Biomedical subjects

Sanjay K Nigam

Publications and source records attributed to Sanjay K Nigam.

At least 19 recordsLinked to original sources

PubMed2007

The developmental nephrome: systems biology in the developing kidney.

PURPOSE OF REVIEW: A set of important genes and signaling pathways involved in kidney development is emerging from analyses of mutant mice, in-vitro models, and global gene expression patterns. Conversion of data into dynamic models or networks through the synthesis of information at multiple levels is crucial for a better understanding of kidney development. RECENT FINDINGS: Genetic and in-vitro evidence is beginning to provide a limited sense of the network topology in stages of kidney development. Intriguing data from other fields suggest how, with the aid of large-scale gene expression studies, these stages might be represented as dynamic attractor states. It is also suggested how branching morphogenesis of the epithelial ureteric bud may be sustained by an autocatalytic set of proteins whose interactions lead to repeated rounds of tip and stalk generation. Accumulating data in lower organisms suggest network topologies may be quite flexible, and the implications of these results for varieties of tubulogenesis and renal regeneration after acute injury are discussed. SUMMARY: Currently it may be feasible to build tentative dynamic multistage models of nephrogenesis that facilitate experimental thinking. As data accumulate, it may become possible to test their predictive value.

Animals↗

PubMed2006

Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high affinity interactions with odorant organic anions.

We have characterized the expression of organic anion transporter 6, Oat6 (slc22a20), in olfactory mucosa, as well as its interaction with several odorant organic anions. In situ hybridization reveals diffuse Oat6 expression throughout olfactory epithelium, yet olfactory neurons laser-capture microdissected from either the main olfactory epithelium (MOE) or the vomeronasal organ (VNO) did not express Oat6 mRNA. These data suggest that Oat6 is expressed in non-neuronal cells of olfactory tissue, such as epithelial and/or other supporting cells. We next investigated interaction of Oat6 with several small organic anions that have previously been identified as odortype components in mouse urine. We find that each of these compounds, propionate, 2- and 3-methylbutyrate, benzoate, heptanoate, and 2-ethylhexanoate, inhibits Oat6-mediated uptake of a labeled tracer, estrone sulfate, consistent with their being Oat6 substrates. Previously, we noted defects in the renal elimination of odortype and odortype-like molecules in Oat1 knockout mice. The finding that such molecules interact with Oat6 raises the possibility that odorants secreted into the urine through one OAT-mediated mechanism (Eraly et al., JBC 2006) are transported through the olfactory mucosa through another OAT-mediated mechanism. Oat6 might play a direct or indirect role in olfaction, such as modulation of the availability of odorant organic anions at the mucosal surface for presentation to olfactory neurons or facilitation of delivery to a distal site of chemosensation, among other possibilities that we discuss.

Animals↗

PubMed2006

Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system.

Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.

Actins↗

PubMed2006

Analyses of 5' regulatory region polymorphisms in human SLC22A6 (OAT1) and SLC22A8 (OAT3).

Kidney excretion of numerous organic anionic drugs and endogenous metabolites is carried out by a family of multispecific organic anion transporters (OATs). Two closely related transporters, SLC22A6, initially identified by us as NKT and also known as OAT1, and SLC22A8, also known as OAT3 and ROCT, are thought to mediate the initial steps in the transport of organic anionic drugs between the blood and proximal tubule cells of the kidney. Coding region polymorphisms in these genes are infrequent and pairing of these genes in the genome suggests they may be coordinately regulated. Hence, 5' regulatory regions of these genes may be important factors in human variation in organic anionic drug handling. We have analyzed novel single nucleotide polymorphisms in the evolutionarily conserved 5' regulatory regions of the SLC22A6 and SLC22A8 genes (phylogenetic footprints) in an ethnically diverse sample of 96 individuals (192 haploid genomes). Only one polymorphism was found in the SLC22A6 5' regulatory region. In contrast, seven polymorphisms were found in the SLC22A8 5' regulatory region, two of which were common to all ethnic groups studied. Computational analysis permitted phase and haplotype reconstruction. Proximity of these non-coding polymorphisms to transcriptional regulatory elements (including potential sex steroid response elements) suggests a potential influence on the level of transcription of the SLC22A6 and/or SLC22A8 genes and will help define their role in variation in human drug, metabolite and toxin excretion. The clustering of OAT genes in the genome raises the possibility that nucleotide polymorphisms in SLC22A6 could also effect SLC22A8 expression, and vice versa.

