Human Ontogeny of Drug Transporters: Review and Recommendations of the Pediatric Transporter Working Group.

The critical importance of membrane-bound transporters in pharmacotherapy is widely recognized, but little is known about drug transporter activity in children. In this white paper, the Pediatric Transporter Working Group presents a systematic review of the ontogeny of clinically relevant membrane transporters (e.g., SLC, ABC superfamilies) in intestine, liver, and kidney. Different developmental patterns for individual transporters emerge, but much remains unknown. Recommendations to increase our understanding of membrane transporters in pediatric pharmacotherapy are presented.

How much do we know about drug handling by SLC and ABC drug transporters in children?

Although solute carrier (SLC) and ATP-binding cassette (ABC) transporters are critical to the absorption, distribution, and elimination of many small-molecule drugs in children, how these transporters regulate pediatric drug handling remains unclear. For proper dosing and to diminish toxicity, we need a better understanding of how organ development and functional maturation, as well as developmental changes in systemic physiology, impact transporter-mediated drug handling at pediatric developmental stages from the preterm infant through adolescence.

Staged in vitro reconstitution and implantation of engineered rat kidney tissue.

A major hurdle for current xenogenic-based and other approaches aimed at engineering kidney tissues is reproducing the complex three-dimensional structure of the kidney. Here, a stepwise, in vitro method of engineering rat kidney-like tissue capable of being implanted is described. Based on the fact that the stages of kidney development are separable into in vitro modules, an approach was devised that sequentially induces an epithelial tubule (the Wolffian duct) to undergo in vitro budding, followed by branching of a single isolated bud and its recombination with metanephric mesenchyme. Implantation of the recombined tissue results in apparent early vascularization. Thus, in principle, an unbranched epithelial tubular structure (potentially constructed from cultured cells) can be induced to form kidney tissue such that this in vitro engineered tissue is capable of being implanted in host rats and developing glomeruli with evidence of early vascularization. Optimization studies (of growth factor and matrix) indicate multiple suitable combinations and suggest both a most robust and a minimal system. A whole-genome microarray analysis suggested that recombined tissue recapitulated gene expression changes that occur in vivo during later stages of kidney development, and a functional assay demonstrated that the recombined tissue was capable of transport characteristic of the differentiating nephron. The approach includes several points where tissue can be propagated. The data also show how functional, 3D kidney tissue can assemble by means of interactions of independent modules separable in vitro, potentially facilitating systems-level analyses of kidney development.

Organic anion and cation transporter expression and function during embryonic kidney development and in organ culture models.

Organic anion and cation transporters (OATs, OCTs, and OCTNs) mediate the proximal tubular secretion of numerous clinically important compounds, including various commonly prescribed pharmaceuticals. Here, we report determination of the ontogeny of these transporters and of NaP(i)2 and SGLT1, using quantitative polymerase chain reaction (QPCR) to determine expression levels of transporter genes in rat embryonic kidneys on each day of gestation from embryonic day (ed) 13 to ed18, in cultures of induced and uninduced metanephric mesenchyme (MM), and on each day of 1 week of whole embryonic kidney (WEK) culture. We also examined ontogeny of Oat1 protein expression in rat embryonic kidney by immunohistochemistry. Finally, we used uptake of fluorescein (FL) as a novel in vitro functional assay of OAT expression in WEK and MM. Developmental induction of OAT and OCT genes does not occur uniformly: some genes are induced early (e.g., Oat1 and Oat3, potential early markers of proximal tubulogenesis), and others after kidney development is relatively advanced (e.g., Oct1, a potential marker of terminal differentiation). The ontogeny of transporter genes in WEK and MM is similar to that observed in vivo, indicating that these organ culture systems may represent convenient in vitro models to study the developmental induction of OATs, OCTs, and OCTNs. Functional transport was evidenced by accumulation of FL in the developing tubule in WEK and MM organ cultures. Our findings on the renal ontogeny of OATs and OCTs could carry implications both for the development of more rational therapeutics for premature infants, as well as for our understanding of proximal tubule differentiation.

