Doris Herzlinger - US grants

Renal function is dependent on the correct association and alignment of at least 14 unique transporting epithelial cell types into glomerular and tubular segments. These hetrrogeneous cell types are derived from the undifferentiated mesenchymal cells present in the embryonic metanephric kidney anlage. Mesenchymal cells could be stem cells that generate all renal cell types or mesenchymal ells might be a heterogeneous group of cells each differentiating directly into unique renal cell types. To distinguish between these models, the lac-Z gene was introduced into individual mephrogenic mesenchymal cells (rat, gestation cay 13) using replication defective retroviruses and their progeny were identified after differentiation in vitro. The clonal progeny of individual tagged cells were found to reside in glomerulus and proximal tubule segments of single nephron. These results demonstrate that the metanephric mesenchyme contains multipotent nephrogenic stem cells. A library of stem ell specific monoclonal antibodies will be prepared to characterize this newly ientified stem cell, and used to follow its fate during differentiation. Cell culture conditions will be defined that support the proliferation of these stem cells. In addition these cells will be immortalized and clonal cell lines established. These reagents and cell lines will be used to identify and characterize the regulatory mechanisms mediating the conversion of an undifferentiated mesenchymal cell into diverse renal epithelial celll types.

DESCRIPTION (provided by applicant): The signaling pathways controlling Ureteric Bud (UB) segmentation into the renal collecting system and ureter during embryogenesis remain poorly understood despite the high incidence of human congenital defects localized to the ureter and its junction with the kidney. Our preliminary data suggest that a unique mesenchymal cell population derived from paraxial mesoderm selectively surrounds the distal UB and secretes factors such as Bone Morphogenetic Factor 4 that are required for patterning the ureter. Specifically, we propose that BMP4 controls ureter structure by activating an antagonist of receptor tyrosine kinase signaling which is known to induce branching morphogenesis. In addition, we show that Bone Morphogenetic Protein 5, another factor secreted by periureteral mesenchyme, induces UB epithelia to terminally differentiate. The aims of this proposal will test these hypotheses describing the molecular mechanisms controlling ureter morphogenesis using established fate mapping, tissue removal, immunohistochemical, morphological, and transgenic techniques. Aim 1) The differentiated fate of ureteral connective tissue cells derived from paraxial mesoderm will be determined and ureter morphogenesis in the absence of these cells analyzed. Aim 2) The expression of membrane-associated Sprouty 1, an active receptor tyrosine kinase antagonist, will be correlated with sites of BMP signaling in the developing ureter. Ureter structure and the cellular distribution of Sprouty 1 will be analyzed in the developing mouse in the absence of BMP signaling. Aim 3) Terminal urothelial cell differentiation will be analyzed in vitro and in vivo in the absence of BMP5 signaling and in the presence of ectopic BMP signaling. Results of proposed experiments will provide much needed insight into the signaling pathways mediating ureter morphogenesis, a process that is prone to abnormalities in humans.

DESCRIPTION: The origin of activated stromal cells in the diseased kidney and the role that these cells play in precipitating end stage renal disease remains elusive, This recalcitrant clinical problem is difficult to address since the origin, differentiation and function of renal stroma in the healthy kidney remains poorly understood. We show that the Winged Helix transcription factor, BF-2, is selectively expressed by renal stromal progenitors and that BF-2 expressing stromal progenitors are essential for renal epithelial growth and differentiation. In this proposal we will use BF-2 mRNA as a molecular marker to identify renal stromal progenitors and test hypotheses describing the origin, differentiation, and fate of this cell population. In Specific Aim 1, the origin of renal stromal cells will be analyzed by retroviral mediated gene transfer lineage tracing techniques. The differentiated fate of lineage tagged, BF-2 expressing cells present in the early chick embryo will be characterized by morphological and molecular marker analyses. Specific Aim 2 will probe BF-2 function and the cellular processes perturbed by BF2 deletion. We show BF-2 null stromal cells undergo precocious differentiation while control cells down-regulate BF-2 expression coincident with differentiation. We will determine if adenovirus mediated constitutive, high level BF-2 expression delays stromal differentiation in vitro. We will test the hypothesis that precocious differentiation caused by BF-2 deletion perturbs the temporal expression patterns of stromal factors regulating ureteric bud growth. Data generated will provide the basis for future studies on the origin of activated stromal cells in the diseased kidney and their role in nephron degeneration and/or repair.

