Chengjin Li

Angiopoietin-1 is essential in mouse vasculature during development and in response to injury

Angiopoietin-1/Tek signaling is a critical regulator of blood vessel development, with conventional knockout of angiopoietin-1 or Tek in mice being embryonically lethal due to vascular defects. In addition, angiopoietin-1 is thought to be required for the stability of mature vessels. Using a Cre-Lox conditional gene targeting approach, we have studied the role of angiopoietin-1 in embryonic and adult vasculature. We report here that angiopoietin-1 is critical for regulating both the number and diameter of developing vessels but is not required for pericyte recruitment. Cardiac-specific knockout of angiopoietin-1 reproduced the phenotype of the conventional knockout, demonstrating that the early vascular abnormalities arise from flow-dependent defects. Strikingly, deletion in the entire embryo after day E13.5 produced no immediate vascular phenotype. However, when combined with injury or microvascular stress, angiopoietin-1 deficiency resulted in profound organ damage, accelerated angiogenesis, and fibrosis. These findings redefine our understanding of the biological roles of angiopoietin-1: it is dispensable in quiescent vessels but has a powerful ability to modulate the vascular response after injury.

Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice.

Rapidly progressive glomerulonephritis (RPGN) is a clinical syndrome characterized by loss of renal function within days to weeks and by glomerular crescents on biopsy. The pathogenesis of this disease is unclear, but circulating factors are believed to have a major role. Here, we show that deletion of the Von Hippel–Lindau gene (Vhlh) from intrinsic glomerular cells of mice is sufficient to initiate a necrotizing crescentic glomerulonephritis and the clinical features that accompany RPGN. Loss of Vhlh leads to stabilization of hypoxia-inducible factor α subunits (HIFs). Using gene expression profiling, we identified de novo expression of the HIF target gene Cxcr4 (ref. 3) in glomeruli from both mice and humans with RPGN. The course of RPGN is markedly improved in mice treated with a blocking antibody to Cxcr4, whereas overexpression of Cxcr4 alone in podocytes of transgenic mice is sufficient to cause glomerular disease. Collectively, these results indicate an alternative mechanism for the pathogenesis of RPGN and glomerular disease in an animal model and suggest novel molecular pathways for intervention in this disease.

Inhibition of MTOR Disrupts Autophagic Flux in Podocytes

Inhibitors of the mammalian target of rapamycin (MTOR) belong to a family of drugs with potent immunosuppressive, antiangiogenic, and antiproliferative properties. De novo or worsening proteinuria can occur during treatment with these agents, but the mechanism by which this occurs is unknown. We generated and characterized mice carrying a podocyte-selective knockout of the Mtor gene. Although Mtor was dispensable in developing podocytes, these mice developed proteinuria at 3 weeks and end stage renal failure by 5 weeks after birth. Podocytes from these mice exhibited an accumulation of the autophagosome marker LC3 (rat microtubule-associated protein 1 light chain 3), autophagosomes, autophagolysosomal vesicles, and damaged mitochondria. Similarly, human podocytes treated with the MTOR inhibitor rapamycin accumulated autophagosomes and autophagolysosomes. Taken together, these results suggest that disruption of the autophagic pathway may play a role in the pathogenesis of proteinuria in patients treated with MTOR inhibitors.

Soluble FLT1 Binds Lipid Microdomains in Podocytes to Control Cell Morphology and Glomerular Barrier Function

Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.

