the injured kidney, expressed the mature endothelial cell marker CD31, and replaced injured endothelial cells. Moreover, infused iEPCs produced abundant proangiogenic proteins, which entered into circulation. In AKI mice, blood urea nitrogen and plasma creatinine levels increased 2?days after I/R injury and reduced after the infusion of iEPCs. Tubular injury, cell apoptosis, and peritubular capillary rarefaction in injured kidneys were attenuated accordingly. In the AKI mice, iEPC therapy also ameliorated apoptosis of cardiomyocytes and cardiac dysfunction, as indicated by echocardiography. The therapy also ameliorated an increase in serum brain natriuretic peptide. Regarding the relevant mechanisms, indoxyl sulfate and interleukin-1? synergistically induced apoptosis of cardiomyocytes. Systemic iEPC therapy downregulated the proapoptotic protein caspase-3 and upregulated the anti-apoptotic protein Bcl-2 in the hearts of the AKI mice, possibly through the reduction of indoxyl sulfate and interleukin-1?. CONCLUSIONS:Therapy using human iPS cell-derived iEPCs provided a protective effect against ischemic AKI and remote cardiac dysfunction through the repair of endothelial cells and the attenuation of cardiomyocyte apoptosis.
Project description:Decreased renal blood flow following an ischemic insult contributes to a reduction in glomerular filtration. However, little is known about the underlying cellular or subcellular mechanisms mediating reduced renal blood flow in human ischemic acute kidney injury (AKI) or acute renal failure (ARF). To examine renal vascular injury following ischemia, intraoperative graft biopsies were performed after reperfusion in 21 cadaveric renal allografts. Confocal fluorescence microscopy was utilized to examine vascular smooth muscle and endothelial cell integrity as well as peritubular interstitial pericytes in the biopsies. The reperfused, transplanted kidneys exhibited postischemic injury to the renal vasculature, as demonstrated by disorganization/disarray of the actin cytoskeleton in vascular smooth muscle cells and disappearance of von Willebrand factor from vascular endothelial cells. Damage to peritubular capillary endothelial cells was more severe in subjects destined to have sustained ARF than in those with rapid recovery of their graft function. In addition, peritubular pericytes/myofibroblasts were more pronounced in recipients destined to recover than those with sustained ARF. Taken together, these data suggest damage to the renal vasculature occurs after ischemia-reperfusion in human kidneys. Preservation of peritubular capillary endothelial integrity and increasing pericytes may be critical to recovery from postischemic AKI.
Project description:Microvascular pericytes and perivascular fibroblasts have recently been identified as the source of scar-producing myofibroblasts that appear after injury of the kidney. We show that cross talk between pericytes and endothelial cells concomitantly dictates development of fibrosis and loss of microvasculature after injury. When either platelet-derived growth factor receptor (R)-? signaling in pericytes or vascular endothelial growth factor (VEGF)R2 signaling in endothelial cells was blocked by circulating soluble receptor ectodomains, both fibrosis and capillary rarefaction were markedly attenuated during progressive kidney injury. Blockade of either receptor-mediated signaling pathway prevented pericyte differentiation and proliferation, but VEGFR2 blockade also attenuated recruitment of inflammatory macrophages throughout disease progression. Whereas injury down-regulated angiogenic VEGF164, the dys-angiogenic isomers VEGF120 and VEGF188 were up-regulated, suggesting that pericyte-myofibroblast differentiation triggers endothelial loss by a switch in secretion of VEGF isomers. These findings link fibrogenesis inextricably with microvascular rarefaction for the first time, add new significance to fibrogenesis, and identify novel therapeutic targets.
Project description:Pericytes are tissue-resident mesenchymal progenitor cells anatomically associated with the vasculature that have been shown to participate in tissue regeneration. Here, we tested the hypothesis that kidney pericytes, derived from FoxD1+ mesodermal progenitors during embryogenesis, are necessary for postnatal kidney homeostasis. Diphtheria toxin delivery to FoxD1Cre::RsDTR transgenic mice resulted in selective ablation of >90% of kidney pericytes but not other cell lineages. Abrupt increases in plasma creatinine, blood urea nitrogen, and albuminuria within 96 h indicated acute kidney injury in pericyte-ablated mice. Loss of pericytes led to a rapid accumulation of neutral lipid vacuoles, swollen mitochondria, and apoptosis in tubular epithelial cells. Pericyte ablation led to endothelial cell swelling, reduced expression of vascular homeostasis markers, and peritubular capillary loss. Despite the observed injury, no signs of the acute inflammatory response were observed. Pathway enrichment analysis of genes expressed in kidney pericytes in vivo identified basement membrane proteins, angiogenic factors, and factors regulating vascular tone as major regulators of vascular function. Using novel microphysiological devices, we recapitulated human kidney peritubular capillaries coated with pericytes and showed that pericytes regulate permeability, basement membrane deposition, and microvascular tone. These findings suggest that through the active support of the microvasculature, pericytes are essential to adult kidney homeostasis.