Animals↗

PubMed2006

Activin A is an endogenous inhibitor of ureteric bud outgrowth from the Wolffian duct.

Development of metanephric kidney begins with ureteric bud outgrowth from the Wolffian duct (WD). GDNF is believed to be a crucial positive signal in the budding process, but the negative regulation of this process remains unclear. Here, we examined the role of activin A, a member of TGF-beta family, in bud formation using an in vitro WD culture system. When cultured with the surrounding mesonephros, WDs formed many ectopic buds in response to GDNF. While the activin signaling pathway is normally active along the non-budding WD (as measured by expression of activin A and phospho-Smad2/3), activin A was absent and phospho-Smad2/3 was undetectable in the ectopic buds induced by GDNF. To examine the role of activin A in bud formation, we attempted to inactivate activin action. Interestingly, the addition of neutralizing anti-activin A antibody potentiated GDNF action. To further clarify the role of activin A, we also tested the effect of activin blockade on the WD cultured in the absence of mesonephros. WDs without mesonephros did not form ectopic buds even in the presence of GDNF. In contrast, blockade of activin action with a variety of agents acting through different mechanisms (natural antagonist, neutralizing antibodies, siRNA) enabled GDNF to induce ectopic buds. Inhibition of GDNF-induced bud formation by activin A was accompanied by inhibition of cell proliferation, reduced expression of Pax-2, and decreased phosphorylation of PI3-kinase and MAP kinase in the WD. Our data suggest that activin A is an endogenous inhibitor of bud formation and that cancellation of activin A autocrine action may be critical for the initiation of this process.

Activins↗

PubMed2006

Rho kinase acts at separate steps in ureteric bud and metanephric mesenchyme morphogenesis during kidney development.

In this study, five different in vitro assays, which together recapitulate much of kidney development, were used to examine the role of the Rho-associated protein serine/threonine kinase (ROCK) in events central to ureteric bud (UB) and metanephric mesenchyme (MM) morphogenensis, in isolation and together. ROCK activity was found to be critical for (1) cell proliferation, growth, and development of the whole embryonic kidney in organ culture, (2) tip and stalk formation in cultures of isolated UBs, and (3) migration of MM cells (in a novel MM migration assay) during their condensation at UB tips (in a UB/MM recombination assay). Together, the data indicate selective involvement of Rho/ROCK in distinct morphogenetic processes necessary for kidney development and that the coordination of these events by Rho/ROCK provides a potential mechanism to regulate overall branching patterns, nephron formation, and thus, kidney architecture.

Animals↗

PubMed2005

Decreased renal organic anion secretion and plasma accumulation of endogenous organic anions in OAT1 knock-out mice.

The "classical" organic anion secretory pathway of the renal proximal tubule is critical for the renal excretion of the prototypic organic anion, para-aminohippurate, as well as of a large number of commonly prescribed drugs among other significant substrates. Organic anion transporter 1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478), has physiological properties consistent with a role in this pathway. However, several other transporters (e.g. OAT2, OAT3, and MRP1) have also been proposed as important PAH transporters on the basis of in vitro studies; therefore, the relative contribution of OAT1 has remained unclear. We have now generated a colony of OAT1 knock-out mice, permitting elucidation of the role of OAT1 in the context of these other potentially functionally redundant transporters. We find that the knock-out mice manifest a profound loss of organic anion transport (e.g. para-aminohippurate) both ex vivo (in isolated renal slices) as well as in vivo (as indicated by loss of renal secretion). In the case of the organic anion, furosemide, loss of renal secretion in the knock-out results in impaired diuretic responsiveness to this drug. These results indicate a critical role for OAT1 in the functioning of the classical pathway. In addition, we have determined the levels of approximately 60 endogenous organic anions in the plasma and urine of wild-type and knock-out mice. This has led to identification of several compounds with significantly higher plasma concentrations and/or lower urinary concentrations in knock-out mice, suggesting the involvement of OAT1 in their renal secretion. We have also demonstrated in xenopus oocytes that some of these compounds interact with OAT1 in vitro. Thus, these latter compounds might represent physiological substrates of OAT1.

