Kenneth Gross

Research Overview:

The Renin-expressing Cell and Development of the Renal Vasculature

Our research program encompasses several projects focused on elucidation of the function of the renin-angiotensin system(RAS) and its regulation. Classically, the RAS is known for its regulation of blood pressure and electrolyte homeostasis through renin release from juxtaglomerular (JG) cells. More recently it has become apparent that the RAS is required for normal renal development and that renin expression is evident throughout the developing renal vasculature, being restricted to the JG cell only upon maturation.

A long term project centers on understanding transcriptional regulation of the renin gene. Our approach in these studies involved development of a renin-expressing cell line by transgene-targeted tumorigenesis in mice. Transient transfection assays in this cell line were used as a first pass assay to delineate functionally important regions of the promoter. Following identification of specific DNA recognition sequences and interacting transcription factors by a variety of in vitro approaches, validation is currently being undertaken in vivo employing transgenesis assays. For these studies renin BAC constructs are modified by homologous recombination in bacteria. These studies have unexpectedly revealed that renin gene transcription is regulated as an immediate downstream target of class I Hox genes and the Notch pathway. These classical developmental pathways appear to play key roles in directing tissue specific expression and facilitating interaction of an evolutionarily conserved downstream enhancer with the basal transcriptional machinery. Their involvement in regulation of a “blood pressure gene” raises interesting issues as regards the origins and role of the RAS.

A second project focuses on rigorously identifying components of the RAS in more primitive vertebrates, particularly those utilizing evolutionarily more primitive kidneys, the pronephros and mesonephros, which are not high pressure kidneys. While many components of the RAS had been verified as present in lower vertebrates, unequivocal evidence for the presence of renin was unavailable outside mammals. Molecular hybridization approaches lacked ability to discriminate renin from other more abundant aspartyl protease members. In collaboration with Ping Liang we identified multiple exons for the putative zebrafish and pufferfish orthologs in silico using the rapidly expanding sequence databases for these species. The in silico approach allowed introduction of an enhanced degree of specificity into the homology search, or ‘virtual hybridization reaction and effectively ‘normalized’ for abundance relative to the est databases. The zebrafish gene was recovered within a BAC and direct sequencing was undertaken to determine the sequence of the remainder of the exons and the homologous cDNA probe was recovered from opisthonephric (mesonephric) tissue of adult fish, confirming the utilization of the encoded exons. This zebrafish renin probe was used in additional whole mount hybridization studies to identify the earliest time and pattern of expression. This led to the interesting finding that the renin expression first observed in association with the larval pronephric kidney is in fact localized to intermingled adrenocortical tissue. Our goal is to use a similar approach to identify other components of the RAS which will ultimately enable analysis of the functional role of this signaling system in these evolutionarily more primitive vertebrates which are so amenable to developmental genetic analysis.

A third project comprises the main thrust of our laboratory’s efforts to understand the functional role of the renin-expressing cell during mammalian kidney organogenesis. In these studies we are using BAC transgenics in which the renin promoter is being used to drive expression of an enhanced green fluorescent protein reporter to isolate renin-expressing cells from different stages of mouse kidney development by flow cytometry. In collaboration with Ping Liang the isolated cells are being expression- profiled using several platforms, including Affymetrix microarrays and Massively Parallel Signature Sequencing. The microarray studies have been designed in such a way that one can discern what expressions are co- or de-enriching with renin. The results of these studies strongly support the suggestion that the renin-expressing cell that is transiently found in association with the developing renal arterial system is in fact an ‘activated pericyte’, i.e. has the phenotype of a mural cell that is actively engaged in communicating with endothelial and other cells to build blood vessels. Once the vessel is established this expression signature is extinguished and a quiescent status characteristic of the mature vessel pertains. These findings nicely account for a number of features of renin expression and the impact of perturbations of RAS signaling on vessel elaboration. Defects in pericyte function have been postulated to underlie vascular pathology associated with hypertension, atherosclerosis and diabetes. We are currently analyzing the cellular lineage and expression profile of the renin-expressing pericyte of kidney following pathophysiological perturbations with the aim of gaining insight into aberrant cellular and vascular function in the indicated disease states.

Finally, during the course of these studies on angiogenesis associated with normal kidney development it was noted that a number of gene expressions previously found to be associated with the expression profile for Clear Cell Renal Cell Carcinoma were evident within the renin-expressing cell expression signature. We hypothesize that this reflects the re-activation of the developmental program for renal vascular development in adult kidney by the ‘wounding process’ associated with tumorigenesis. There is considerable interest in developing anti-angiogenic approaches for treatment of this highly vascularized tumor. Given the recent realization that such approaches may need to target the pericyte compartment as well as the endothelial compartment, we are engaged in discerning in depth the human pericyte-specific expression profile with the aim of identifying markers with diagnostic or prognostic utility, or as potential targets for therapeutic intervention.