ABSTRACT Mammalian renal development is dependent on the ureteric bud. Its proximal tip induces nephron formation in the developing kidney and undergoes branching morphogenesis forming the intra-renal collecting system whereas its distal or trunk domain differentiates into the ureter. Abnormal regulation of ureteric bud morphogenesis can result in a spectrum of congenital defects, the most common being ureteral duplications and obstructions. Yet the tissue interactions and signaling pathways that limit ureter number and control ureteral smooth muscle differentiation required for the un-obstructed flow of urine to the bladder remain poorly understood. This proposal is focused on ureter morphogenesis. In Aim 1, the role of Bmp signaling in restricting ureteric bud outgrowth to a single site along the urinary tract and in controlling ureteral smooth muscle differentiation will be analyzed in mice with a severe knockdown in Bmp4 gene dosage that supports embryonic viability to birth, but not beyond. Inducible Bmp4 knockdown will be accomplished using Cre-lox technology and existing mouse lines. Bmp4 will be knocked down at different stages of development to analyze the role of this signaling factor in regulating ureter number separately, from its role in controlling terminal ureter differentiation. In Aim 2, we will analyze a mouse line lacking expression of Pbx1, a hox gene cofactor. We have discovered that Pbx1 is essential for restricting Bmp4 signaling and smooth muscle formation to the ureter. This mutant line will be used to dissect the genetic pathways and cell type (s) that control the organization of smooth muscle at the border between the kidney and the ureter, the most common site of urinary tract obstructions in newborns. The successful completion of these experiments will provide insight into the susceptibility of ureter morphogenesis to congenital defects.

DESCRIPTION (provided by applicant): Coordinated contractions of the smooth muscle coat surrounding the kidney outflow tract (OT), including the renal calyces, pelvis and ureter, are essential for draining urine out of the kidney. Congenital defects that impair this peristaltic process are common, and the leading cause of renal failure in children. However, the signaling pathways that control the differentiation of the cell types mediating urinary tract peristalsis remain poorly understood. In the previous funding period we showed that Bone Morphogenetic Protein signaling during the early stages of development is essential for the formation of the OT smooth muscle coat. In this project we will study the differentiated cell types that coordinate contraction in this musculature. We recently discovered that the normal initiation and coordination of OT smooth muscle contraction is dependent on Hyperpolarizing Cation Channel (HCN) activity. In Aim 1, we will determine if HCN expressing OT cells exhibit the electrophysiological and structural properties required for triggering smooth muscle contraction using our recently developed high resolution video-microscopic and optical mapping protocols and electron-microscopic techniques. Moreover, we will determine if HCN channel activity is essential for the efficient flow of urine from the kidney to the bladder by analyzing OT structure, smooth muscle contractile and electrical activity in mice with targeted HCN gene deletions. We have also uncovered a role for the tyrosine kinase receptor C-kit in controlling OT contractile activity. In Aim 2 we will analyze OT architecture and smooth muscle function in mice with loss-of-function mutant C-Kit alleles using morphological and functional assays. Aim 3 is based on our preliminary data demonstrating that mice harboring a constitutively active C-kit allele develop fetal hydronephrosis. We will determine if this defect due to C-kit gain-of-function is caused by a structural occlusion of the OT tract or as we predict, a defect in coordinated peristalsis. Collectively, the results of proposed experiments identifying the cell types required for triggering coordinated OT peristalsis will open up a new area of investigation focused on the signaling pathways controlling the differentiation of these pacemaker cells. Ultimately, our studies will reveal drug targets for the modification of ureter peristalsis and may reveal unappreciated causes of hydronephrosis.