Animals↗

PubMed2005

Adult kidney tubular cell population showing phenotypic plasticity, tubulogenic capacity, and integration capability into developing kidney.

Using in vivo bromodeoxyuridine (BrdU) labeling, a tubular cell population (label-retaining tubular cells [LRTC]) was identified recently in normal adult kidneys, which contributes actively to the regeneration process of the kidney after injury. Here, these LRTC are characterized in vitro. The LRTC population was isolated from BrdU-treated rat kidney by FACS. Both LRTC and non-LRTC underwent proliferation and maintained an epithelial phenotype in the presence of tubulogenic growth factors such as EGF, TGF-alpha, IGF-I, and hepatocyte growth factor. It is interesting that LRTC also proliferated without epithelial markers expression in the presence of soluble factors derived from an embryonic kidney metanephric mesenchyme cell line. The type of extracellular matrix strongly influenced the phenotype of LRTC. Furthermore, in three-dimensional collagen gel culture, LRTC formed tubule-like or tubulocystic structures in response to growth factors (hepatocyte growth factor and fibroblast growth factor) that are known to induce kidney cell tubulogenesis in vitro and/or participate in renal regeneration in vivo. In contrast, non-LRTC did not form these structures. When transplanted into the metanephric kidney, LRTC but not non-LRTC were integrated into epithelial components of nephron, including the proximal tubular cells and the ureteric bud. They also differentiated into fibroblast-like cells. Collectively, these findings suggest that LRTC are an adult kidney tubular cell population that shows phenotypic plasticity, tubulogenic capacity, and integration capability into the developing kidney.

Animals↗

PubMed2005

Heregulin induces glial cell line-derived neurotrophic growth factor-independent, non-branching growth and differentiation of ureteric bud epithelia.

We have purified a protein present in a conditioned medium derived from the metanephric mesenchyme that supports non-branching growth and epithelial differentiation of the isolated ureteric bud (UB) independent of glial cell line-derived neurotrophic growth factor (GDNF). By sequential liquid chromatography, together with protein microsequencing, the protein was identified as heregulin (HRG)alpha. The addition of recombinant HRG to the isolated UB grown in three-dimensional culture confirmed the proliferative activity of HRG. In branching UBs induced by whole metanephric mesenchyme cell-conditioned medium, proliferating cells were localized at ampullae, where a binding receptor for GDNF, GFRalpha1, was found. In HRG-induced UBs, however, the expression of GFRalpha1 was down-regulated, and proliferating cells were distributed throughout the structure. Electron microscopic examination of the HRG-induced UB revealed the presence of structurally mature and polarized epithelial cells reminiscent of the epithelial cells found in the stalk portion of the branching UB. cDNA array analysis further revealed that genes ontologically classified as developmental were down-regulated by HRG, whereas those involved in transport were up-regulated. For example, the mRNA for the GDNF receptors, GFRalpha1 and ret9, was down-regulated, whereas the mRNA for collecting duct transporters, such as urea transporter2, aquaporin3, and sodium-hydrogen exchanger2 was up-regulated in HRG-treated UBs compared with UBs grown in the presence of branch-promoting factors. Moreover, HRG promoted growth of UBs cultured in the absence of GDNF. Taken together, the data suggest that HRG supports UB epithelial cell differentiation and non-GDNF-dependent growth, raising the possibility that this kind of activity plays a role in the growth and differentiation of the stalk portion of the branching epithelial UB.

Animals↗

PubMed2005

Analyses of coding region polymorphisms in apical and basolateral human organic anion transporter (OAT) genes [OAT1 (NKT), OAT2, OAT3, OAT4, URAT (RST)].