Rapid Isolation of Glomeruli Coupled with Gene Expression Profiling Identifies Downstream Targets in Pod1 Knockout Mice

Mouse mutations have provided tremendous insights into the molecular basis of renal and glomerular development. However, genes often play important roles during multiple stages of nephrogenesis, making it difficult to determine the role of a gene in a specific cell lineage such as the podocyte. Conditional gene targeting and chimeric analysis are two possible approaches to dissect the function of genes in specific cell populations. However, these are labor-intensive and costly and require the generation, validation, and analysis of additional transgenic lines. For overcoming these shortcomings and, specifically, for studying the role of gene function in developing glomeruli, a technique to isolate and purify glomeruli from murine embryos was developed. Combined with gene expression profiling, this method was used to identify differentially expressed genes in glomeruli from Pod1 knockout (KO) mice that die in the perinatal period with multiple renal defects. Glomeruli from early developing stages (late S-shape/early capillary loop) onward can be isolated successfully from wild-type and KO kidneys at 18.5 d postcoitus, and RNA can readily be obtained and used for genome-wide microarray analysis. With this approach, 3986 genes that are differently expressed between glomeruli from Pod1 KO and wild-type mice were identified, including a four-fold reduction of alpha 8 integrin mRNA in glomeruli from Pod1 KO mice that was confirmed by immunostaining. This procedure may be adapted to any transgenic strain, providing a rapid and efficient method to dissect the function of specific genes in glomerular development.

Functions of Type II Pneumocyte-Derived Vascular Endothelial Growth Factor in Alveolar Structure, Acute Inflammation, and Vascular Permeability

Vascular endothelial growth factor-A (VEGF) is a potent regulator of vascular permeability, inflammatory response, and cell survival in the lung. To explore the functions of VEGF produced locally in type II pneumocytes, we generated mice with a conditional deletion of VEGF-A using Cre recombinase driven by the human surfactant protein C (SPC) promoter. In 7- to 10-week-old VEGF-knockout (SPC-VEGF-KO) mice, lung histology and physiology were essentially normal, except for higher dynamic lung compliance and lower pulmonary vascular permeability. Emphysema was seen in 28- to 32-week-old animals. To investigate the role of type II pneumocyte-derived VEGF in acute lung injury, we challenged 7- to 10-week-old SPC-VEGF-KO mice and their wild-type littermates with intestinal ischemia-reperfusion. Bronchoalveolar lavage fluid total cell count, pulmonary permeability, and lung injury score were significantly attenuated, and total lung VEGF levels were significantly lower in SPC-VEGF-KO mice compared with wild-type controls. In SPC-VEGF-KO mice, activated caspase 3-positive type II epithelial cells were increased after intestinal ischemia-reperfusion, even though there was no significant difference in the total number of cells positive for terminal deoxynucleotidyl transferase dUTP nick-end labeling. We conclude that VEGF in type II cells helps protect alveolar epithelial cells from caspase-dependent apoptosis. However, VEGF produced from type II cells may contribute to increased vascular permeability during acute lung injury.

A new Cre driver mouse line, Tcf21/Pod1-Cre, targets metanephric mesenchyme

Conditional gene targeting in mice has provided great insight into the role of gene function in kidney development and disease. Although a number of Cre-driver mouse strains already exist for the kidney, development of additional strains with unique expression patterns is needed. Here we report the generation and validation of a Tcf21/Pod1-Cre driver strain that expresses Cre recombinase throughout the condensing and stromal mesenchyme of developing kidneys and in their derivatives including epithelial components of the nephron and interstitial cells. To test the efficiency of this line, we crossed it to mice transgenic for either loss or gain of function β-catenin conditional alleles. Mice with deletion of β-catenin from Tcf21-expressing cells are born with hypoplastic kidneys, hydroureters and hydronephrosis. By contrast, Tcf21-Cre driven gain of function for β-catenin in mice results in fused midline kidneys and hypoplastic kidneys. Finally, we report the first renal mesenchymal deletion of Patched1 (Ptch1), the receptor for sonic hedgehog (Shh), which results in renal cysts demonstrating a functional role of Shh signaling pathway in renal cystogensis. In summary, we report the generation and validation of a new Cre driver strain that provides robust excision in metanephric mesenchyme.