Multiple fibroblast growth factors support growth of the ureteric bud but have different effects on branching morphogenesis.

Together with glial-derived neurotrophic factor (GDNF), soluble factors present in a metanephric mesenchyme (MM) cell conditioned medium (BSN-CM) are necessary to induce branching morphogenesis of the isolated ureteric bud (UB) in vitro (Proc. Natl. Acad. Sci. USA 96 (1999) 7330). Several lines of evidence are presented here in support of a modulating role for fibroblast growth factors (FGFs) in this process. RT-PCR revealed the expression of two FGF receptors, FGFR1(IIIc) and FGFR2(IIIb), in isolated embryonic day 13 rat UBs, which by indirect immunofluorescence displayed a uniform distribution. Rat kidney organ culture experiments in the presence of a soluble FGFR2(IIIb) chimera or a neutralizing antibody to FGF7 suggested an important contribution of FGFs other than FGF7 to the branching program. Several FGFs, including FGF1, FGF2, FGF7 and FGF10, in combination with GDNF and BSN-CM were found to affect growth and branching of the isolated UB, albeit with very different effects. FGF1 and FGF7 were at extreme ends of the spectrum, with FGF10 (more FGF1-like) and FGF2 (more FGF7-like) falling in between. FGF1 induced the formation of elongated UB branching stalks with distinct proliferative ampullary tips, whereas FGF7 induced amorphous buds displaying nonselective proliferation with little distinction between stalks and ampullae. Electron microscopic examination demonstrated that FGF1 treatment induced cytoskeletal organization, intercellular junctions and lumens along the stalk portion of the developing tubules, while the ampullary regions contained 'less differentiated' cells with an abundant secretory apparatus. In contrast, FGF7-induced UBs displayed this 'less differentiated' morphology regardless of position on the structure and were virtually indistinguishable from FGF1-induced ampullae. Consistent with this, GeneChip array analysis (employing a novel nanogram-scale assay consisting of two rounds of amplification and in vitro transcription for analyzing small quantities of RNA) revealed that FGF7-induced UBs expressed more markers of cell proliferation than FGF1, which caused the UB to express cytoskeletal proteins, extracellular matrix proteins, and at least one integrin, some of which may be important in UB branch elongation. Thus, while the various FGFs examined all support UB growth, FGF1 and FGF10 appear to be more important for branching and branch elongation, and may thus play a role in determination of nephron number and patterning in the developing kidney. These in vitro data may help to explain results from knockout and transgenic studies and suggest how different FGFs may, together with GDNF and other factor(s) secreted by MM cells, regulate branching morphogenesis of the UB by their relative effects on its growth, branching and branch elongation and differentiation, thereby affecting patterning in the developing kidney.

EEG1, a putative transporter expressed during epithelial organogenesis: comparison with embryonic transporter expression during nephrogenesis.

A screen for genes differentially regulated in a model of kidney development identified the novel gene embryonic epithelia gene 1 (EEG1). EEG1 exists as two transcripts of 2.4 and 3.5 kb that are most highly expressed at embryonic day 7 and later in the fetal liver, lung, placenta, and kidney. The EEG1 gene is composed of 14 exons spanning a 20-kb region at human chromosome 11p12 and the syntenic region of mouse chromosome 2. Six EEG1 exons have previously been assigned to a longer isoform of eosinophil major basic protein termed proteoglycan 2. Another gene distantly related to EEG1, POV1/PB39, is located 88 kb upstream from the EEG1 gene on chromosome 11. Temporal expression of 65 members of the solute carrier (SLC)-class of transport proteins was followed during kidney development using DNA arrays. POV-1 and EEG1, like glucose transporters, displayed very early maximal gene expression. In contrast, other SLC genes, such as organic anion and cation transporters, amino acid permeases, and nucleoside transporters, had maximal expression later in development. Thus, although the bulk of transporters are expressed late in kidney development, a fraction are expressed near the onset of nephrogenesis. The data raise the possibility that EEG1 and POV1 may define a new family of transport proteins involved in the transport of nutrients or metabolites in rapidly growing and/or developing tissues.