DESCRIPTION (provided by applicant): Lower urinary tract (LUT) dysfunction is the most common cause of end-stage renal disease in children. Although several genetic pathways controlling LUT structure have been identified, the genetic pathways controlling the coordinated contraction of LUT musculature, a process essential for moving wastes out of the body, remain poorly understood. This is due, in large part, to the absence of techniques to assess LUT motility in real time in intact tissues such as the ureter, bladder and urethra. We have developed a novel ureter explant system and video microscopic and optical mapping protocols to analyze ureteral smooth muscle contractile and excitation patterns and discovered that the initiation of electrical activity driving coordinated, proximal to distal contraction is dependent on pacemaker cells that express hyperpolization activated cation channels. This Resource Center will apply our state-of-the-art functional imaging techniques to existing mouse mutants exhibiting LUT dysfunction to determine if aberrant smooth muscle function plays a causative, pathological role. Results of these studies will provide much needed model systems to identify the molecular mechanisms underlying coordinated LUT motility and ultimately yield novel drug therapies to treat inborn or acquired defects in this process.

The research component of this Center will employ novel intact lower urinary tract (LUT) explants, video microscopy, and optical mapping techniques to provide the urological community, and investigators outside the realm of this discipline, with the tools for real---time systems level analysis of UT smooth muscle activity, which is currently lacking. Video microscopy and optical mapping analyses of intact ureter and bladder explants will be used to document the contractile and electrical excitation patterns within intact tissues and within specified regions of these urinary tract segments in wild type and mutant murine models of hydronephrosis and overactive bladder syndrome. Data will be processed and analysed using custom written software that incorporates advanced mathematical algorithms that enable the differentiation of membrane voltage signals and experimental noise, as well as the representation of membrane depolarization data as spatial temporal excitation maps and action potential contours. High---resolution movies and time lapse images of representative real---time video microscopy will be performed to correlate electrical and contractile activity. Thus, our core will provide a comprehensive analysis of large scale LUT motility patterns and pave the way for the generation of novel diagnostics and drugs to alleviate UT motility defects such as in---born functional UT obstructions and overactive active bladder syndrome.

SUMMARY Hemodynamic forces play a crucial role in renal physiology and pathophysiology but the cellular and molecular mechanisms controlling the development of the specialized properties of the renal vascular bed remain poorly understood. We recently discovered that the Foxd1 lineage, which differentiates into pericytes, vascular smooth muscle, and the glomerular mesangium, plays a fundamental role in this process. Specifically, conditional ablation of the TALE transcription factor, Pbx1, in this lineage results in gross renal arterial patterning defects and premature death of mutant pups. Strikingly, conditional Pbx1 ablation does not markedly perturb renal epithelial morphogenesis. These data suggest that ablation of Pbx1 in the Foxd1 lineage reveals vascular patterning defects arising solely from the abnormal function of cells derived from the Foxd1 lineage. Using murine genetics and state-of-the art functional imaging techniques in collaboration with Dr. Peti-Peterdi, we will investigate the role of Pbx1 transcriptional regulation in the Foxd1 lineage. In Aim 1, we will determine how conditional Pbx1 ablation perturbs renal hemodynamics. Aim 2 will investigate the role of Pbx1-TR regulation in controlling the secretion of angiogenic factors by Foxd1-derivatives. Finally, in Aim 3 we will test whether Pbx1 transcriptional regulation in the mature kidney plays a role in its response to injury. Results of these studies investigating the mechanisms of gross renal vascular patterning will provide important clues for the design of protocols to functionally vascular renal tissues engineered in vitro and may inspire novel techniques to attenuate renal fibrosis, a major cause of end stage renal disease.