BACKGROUND: Excretion by the kidney of a variety of organic anionic drugs and metabolites is mediated by a family of multispecific organic anion transporters (OAT genes) that are part of the SLC22 family of solute carriers. Different OATs localize to the apical (OAT2, OAT4, and RST/URAT) or basolateral (OAT1/NKT and OAT3) membranes of the renal proximal tubule; the net transport of organic anions from blood to urine is believed to require both apical and basolateral OATs. These genes are also thought to mediate transport of organic anionic drugs and metabolites (e.g., urate) across choroid plexus, retina, placenta, and possibly olfactory mucosa. The extent of functional redundancy among OATs remains uncertain, but closely related OAT genes are tightly linked in the genome. Hence, a better understanding of human variation in organic anionic drug excretion may be obtained by studying OAT genes in combination rather than individually. METHODS: We have analyzed single nucleotide polymorphisms (SNPs) in OAT1 (NKT), OAT2, OAT3, OAT4, and URAT1 (human homologue of RST) in an ethnically diverse sample of 96 individuals (192 haploid genomes). Ka/Ks analysis was also performed as well as haplotype reconstruction using the software program Arelquin. RESULTS: The data indicate that (1) nonsynonymous SNPs in OAT1 and OAT3 may not be frequent so it will be important to consider promoter region SNPs that regulate gene expression; (2) certain ethnic groups may have a high prevalence of nonsynonymous SNPs in particular OATs (e.g., OAT4 in Sub-Saharan Africans); (3) there are individuals who have nonsynonymous SNPs in apical and basolateral OATs; (4) nonsynonymous OAT4 SNPs may be more frequent, raising the possibility of altered maternofetal transport of drugs and metabolites; and (5) combinations of synonymous SNPs in OAT1 and OAT3 also occur in certain individuals. In addition, Ka/Ks analysis of human, chimp and rodent genes suggests that OAT4 is under accelerated selection pressure, perhaps reflecting specific human environmental exposures during evolution. In contrast, Ka/Ks analysis for URAT1 suggests decelerated selection pressure. Haplotype reconstruction also supports this view. CONCLUSION: Together, these data suggest that, in order to understand the effect of SNPs in genes of the SLC22 family on drug handling as well as excretion of metabolites like uric acid, it is important to consider the entire set of organic anion transporters. It will be particularly interesting to determine if individuals with nonsynonymous apical and basolateral SNPs have altered handling (and toxicity) of organic anionic drugs and metabolites. Certain OAT family members appear to be under greater evolutionary selection pressure.

Carrier Proteins↗

PubMed2004

Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney.

In search of guiding principles involved in the branching of epithelial tubes in the developing kidney, we analyzed branching of the ureteric bud (UB) in whole kidney culture as well as in isolated UB culture independent of mesenchyme but in the presence of mesenchymally derived soluble factors. Microinjection of the UB lumen (both in the isolated UB and in the whole kidney) with fluorescently labeled dextran sulfate demonstrated that branching occurred via smooth tubular epithelial outpouches with a lumen continuous with that of the original structure. Epithelial cells within these outpouches cells were wedge-shaped with actin, myosin-2 and ezrin localized to the luminal side, raising the possibility of a "purse-string" mechanism. Electron microscopy and decoration of heparan sulfates with biotinylated FGF2 revealed that the basolateral surface of the cells remained intact, without the type of cytoplasmic extensions (invadopodia) that are seen in three-dimensional MDCK, mIMCD, and UB cell culture models of branching tubulogenesis. Several growth factor receptors (i.e., FGFR1, FGFR2, c-Ret) and metalloproteases (i.e., MT1-MMP) were localized toward branching UB tips. A large survey of markers revealed the ER chaperone BiP to be highly expressed at UB tips, which, by electron microscopy, are enriched in rough endoplasmic reticulum and Golgi, supporting high activity in the synthesis of transmembrane and secretory proteins at UB tips. After early diffuse proliferation, proliferating and mitotic cells were mostly found within the branching ampullae, whereas apoptotic cells were mostly found in stalks. Gene array experiments, together with protein expression analysis by immunoblotting, revealed a differential spatiotemporal distribution of several proteins associated with epithelial maturation and polarization, including intercellular junctional proteins (e.g., ZO-1, claudin-3, E-cadherin) and the subapical cytoskeletal/microvillar protein ezrin. In addition, Ksp-cadherin was found at UB ampullary cells next to developing outpouches, suggesting a role in epithelial-mesenchymal interactions. These data from the isolated UB culture system support a model where UB branching occurs through outpouching possibly mediated by wedge-shaped cells created through an apical cytoskeletal purse-string mechanism. Additional potential mechanisms include (1) differential localization of growth factor receptors and metalloproteases at tips relative to stalks; (2) creation of a secretory epithelium, in part manifested by increased expression of the ER chaperone BiP, at tips relative to stalks; (3) after initial diffuse proliferation, coexistence of a balance of proliferation vs. apoptosis favoring tip growth with a very different balance in elongating stalks; and (4) differential maturation of the tight and adherens junctions as the structures develop. Because, without mesenchyme, both lateral and bifid branching occurs (including the ureter), the mesenchyme probably restricts lateral branching and provides guidance cues in vivo for directional branching and elongation as well as functioning to modulate tubular caliber and induce differentiation. Selective cadherin, claudin, and microvillar protein expression as the UB matures likely enables the formation of a tight, polarized differentiated epithelium. Although, in vivo, metanephric mesenchyme development occurs simultaneously with UB branching, these studies shed light on how (mesenchymally derived) soluble factors alone regulate spatial and temporal expression of morphogenetic molecules and processes (proliferation, apoptosis, etc.) postulated to be essential to the UB branching program as it forms an arborized structure with a continuous lumen.

Animals↗

PubMed2004

Identification of a novel murine organic anion transporter family member, OAT6, expressed in olfactory mucosa.

The organic anion and cation transporters (OATs and OCTs) are a large family (SLC22) of transmembrane proteins that are able to transport a variety of compounds including drugs, environmental toxins, and endogenous metabolites. OATs are expressed in various tissues, primarily kidney and liver, but also in placenta, small intestine, and choroid plexus, which are all epithelial tissues that transport xenobiotics. The upper airway, particularly the nose, is also a site of frequent exposure to environmental toxins. Many drugs are administered intranasally. This raises the possibility that the olfactory epithelium contains OATs and OCTs. Here, we report the identification of a novel putative transporter, mouse OAT6, expressed predominantly in olfactory mucosa but not in kidney or brain. Sequence comparisons and intron phasing analysis indicated that OAT6 is closely related to OAT1 and OAT3. Unlike many other slc22 genes, OAT6 is unpaired in the genome, although it is in proximity to the OAT1/OAT3 gene pair. Expression of OAT6 was also observed in testis. Embryonic expression was observed at day 7, but not later in embryogenesis. This might be due to the need for a key metabolite transported by OAT6. The data raise the possibility that the olfactory mucosa may have a significant transport apparatus which could be important in the design of new therapeutic approaches for direct nose-to-brain transfer of drugs and olfaction. Supporting this possibility, we have demonstrated that OAT1, OCT1-2, and OCTN1-3 are also expressed in olfactory mucosa. Furthermore, e-blot data suggest very different expression of individual OATs, OCTs and OCTNs in kidney, brain, liver, and eye.

Aging↗

PubMed2004

Tunicamycin preserves intercellular junctions, cytoarchitecture, and cell-substratum interactions in ATP-depleted epithelial cells.

Pretreatment with the nucleoside antibiotic tunicamycin was found to protect cultured renal epithelial cells in the face of ATP-depletion, in large part by preserving junctional and cellular architecture. Tunicamycin pretreatment of Madin-Darby canine kidney cells not only preserved E-cadherin staining at the plasma membrane, but also inhibited ATP-depletion-mediated E-cadherin degradation. Electron microscopic analysis, together with the preservation of the staining patterns of the tight junction marker ZO-1, the apical/microvillar marker gp135, and basolateral marker Na/K-ATPase suggested that tunicamycin preserved the junctional complex and the polarized epithelial cell phenotype. Tunicamycin pretreatment also prevented reductions in the filamentous actin content of the cells, as well as preserving Golgi architecture. Moreover, a quantitative measure of cell adhesion demonstrated that tunicamycin pretreatment resulted in a fivefold increase in attachment of cells to the substratum (77% versus 16%). Thus, pretreatment with tunicamycin protects polarized epithelial cells from ischemic injury through the preservation of epithelial cell architecture, intercellular junctions, and cell-substratum interactions in the setting of intracellular ATP-depletion.

Adenosine Triphosphate↗

PubMed2004

Regulation of ureteric bud branching morphogenesis by sulfated proteoglycans in the developing kidney.

Glycosaminoglycans in the form of heparan sulfate proteoglycans (HSPG) and chondroitin sulfate proteoglycans (CSPG) are required for normal kidney organogenesis. The specific roles of HSPGs and CSPGs on ureteric bud (UB) branching morphogenesis are unclear, and past reports have obtained differing results. Here we employ in vitro systems, including isolated UB culture, to clarify the roles of HSPGs and CSPGs on this process. Microarray analysis revealed that many proteoglycan core proteins change during kidney development (syndecan-1,2,4, glypican-1,2,3, versican, decorin, biglycan). Moreover, syndecan-1, syndecan-4, glypican-3, and versican are differentially expressed during isolated UB culture, while decorin is dynamically regulated in cultured isolated metanephric mesenchyme (MM). Biochemical analysis indicated that while both heparan sulfate (HS) and chondroitin sulfate (CS) are present, CS accounts for approximately 75% of the glycosaminoglycans (GAG) in the embryonic kidney. Selective perturbation of HS in whole kidney rudiments and in the isolated UB resulted in a significant reduction in the number of UB branch tips, while CS perturbation has much less impressive effects on branching morphogenesis. Disruption of endogenous HS sulfation with chlorate resulted in diminished FGF2 binding and proliferation, which markedly altered kidney area but did not have a statistically significant effect on patterning of the ureteric tree. Furthermore, perturbation of GAGs did not have a detectable effect on FGFR2 expression or epithelial marker localization, suggesting the expression of these molecules is largely independent of HS function. Taken together, the data suggests that nonselective perturbation of HSPG function results in a general proliferation defect; selective perturbation of specific core proteins and/or GAG microstructure may result in branching pattern defects. Despite CS being the major GAG synthesized in the whole developing kidney, it appears to play a lesser role in UB branching; however, CS is likely to be integral to other developmental processes during nephrogenesis, possibly involving the MM. A model is presented of how, together with growth factors, heterogeneity of proteoglycan core proteins and glycosaminoglycan sulfation act as a switching mechanism to regulate different stages of the branching process. In this model, specific growth factor-HSPG combinations play key roles in the transitioning between stages and their maintenance.

Animals↗

PubMed2004

Novel slc22 transporter homologs in fly, worm, and human clarify the phylogeny of organic anion and cation transporters.

Slc22 family organic anion and cation transporters (OATs, OCTs, and OCTNs) are transmembrane proteins expressed predominantly in kidney and liver. These proteins mediate the uptake or excretion of numerous physiologically (and pharmacologically) important compounds, and accordingly have been the focus of intensive study. Here we investigate the molecular phylogeny of the slc22 transporters, identifying homologs in Drosophila and C. elegans, several of which are developmentally regulated, as well as reporting the cloning of a novel human family member, UST6, expressed exclusively in liver in both embryo and adult. The latter helps define a subfamily within the OATs, which appears to have human- and rodent-specific members, raising potential issues with respect to the use of rodents as models for the transport of organic anions (which include many pharmaceuticals) in humans. Although this phylogenetic inference could not be made on the basis of sequence alignment, analysis of intron phasing suggests that the OAT, OCT, and OCTN lineages of the slc22 family formed after the divergence of vertebrates and invertebrates. Subsequently, these lineages expanded through independent tandem duplications to produce multiple gene pairs. After analyzing over 200 other transporter genes, we find such pairing to be relatively specific to vertebrate organic anion and cation transporters, suggesting selection for gene pairing operating within this family in particular. This might reflect a requirement for redundancy or broader substrate specificity in vertebrates (compared to invertebrates), due to their greater physiological complexity and thus potentially broader exposure to organic ions.