Identification of pleiotrophin as a mesenchymal factor involved in ureteric bud branching morphogenesis.

Branching morphogenesis is central to epithelial organogenesis. In the developing kidney, the epithelial ureteric bud invades the metanephric mesenchyme, which directs the ureteric bud to undergo repeated branching. A soluble factor(s) in the conditioned medium of a metanephric mesenchyme cell line is essential for multiple branching morphogenesis of the isolated ureteric bud. The identity of this factor had proved elusive, but it appeared distinct from factors such as HGF and EGF receptor ligands that have been previously implicated in branching morphogenesis of mature epithelial cell lines. Using sequential column chromatography, we have now purified to apparent homogeneity an 18 kDa protein, pleiotrophin, from the conditioned medium of a metanephric mesenchyme cell line that induces isolated ureteric bud branching morphogenesis in the presence of glial cell-derived neurotrophic factor. Pleiotrophin alone was also found to induce the formation of branching tubules in an immortalized ureteric bud cell line cultured three-dimensionally in an extracellular matrix gel. Consistent with an important role in ureteric bud morphogenesis during kidney development, pleiotrophin was found to localize to the basement membrane of the developing ureteric bud in the embryonic kidney. We suggest that pleiotrophin could act as a key mesenchymally derived factor regulating branching morphogenesis of the ureteric bud and perhaps other embryonic epithelial structures.

Expanding role of G proteins in tight junction regulation: Galpha(s) stimulates TJ assembly.

Multiple signaling mechanisms regulate epithelial cell tight junction (TJ) assembly and maintenance. Several G proteins are likely to regulate these processes, but only G(i/o) have been specifically tested. Treatment of MDCK cells with cholera toxin, a Galpha(s) activator, accelerated TJ development in the calcium switch as measured by the time to half-maximal [T(50) (H)] transepithelial resistance (TER). Galpha(s) was predominantly localized in the lateral membrane, but a fraction colocalizes with ZO-1 in the TJ. MDCK cell lines expressing epitope-tagged Galpha(s) and constitutively active (R201Calpha(s)) showed a similar localization. TJ assembly was significantly faster in R201Calpha(s)-MDCK cell lines (T(50) (H) of 1.7 versus 3.3 h for controls) without detectable differences in cAMP levels. Confocal studies showed R201Calpha(s)-MDCK cells more rapidly localized ZO-1 and occludin into the developing TJ without affecting E-cadherin or Na(+)/K(+) ATPase localization. Endogenous Galpha(s) and R201Calpha(s) were immunoprecipitated with ZO-1 at baseline and during TJ assembly. The data supports a model of multiple Galpha subunits interacting with TJ proteins to regulate the assembly and maintenance of the TJ.

The organic anion transporter family: from physiology to ontogeny and the clinic.

The organic anion transporter (OAT) family handles a wide variety of clinically important compounds (antibiotics, nonsteriodal anti-inflammatory drugs, etc.) and toxins. However, little is known about their appearance during development despite documented differences in the handling of anionic drugs among neonates, children, and adults. A similar spatiotemporal pattern of mRNA expression of the OATs (OAT1-4) during kidney development suggests that OAT genes may be useful in understanding the mechanisms of proximal tubule maturation. Moreover, OAT expression in unexpected extrarenal sites (e.g., spinal cord, bone, skin) has also been detected during development, possibly indicating a role for these transporters in the formation or preservation of extrarenal tissues. The cloning of these transporters also paves the way for computer-based modeling of drug-transporter interactions at the molecular level, potentially aiding in the design and assessment of new drugs. Additionally, increased understanding of single nucleotide polymorphisms in OATs and other transporters may eventually allow the use of a patient's expression profile and polymorphisms to individualize drug therapy.