Loss of the Podocyte-Expressed Transcription Factor Tcf21/Pod1 Results in Podocyte Differentiation Defects and FSGS

Podocytes are terminally differentiated cells with an elaborate cytoskeleton and are critical components of the glomerular barrier. We identified a bHLH transcription factor, Tcf21, that is highly expressed in developing and mature podocytes. Because conventional Tcf21 knockout mice die in the perinatal period with major cardiopulmonary defects, we generated a conditional Tcf21 knockout mouse to explore the role of this transcription factor in podocytes in vivo. Tcf21 was deleted from podocytes and podocyte progenitors using podocin-cre (podTcf21) and wnt4-cre (wnt4creTcf21) driver strains, respectively. Loss of Tcf21 from capillary-loop stage podocytes (podTcf21) results in simplified glomeruli with a decreased number of endothelial and mesangial cells. By 5 weeks of age, 40% of podTcf21 mice develop massive proteinuria and lesions similar to FSGS. Notably, the remaining 60% of mice do not develop proteinuria even when aged to 8 months. By contrast, earlier deletion of Tcf21 from podocyte precursors (wnt4creTcf21) results in a profound developmental arrest of podocyte differentiation and renal failure in 100% of mice during the perinatal period. Taken together, our results demonstrate a critical role for Tcf21 in the differentiation and maintenance of podocytes. Identification of direct targets of this transcription factor may provide new therapeutic avenues for proteinuric renal disease, including FSGS.

MTOR regulates autophagic flux in the glomerulus.

Sirolimus (rapamycin), an inhibitor of the mechanistic target of rapamycin (MTOR), was originally proposed as an immunosuppressant to prevent rejection of solid organ transplants. There were expectations that MTOR inhibitors would replace nephrotoxic calcineurin inhibitors (CNIs). Despite its potential advantages, evidence that sirolimus causes de novo or worsening proteinuria is unequivocal. Given the well-recognized proteinuric effect of MTOR inhibitors, we were interested in understanding its role in maintaining the glomerular filtration barrier. To investigate this in vivo, we developed a mouse model with a podocyte selective deletion of the Mtor gene (Mtor pod-KO).

Context-dependent functions of angiopoietin 2 are determined by the endothelial phosphatase VEPTP

Significance Reducing vascular leakage and stabilizing the endothelium through activation of the angiopoietin (ANGPT)–TIE2 receptor tyrosine kinase pathway is a promising therapeutic strategy for vascular diseases. ANGPT2 is one of two major ligands for the TIE2 receptor. Uniquely, ANGPT2 possesses an agonistic role in lymphatic endothelium, but acts as a competitive antagonist in blood endothelium. The molecular basis for the opposing actions of ANGPT2 in these two vascular beds is poorly understood. Here we demonstrate that the absence of VEPTP expression in the lymphatic endothelium confers an agonist function of ANGPT2 on TIE2 receptor, but VEPTP expression in blood endothelium abrogates its activity. Our findings provide mechanistic insights needed to advance therapeutic targeting of this pathway. The angiopoietin (ANGPT)–TIE2/TEK signaling pathway is essential for blood and lymphatic vascular homeostasis. ANGPT1 is a potent TIE2 activator, whereas ANGPT2 functions as a context-dependent agonist/antagonist. In disease, ANGPT2-mediated inhibition of TIE2 in blood vessels is linked to vascular leak, inflammation, and metastasis. Using conditional knockout studies in mice, we show TIE2 is predominantly activated by ANGPT1 in the cardiovascular system and by ANGPT2 in the lymphatic vasculature. Mechanisms underlying opposing actions of ANGPT2 in blood vs. lymphatic endothelium are poorly understood. Here we show the endothelial-specific phosphatase VEPTP (vascular endothelial protein tyrosine phosphatase) determines TIE2 response to ANGPT2. VEPTP is absent from lymphatic endothelium in mouse in vivo, permitting ANGPT2/TIE2-mediated lymphangiogenesis. Inhibition of VEPTP converts ANGPT2 into a potent TIE2 activator in blood endothelium. Our data support a model whereby VEPTP functions as a rheostat to modulate ANGPT2 ligand effect on TIE2.