Amino Acid Sequence↗

PubMed2004

TGF-beta superfamily members modulate growth, branching, shaping, and patterning of the ureteric bud.

Protein-rich fractions inhibitory for isolated ureteric bud (UB) growth were separated from a conditioned medium secreted by cells derived from the metanephric mesenchyme (MM). Elution profiles and immunoblotting indicated the presence of members of the transforming growth factor-beta (TGF-beta) superfamily. Treatment of cultured whole embryonic kidney with BMP2, BMP4, activin, or TGF-beta1 leads to statistically significant differences in the overall size of the kidney, the number of UB branches, the length and angle of the branches, as well as in the thickness of the UB stalks. Thus, the pattern of the ureteric tree is altered. LIF, however, appeared to have only minimal effect on growth and development of the whole embryonic kidney in organ culture. The factors all directly inhibited, in a concentration-dependent fashion, the growth and branching of the isolated UB, albeit to different extents. Antagonists of some of these factors reduced their inhibitory effect. Detailed examination of TGF-beta1-treated UBs revealed only a slight increase in the amount of apoptosis in tips by TUNEL staining, but diminished proliferation throughout by Ki67 staining. These data suggest an important direct modulatory role for BMP2, BMP4, LIF, TGF-beta1, and activin (as well as their antagonists) on growth and branching of the UB, possibly in shaping the growing UB by playing a role in determining the number of branches, as well as where and how the branches occur. In support of this notion, UBs cultured in the presence of fibroblast growth factor 7 (FGF7), which induces the formation of globular structures with little distinction between the stalk and ampullae [Mech. Dev. 109 (2001) 123], and TGF-beta superfamily members lead to the formation of UBs with clear stalks and ampullae. This indicates that positive (i.e., growth and branch promoting) and negative (i.e., growth and branch inhibiting) modulators of UB morphogenesis can cooperate in the formation of slender arborized UB structures similar to those observed in the intact developing kidney or in whole embryonic kidney organ culture. Finally, purification data also indicate the presence of an as yet unidentified soluble non-heparin-binding activity modulating UB growth and branching. The data suggest how contributions of positive and negative growth factors can together (perhaps as local bipolar morphogenetic gradients existing within the mesenchyme) modulate the vectoral arborization pattern of the UB and shape branches as they develop, thereby regulating both nephron number and tubule/duct caliber. We suggest that TGF-beta-like molecules and other non-heparin-binding inhibitory factors can, in the appropriate matrix context, facilitate "braking" of the branching program as the UB shifts from a rapid branching stage (governed by a feed-forward mechanism) to a stage where branching slows down (negative feedback) and eventually stops.

Animals↗

PubMed2004

The molecular pharmacology of organic anion transporters: from DNA to FDA?

Renal organic anion secretion has been implicated in numerous clinically significant drug interactions and adverse reactions, indicating the importance of a detailed understanding of this pathway for the development of optimum therapeutics. With the cloning of multiple genes encoding organic anion transporters (OATs), the study of organic anion secretion has entered the molecular age. In this review, we focus on various aspects of the molecular biology and pharmacology of the OATs, including discussion of their structural biology, genomic organization in pairs, developmental regulation, toxicology, and pharmacogenetics. We propose functional, pathophysiological, and evolutionary hypotheses to help explain recent experimental and genomic data.

Animals↗

PubMed2004

Developmental approaches to kidney tissue engineering.

Recent advances in our understanding of the developmental biology of the kidney, as well as the establishment of novel in vitro model systems, have potential implications for kidney tissue engineering. These advances include delineation of the roles of a number of growth factors in the developmental programs of branching morphogenesis and mesenchymal differentiation, a new understanding of the roles of the extracellular matrix, identification of potential "renal" stem cells, the ex vivo propagation and subsequent recombination of isolated components of the kidney, and successful transplantation of renal primordia into adult hosts. This review will examine these advances in the context of approaches to tissue engineering. Finally, novel approaches that synthesize advances in both cell-based and organ-based approaches are proposed.

Animals↗