Effect of sensitized macrophages in transplantable sarcoma in mice. synergistic effect of hyperimmunized serum, sensitized spleen cells and macrophages in tumor transplantation.

BACKGROUND: Immunosuppression cannot develop tumors by itself. It may induce tumors under appropriate conditions and may accelerate tumor development which may be reversed by sensitized spleen cells. This study concerns the effect of sensitized macrophages in murine-transplantable sarcomas by a combination of hyperimmune serum, sensitized spleen cells and macrophages. METHODS: The main technique adopted was intraperitoneal (ip) injection of 2 mL of 10% protease peptone broth, followed 2 days later by inoculation into the peritoneum with 10-mL Hank isotonic solution. The cells from the pooled peritoneal fluid were tested by dye exclusion test to ascertain the percentage of live cells. They were tried in whole body-irradiated mice with a combination of immune serum and sensitized spleen cells to ascertain whether a suppression of growth of solid tumors could be achieved when subcutaneously (sc) administered with the previously mentioned combinations. RESULTS: The addition of immunomacrophages from transplanted tumor-bearing mice significantly suppressed the growth of subcutaneous solid tumors when the number of tumor cells was kept constant. A change in number of immunomacrophages from hyperimmunized mice at a ratio of 4:l showed a direct relationship in suppression of tumor growth. Experiments were initiated in which tumor cells were injected sc and peritoneal macrophages were injected either intravenously (iv) or ip. Experiments were then initiated to prove that cell-to-cell contact is essential for tumor suppression. In experiments in which tumor cells were administered sc and macrophages injected either iv or ip, a significant immunosuppressive effect was not shown, thus also indicating that regardless of which, cell-to-cell contact is an absolutely essential factor involved in tumor suppression. A combination of hyperimmune serum and macrophages was found to act synergistically. Macrophages and hyperimmune serum at a lesser proportion did not suppress tumor growth. Sensitized macrophages and spleen cells together significantly suppressed tumor growth in a pure isogenic strain of irradiated mice. The sensitized macrophages injected iv prolonged the survival period and retarded tumor growth. CONCLUSIONS: Tumor suppression by macrophages was found to be due to its contact with tumor cells that enables the effective transfer of immunity. Hyperimmune serum and other cells (macrophages, spleen lymphocytes) act synergistically toward each other and prolong the survival period.

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-alpha, TGF-beta2, 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.

Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity.

Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-D-glucose) did not induce these changes. The intracellular hydrogen peroxide scavenger pyruvate protected Madin-Darby canine kidney cells from loss of transepithelial resistance as did the heavy metal scavenger N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the lateral membrane, was detected by immunofluorescence. The tyrosine kinase inhibitors genistein and PP-2 inhibited the recovery of transepithelial resistance and perturbed the relocalization of ZO-1 and occludin to the tight junction, indicating that tyrosine kinases, possibly members of the Src family, are critical for reassembly after oxidative stress.

Effect of modified tumor antigen on experimentally induced sarcomas.

OBJECTIVE: To explore the possibility of controlling established tumors by active immunization through specific and nonspecific methods. METHODS: By subcutaneous methylcholanthrene, a fibrosarcoma was produced in adult Swiss male mice. The fibrosarcoma was transplanted into the isogenic strain. The cleared tumor cells were injected subcutaneous into the hind leg of the 1st sarcoma group. The 2nd group received intraperitoneally sensitized spleen cells. One section of the irradiated 3rd tumor group received intraperitoneally sensitized spleen cells and subsequently a mild dose of modified tumor antigen. The 2nd section of the 3rd group was irradiated in between the administration of modified tumor antigen. In both the groups, liver of normal and transplanted tumor bearing mice was processed and intraperitoneally injected into the isogenic tumor bearing mice. Histopathology and tumor size by calipers was assessed. RESULTS: The first group showed enhancement of tumor growth in all its 3 fractions injected at different intervals. In the 2nd group, the average survival period was prolonged. In the first section of the 3rd group a decrease in tumor size and protracted survival was noted. In the transplanted tumor bearing mice, complete suppression of tumor growth was observed. In the 2nd fraction of the 3rd group, long survival period with regression of tumor was observed. In the 4th group, attenuated tumor and one mouse was observed to become tumor free after 60 days. CONCLUSION: Active immunosuppression by sensitized spleen cells and vaccines from transplanted sarcomas enabled regression of tumor size and longevity in tumor bearing mice. The modified tumor antigens, processed isogenic liver cells attenuated to zero level in tumor size in isogenic transplanted tumor bearing mice. The results show vaccines from allogenic and syngeneic sources bear the potential to regress tumor and enhance survival period.