Project description:Reductions in renal microvasculature density and increased lymphocyte activity may play critical roles in the progression of chronic kidney disease (CKD) following acute kidney injury (AKI) induced by ischemia/reperfusion injury (IRI). Vitamin D deficiency is associated with tubulointerstitial damage and fibrosis progression following IRI-AKI We evaluated the effect of vitamin D deficiency in sustained IRI-AKI, hypothesizing that such deficiency contributes to the early reduction in renal capillary density or alters the lymphocyte response to IRI Wistar rats were fed vitamin D-free or standard diets for 35 days. On day 28, rats were randomized into four groups: control, vitamin D deficient (VDD), bilateral IRI, and VDD+IRI Indices of renal injury and recovery were evaluated for up to 7 days following the surgical procedures. VDD rats showed reduced capillary density (by cablin staining), even in the absence of renal I/R. In comparison with VDD and IRI rats, VDD+IRI rats manifested a significant exacerbation of capillary rarefaction as well as higher urinary volume, kidney weight/body weight ratio, tissue injury scores, fibroblast-specific protein-1, and alpha-smooth muscle actin. VDD+IRI rats also had higher numbers of infiltrating activated CD4(+) and CD8(+) cells staining for interferon gamma and interleukin-17, with a significant elevation in the Th17/T-regulatory cell ratio. These data suggest that vitamin D deficiency impairs renal repair responses to I/R injury, exacerbates changes in renal capillary density, as well as promoting fibrosis and inflammation, which may contribute to the transition from AKI to CKD.
Project description:Human kidney peritubular capillaries are particularly susceptible to injury, resulting in dysregulated angiogenesis, capillary rarefaction and regression, and progressive loss of kidney function. However, little is known about the structure and function of human kidney microvasculature. Here, we isolated, purified, and characterized human kidney peritubular microvascular endothelial cells (HKMECs) and reconstituted a three-dimensional human kidney microvasculature in a flow-directed microphysiologic system. By combining epithelial cell depletion and cell culture in media with high concentrations of vascular endothelial growth factor, we obtained HKMECs of high purity in large quantity. Unlike other endothelial cells, isolated HKMECs depended on high vascular endothelial growth factor concentration for survival and growth and exhibited high tubulogenic but low angiogenic potential. Furthermore, HKMECs had a different transcriptional profile. Under flow, HKMECs formed a thin fenestrated endothelium with a functional permeability barrier. In conclusion, this three-dimensional HKMEC-specific microphysiologic system recapitulates human kidney microvascular structure and function and shows phenotypic characteristics different from those of other microvascular endothelial cells.
Project description:Peritubular capillary (PTC) rarefaction along with tissue fibrosis is a hallmark of chronic kidney disease (CKD). However, molecular mechanisms of PTC loss have been poorly understood. Previous studies have demonstrated that functional loss of endothelial sirtuin 1 (SIRT1) impairs angiogenesis during development and tissue damage. Here, we found that endothelial SIRT1 dysfunction causes activation of endothelial Notch1 signaling, which leads to PTC rarefaction and fibrosis following kidney injury. In mice lacking functional SIRT1 in the endothelium (Sirt1 mutant), kidney injury enhanced apoptosis and senescence of PTC endothelial cells with impaired endothelial proliferation and expanded myofibroblast population and collagen deposition. Compared to wild-type kidneys, Sirt1 mutant kidneys up-regulated expression of Delta-like 4 (DLL4, a potent Notch1 ligand), Hey1 and Hes1 (Notch target genes), and Notch intracellular domain-1 (NICD1, active form of Notch1) in microvascular endothelial cells (MVECs) post-injury. Sirt1 mutant primary kidney MVECs reduced motility and vascular assembly and enhanced senescence compared to wild-type kidney MVECs. This difference in the phenotype was negated with Notch inhibition. Concurrent stimulation of DLL4 and transforming growth factor (TGF)-?1 increased trans-differentiation of primary kidney pericytes into myofibroblast more than TGF-?1 treatment alone. Collectively, these results indicate that endothelial SIRT1 counteracts PTC rarefaction by repression of Notch1 signaling and antagonizes fibrosis via suppression of endothelial DLL4 expression.
Project description:Kidney transplantation (Tx) is considered the only definite treatment for end-stage kidney disease (ESKD) patients. The increasing prevalence of ESKD has necessitated the introduction of transplantation with kidneys from suboptimal donors. There is, however, still a lack of fundamental and longitudinal research on suboptimal kidney transplants. Specifically, there is a demand for accurate pre-Tx predictors of donor kidney function and injury to predict post-Tx outcome. In the present study, we combine rat models of chronic kidney disease (CKD) and renal Tx to dissect the effects of healthy and CKD renal grafts on healthy and CKD recipients. We show that renal function at 6 weeks post-Tx is exclusively determined by donor graft quality. Using cell tracking within enhanced green fluorescent protein-positive (eGFP+) recipients, we furthermore show that most inflammatory cells within the donor kidney originate from the donor. Oxidative and vascular extra-renal damage were, in contrast, determined by the recipient. Post- versus pre-Tx evaluation of grafts showed an increase in glomerular and peritubular capillary rarefaction in healthy but not CKD grafts within a CKD environment. Proliferation of glomerular endothelium was similar in all groups, and influx of eGFP+ recipient-derived cells occurred irrespective of graft or recipient status. Glomerular and peritubular capillary rarefaction, severity of inflammation and macrophage subtype data post-Tx were, however, determined by more complicated effects, warranting further study. Our experimental model could help to further distinguish graft from recipient environment effects, leading to new strategies to improve graft survival of suboptimal Tx kidneys.This article has an associated First Person interview with the first author of the paper.