Aleksandr Vasilyev

Collective cell migration drives morphogenesis of the kidney nephron.

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis.

Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models.

Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to cross-link actin microfilaments into higher-order structures has been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD.

odd skipped related1 reveals a novel role for endoderm in regulating kidney versus vascular cell fate.

The kidney and vasculature are intimately linked both functionally and during development, when nephric and blood/vascular progenitor cells occupy adjacent bands of mesoderm in zebrafish and frog embryos. Developmental mechanisms that underlie the differentiation of kidney versus blood/vascular lineages remain unknown. The odd skipped related1 (osr1) gene encodes a zinc-finger transcription factor that is expressed in the germ ring mesendoderm and subsequently in the endoderm and intermediate mesoderm, prior to the expression of definitive kidney or blood/vascular markers. Knockdown of osr1 in zebrafish embryos resulted in a complete, segment-specific loss of anterior kidney progenitors and a compensatory increase in the number of angioblast cells in the same trunk region. Histology revealed a subsequent absence of kidney tubules, an enlarged cardinal vein and expansion of the posterior venous plexus. Altered kidney versus vascular development correlated with expanded endoderm development in osr1 knockdowns. Combined osr1 loss of function and blockade of endoderm development by knockdown of sox32/casanova rescued anterior kidney development. The results indicate that osr1 activity is required to limit endoderm differentiation from mesendoderm; in the absence of osr1, excess endoderm alters mesoderm differentiation, shifting the balance from kidney towards vascular development.

Invasion of Distal Nephron Precursors Associates with Tubular Interconnection during Nephrogenesis

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.

Mechanical Stretch and PI3K Signaling Link Cell Migration and Proliferation to Coordinate Epithelial Tubule Morphogenesis in the Zebrafish Pronephros

Organ development leads to the emergence of organ function, which in turn can impact developmental processes. Here we show that fluid flow-induced collective epithelial migration during kidney nephron morphogenesis induces cell stretch that in turn signals epithelial proliferation. Increased cell proliferation was dependent on PI3K signaling. Inhibiting epithelial proliferation by blocking PI3K or CDK4/Cyclin D1 activity arrested cell migration prematurely and caused a marked overstretching of the distal nephron tubule. Computational modeling of the involved cell processes predicted major morphological and kinetic outcomes observed experimentally under a variety of conditions. Overall, our findings suggest that kidney development is a recursive process where emerging organ function “feeds back” to the developmental program to influence fundamental cellular events such as cell migration and proliferation, thus defining final organ morphology.

Leiomyosarcoma of the Uterus with Intravascular Tumor Extension and Pulmonary Tumor Embolism

We report the case of a 48-year-old woman presenting with recurrent uterine leiomyosarcoma (LMS) associated with right iliac vein and inferior vena cava (IVC) invasion and left lower lobe pulmonary tumor embolus. Because the prognosis and treatment differ from that of thrombotic pulmonary emboli, the differentiating imaging characteristics of intravascular tumor embolism are reviewed. To our knowledge, only two other cases of intravenous uterine leiomyosarcomatosis have been described in the existing literature, and this is the first reported case of the entity with associated intravascular tumor embolism.

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12.

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer.

Collective Epithelial Migration Drives Kidney Repair after Acute Injury

Acute kidney injury (AKI) is a common and significant medical problem. Despite the kidney’s remarkable regenerative capacity, the mortality rate for the AKI patients is high. Thus, there remains a need to better understand the cellular mechanisms of nephron repair in order to develop new strategies that would enhance the intrinsic ability of kidney tissue to regenerate. Here, using a novel, laser ablation-based, zebrafish model of AKI, we show that collective migration of kidney epithelial cells is a primary early response to acute injury. We also show that cell proliferation is a late response of regenerating kidney epithelia that follows cell migration during kidney repair. We propose a computational model that predicts this temporal relationship and suggests that cell stretch is a mechanical link between migration and proliferation, and present experimental evidence in support of this hypothesis. Overall, this study advances our understanding of kidney repair mechanisms by highlighting a primary role for collective cell migration, laying a foundation for new approaches to treatment of AKI.

Cytoplasmic carboxypeptidase 5 regulates tubulin glutamylation and zebrafish cilia formation and function

Zebrafish have four tubulin deglutamylases: Ccp1, Ccp2, Ccp5, and Ccp6. Except for ccp1, all deglutamylase genes are expressed during ciliogenesis in zebrafish. Only loss of ccp5 induces cilia hyperglutamylation and the complete spectrum of ciliopathy phenotype. ccp5 knockdown can bypass Fleer/Ift70 or Ift88 deficiency in zebrafish to form multicilia.

Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis

Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury.