Kevin T. Bush

Eya protein phosphatase activity regulates Six1–Dach–Eya transcriptional effects in mammalian organogenesis

The precise mechanistic relationship between gene activation and repression events is a central question in mammalian organogenesis, as exemplified by the evolutionarily conserved sine oculis (Six), eyes absent (Eya) and dachshund (Dach) network of genetically interacting proteins. Here, we report that Six1 is required for the development of murine kidney, muscle and inner ear, and that it exhibits synergistic genetic interactions with Eya factors. We demonstrate that the Eya family has a protein phosphatase function, and that its enzymatic activity is required for regulating genes encoding growth control and signalling molecules, modulating precursor cell proliferation. The phosphatase function of Eya switches the function of Six1–Dach from repression to activation, causing transcriptional activation through recruitment of co-activators. The gene-specific recruitment of a co-activator with intrinsic phosphatase activity provides a molecular mechanism for activation of specific gene targets, including those regulating precursor cell proliferation and survival in mammalian organogenesis.

Changes in global gene expression patterns during development and maturation of the rat kidney

We set out to define patterns of gene expression during kidney organogenesis by using high-density DNA array technology. Expression analysis of 8,740 rat genes revealed five discrete patterns or groups of gene expression during nephrogenesis. Group 1 consisted of genes with very high expression in the early embryonic kidney, many with roles in protein translation and DNA replication. Group 2 consisted of genes that peaked in midembryogenesis and contained many transcripts specifying proteins of the extracellular matrix. Many additional transcripts allied with groups 1 and 2 had known or proposed roles in kidney development and included LIM1, POD1, GFRA1, WT1, BCL2, Homeobox protein A11, timeless, pleiotrophin, HGF, HNF3, BMP4, TGF-α, TGF-β2, IGF-II, met, FGF7, BMP4, and ganglioside-GD3. Group 3 consisted of transcripts that peaked in the neonatal period and contained a number of retrotransposon RNAs. Group 4 contained genes that steadily increased in relative expression levels throughout development, including many genes involved in energy metabolism and transport. Group 5 consisted of genes with relatively low levels of expression throughout embryogenesis but with markedly higher levels in the adult kidney; this group included a heterogeneous mix of transporters, detoxification enzymes, and oxidative stress genes. The data suggest that the embryonic kidney is committed to cellular proliferation and morphogenesis early on, followed sequentially by extracellular matrix deposition and acquisition of markers of terminal differentiation. The neonatal burst of retrotransposon mRNA was unexpected and may play a role in a stress response associated with birth. Custom analytical tools were developed including “The Equalizer” and “eBlot,” which contain improved methods for data normalization, significance testing, and data mining.

Branching morphogenesis and kidney disease

Branching morphogenesis in the kidney is a tightly regulated, complex process and its disruption potentially can lead to a broad spectrum of diseases, ranging from rare hereditary syndromes to common conditions such as hypertension and chronic kidney failure. This review synthesizes data on branching during kidney development derived from in vitro and in vivo rodent studies and to apply them to human diseases. It discusses how the broad organization of molecular interactions during kidney development might provide a mechanistic framework for understanding disorders related to aberrant branching.

Genesis and reversal of the ischemic phenotype in epithelial cells

Ischemic injury to predominantly epithelial organs such as the kidney remains a major cause of morbidity and mortality. For example, in hospitalized patients with acute renal failure, it is estimated that around 50% of all cases are ischemic in origin (1). Nevertheless, progress in the medical management of this and other syndromes in which ischemia occurs has been slow. In part, this may be related to our still rudimentary understanding of the cell biology underlying the ischemic epithelial phenotype and the molecular mechanisms behind the recovery of normal cell and tissue organization. Epithelial tissues, such as the kidney and gut, not only provide a physical barrier between biologic compartments, but mediate vectorial and selective transport of ions, water, and macromolecules between blood and the external environment. These functions depend on the integrity of intercellular junctions (e.g., adherens, tight), the arrangement of lipids and proteins in the plasma membrane into strictly maintained apical and basolateral domains, and productive cell-substratum interactions, all of which are severely affected by ischemia/reperfusion. Although other factors, such as oxidative damage and ion and pH changes, likely play important roles in the generation of the ischemic epithelial phenotype, much of the damage is believed to be due to depletion of cellular ATP (2, 3). Thus, cell culture models using agents that deplete cellular ATP have been used extensively to study ischemic injury in polarized epithelial cells (3). Although the fidelity with which these in vitro models reproduce the lesions observed in vivo has been debated, there is little doubt that these ATP depletion/repletion cell culture models provide valuable insights into the molecular mechanisms underlying ischemic injury and recovery, as similar cellular and molecular lesions are often found in cells of the ischemic whole organ. Many of these lesions are remarkably specific, biochemically definable, and potentially regulated; recovery from these lesions after short-term injury appears to be mediated by a combination of both previously elucidated and potentially novel sorting mechanisms that are transduced by “classical” signaling pathways. Among other cellular and molecular lesions, ischemia and/or ATP depletion induces misfolding and/or aggregation of membrane and secreted proteins (4); disruption of the actin-based cytoskeleton (5); disturbances in apical-basolateral protein polarization (6); mislocalization and degradation of protein components of the intercellular junctions (7, 8); upregulation of a number of genes, including molecular chaperones (4, 9), growth factors and their receptors (10); perturbation of integrin-mediated cell-substratum adhesion (11–13); and induction of programmed and nonprogrammed cell death (2). Alterations in the actin cytoskeleton and integrin-mediated cell-substratum interactions have been extensively reviewed elsewhere (5, 13). Here we focus primarily on recent information on lesions affecting the permeability barrier (intercellular junctions), signaling events involved in the recovery of this barrier, and the roles of molecular chaperones in protecting epithelial cells. The establishment and maintenance of a selectively permeable barrier occur through homotypic interactions of the extracellular domains of multiple transmembrane adhesion molecules between adjacent cells. Examples of such proteins include E-cadherin in the adherens junction (AJ) and the occludin/claudin families in the tight junction (TJ). The intracellular domains of these adhesion molecules also interact (directly or indirectly) with a number of cytoplasmic proteins, including α, β, and γ catenin in the AJ, and zonula occludens-1 (ZO-1), ZO-2, ZO-3, and fodrin in the TJ, providing a functional link to the actin-based cytoskeleton. These interactions also modulate the stability of the adhesion proteins either by maintaining their appropriate conformations to recognize extracellular domains in adjoining cells or perhaps by inhibiting internalization and degradation. Under ischemic conditions, it appears that many of these cellular processes/structures are compromised, promoting junctional protein internalization and degradation, thereby disturbing the cell-cell interactions and the permeability barrier. Identifying molecular mechanisms underlying the cascade of events that induce cellular injury and those involved in the cell’s recovery is key to developing rational therapeutic approaches to diminish the morbidity associated with ischemic injury to epithelial tissues.

Endostatin regulates branching morphogenesis of renal epithelial cells and ureteric bud

Endostatin (ES) inhibits endothelial cell migration and has been found to bind to glypicans (Gpcs) on both endothelial cells and renal epithelial cells. We examined the possibility that ES might regulate epithelial cell morphogenesis. The addition of ES to cultured epithelial cells causes an inhibition of both hepatocyte growth factor- and epidermal growth factor-dependent process formation and migration. In contrast, ES does not inhibit epidermal growth factor-dependent morphogenesis in renal epithelial cells derived from Gpc-3 −/mice, whereas expression of Gpc-1 in these cells reconstitutes ES responsiveness. Gpc-3 −/mice have been shown to display enhanced ureteric bud (UB) branching early in development, and cultured UB cells release ES into the media, suggesting that ES binding to Gpcs may regulate UB branching. The addition of ES inhibits branching of the explanted UB, whereas a neutralizing Ab to ES enhances UB outgrowth and branching. Thus, local expression of ES at the tips of the UB may play a role in the regulation of UB arborization.

Handling of Drugs, Metabolites, and Uremic Toxins by Kidney Proximal Tubule Drug Transporters

The proximal tubule of the kidney plays a crucial role in the renal handling of drugs (e.g., diuretics), uremic toxins (e.g., indoxyl sulfate), environmental toxins (e.g., mercury, aristolochic acid), metabolites (e.g., uric acid), dietary compounds, and signaling molecules. This process is dependent on many multispecific transporters of the solute carrier (SLC) superfamily, including organic anion transporter (OAT) and organic cation transporter (OCT) subfamilies, and the ATP-binding cassette (ABC) superfamily. We review the basic physiology of these SLC and ABC transporters, many of which are often called drug transporters. With an emphasis on OAT1 (SLC22A6), the closely related OAT3 (SLC22A8), and OCT2 (SLC22A2), we explore the implications of recent in vitro, in vivo, and clinical data pertinent to the kidney. The analysis of murine knockouts has revealed a key role for these transporters in the renal handling not only of drugs and toxins but also of gut microbiome products, as well as liver-derived phase 1 and phase 2 metabolites, including putative uremic toxins (among other molecules of metabolic and clinical importance). Functional activity of these transporters (and polymorphisms affecting it) plays a key role in drug handling and nephrotoxicity. These transporters may also play a role in remote sensing and signaling, as part of a versatile small molecule communication network operative throughout the body in normal and diseased states, such as AKI and CKD.

Deletion of Multispecific Organic Anion Transporter Oat1/Slc22a6 Protects against Mercury-induced Kidney Injury

The primary site of mercury-induced injury is the kidney due to uptake of the reactive Hg2+-conjugated organic anions in the proximal tubule. Here, we investigated the in vivo role of Oat1 (organic anion transporter 1; originally NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478)) in handling of known nephrotoxic doses of HgCl2. Oat1 (Slc22a6) is a multispecific organic anion drug transporter that is expressed on the basolateral aspects of renal proximal tubule cells and that mediates the initial steps of elimination of a broad range of endogenous metabolites and commonly prescribed pharmaceuticals. Mercury-induced nephrotoxicity was observed in a wild-type model. We then used the Oat1 knock-out to determine in vivo whether the renal injury effects of mercury are mediated by Oat1. Most of the renal injury (both histologically and biochemically as measured by blood urea nitrogen and creatinine) was abolished following HgCl2 treatment of Oat1 knock-outs. Thus, acute kidney injury by HgCl2 was found to be mediated mainly by Oat1. Our findings raise the possibility that pharmacological modulation of the expression and/or function of Oat1 might be an effective therapeutic strategy for reducing renal injury by mercury. This is one of the most striking phenotypes so far identified in the Oat1 knock-out. (Eraly, S. A., Vallon, V., Vaughn, D. A., Gangoiti, J. A., Richter, K., Nagle, M., Monte, J. C., Rieg, T., Truong, D. M., Long, J. M., Barshop, B. A., Kaler, G., and Nigam, S. K. (2006) J. Biol. Chem. 281, 5072–5083).

β1-Integrin is required for kidney collecting duct morphogenesis and maintenance of renal function

Deletion of integrin-beta1 (Itgb1) in the kidney collecting system led to progressive renal dysfunction and polyuria. The defect in the concentrating ability of the kidney was concomitant with decreased medullary collecting duct expression of aquaporin-2 and arginine vasopressin receptor 2, while histological examination revealed hypoplastic renal medullary collecting ducts characterized by increased apoptosis, ectasia and cyst formation. In addition, a range of defects from small kidneys with cysts and dilated tubules to bilateral renal agenesis was observed. This was likely due to altered growth and branching morphogenesis of the ureteric bud (the progenitor tissue of the renal collecting system), despite the apparent ability of the ureteric bud-derived cells to induce differentiation of the metanephric mesenchyme. These data not only support a role for Itgb1 in the development of the renal collecting system but also raise the possibility that Itgb1 links morphogenesis to terminal differentiation and ultimately collecting duct function and/or maintenance.

Linkage of Organic Anion Transporter-1 to Metabolic Pathways through Integrated “Omics”-driven Network and Functional Analysis

The main kidney transporter of many commonly prescribed drugs (e.g. penicillins, diuretics, antivirals, methotrexate, and non-steroidal anti-inflammatory drugs) is organic anion transporter-1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478). Targeted metabolomics in knockouts have shown that OAT1 mediates the secretion or reabsorption of many important metabolites, including intermediates in carbohydrate, fatty acid, and amino acid metabolism. This observation raises the possibility that OAT1 helps regulate broader metabolic activities. We therefore examined the potential roles of OAT1 in metabolic pathways using Recon 1, a functionally tested genome-scale reconstruction of human metabolism. A computational approach was used to analyze in vivo metabolomic as well as transcriptomic data from wild-type and OAT1 knock-out animals, resulting in the implication of several metabolic pathways, including the citric acid cycle, polyamine, and fatty acid metabolism. Validation by in vitro and ex vivo analysis using Xenopus oocyte, cell culture, and kidney tissue assays demonstrated interactions between OAT1 and key intermediates in these metabolic pathways, including previously unknown substrates, such as polyamines (e.g. spermine and spermidine). A genome-scale metabolic network reconstruction generated some experimentally supported predictions for metabolic pathways linked to OAT1-related transport. The data support the possibility that the SLC22 and other families of transporters, known to be expressed in many tissues and primarily known for drug and toxin clearance, are integral to a number of endogenous pathways and may be involved in a larger remote sensing and signaling system (Ahn, S. Y., and Nigam, S. K. (2009) Mol. Pharmacol. 76, 481–490, and Wu, W., Dnyanmote, A. V., and Nigam, S. K. (2011) Mol. Pharmacol. 79, 795–805). Drugs may alter metabolism by competing for OAT1 binding of metabolites.

Analysis of Metagene Portraits Reveals Distinct Transitions During Kidney Organogenesis

Grouping microarray expression data into metagenes, followed by organization of these gene clusters into self-organizing maps, reveals distinct stages of kidney organogenesis. Revealing Kidney Formation Through Metagenes By collecting the more than 30,000 genes into 650 groups called metagenes, Tsigelny et al. uncover genes that may orchestrate the transitions between stages of kidney development. Organization of the metagenes into self-organizing maps revealed up to eight distinct stages of kidney development. Entropy calculations of the self-organizing maps allowed the metagene-defined stages to be correlated with morphometric parameters and with specific gene networks. Genes included those already known for their involvement in kidney development as well as ones previously not implicated in this process of organogenesis, thus yielding new insight. Organogenesis is a multistage process, but it has been difficult, by conventional analysis, to separate stages and identify points of transition in developmentally complex organs or define genetic pathways that regulate pattern formation. We performed a detailed time-series examination of global gene expression during kidney development and then represented the resulting data as self-organizing maps (SOMs), which reduced more than 30,000 genes to 650 metagenes. Further clustering of these maps identified potential stages of development and suggested points of stability and transition during kidney organogenesis that are not obvious from either standard morphological analyses or conventional microarray clustering algorithms. We also performed entropy calculations of SOMs generated for each day of development and found correlations with morphometric parameters and expression of candidate genes that may help in orchestrating the transitions between stages of kidney development, as well as macro- and micropatterning of the organ.