Molecular cloning and functional analysis of mouse C-terminal kinesin motor KifC3.

Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. To study the molecular mechanism of intracellular transport involving microtubule-dependent motors, a cDNA encoding a new kinesin-like protein called KifC3 was cloned from a mouse brain cDNA library. Sequence and secondary structure analysis revealed that KifC3 is a member of the C-terminal motor family. In contrast to other mouse C-terminal motors, KifC3 is apparently ubiquitous and may have a general role in intracellular transport. To understand the in vivo function of the KifC3 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC3 gene. Homozygous mutants of the KifC3 gene are viable, reproduce normally, and apparently develop normally. These results suggest that KifC3 is dispensable for normal development and reproduction in the mouse.

Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development.

Branching morphogenesis of the ureteric bud (UB) [induced by the metanephric mesenchyme (MM)] is necessary for normal kidney development. The role of integrins in this complex developmental process is not well understood. However, the recent advent of in vitro model systems to study branching of UB cells and isolated UB tissue makes possible a more detailed analysis of the integrins involved. We detected integrin subunits alpha3, alpha6, beta1, and beta4 in both the UB and cells derived from the early UB. Blocking the function of each of these integrin subunits individually markedly inhibited branching morphogenesis in cell culture models. However, inhibiting individual integrin function with blocking antibodies in whole kidney and isolated UB culture only partially inhibited UB branching morphogenesis, suggesting that, in these more complex in vitro systems, multiple integrins are involved in the branching program. In whole organ and isolated bud culture, marked retardation of UB branching was observed only when both alpha3 and alpha6 integrin subunits were inhibited. The alpha6 integrin subunit can be expressed as both alpha6beta1 and alpha6beta4, and both of these beta subunits are important for UB branching morphogenesis in both cell and organ culture. Furthermore, laminin-5, a common ligand for integrins alpha3beta1 and alpha6beta4, was detected in the developing UB and shown to be required for normal UB branching morphogenesis in whole embryonic kidney organ culture as well as isolated UB culture. Together, these data from UB cell culture, organ culture, and isolated UB culture models indicate that both integrin alpha3 and alpha6 subunits play a direct role in UB branching morphogenesis, as opposed to being modulators of the inductive effects of mesenchyme on UB development. Furthermore the data are consistent with a role for laminin-5, acting through its alpha3beta1 and/or alpha6beta4 integrin receptors, in UB branching during nephrogenesis. These data may help to partially explain the renal phenotype seen in integrin alpha3 and alpha3/alpha6 subunit-deficient animals.

Matrix metalloproteinases and their inhibitors regulate in vitro ureteric bud branching morphogenesis.

Mammalian kidney development is initiated by the mutual interaction between embryonic metanephric mesenchyme (MM) and the ureteric bud (UB), leading to tightly controlled UB branching morphogenesis. In a three-dimensional cell culture model, which employs MM cell-derived conditioned medium (BSN-CM) to induce UB cell branching morphogenesis in extracellular matrix (ECM) gels (Sakurai H, Barros EJ, Tsukamoto T, Barasch J, and Nigam SK. Proc Natl Acad Sci USA 94: 6279-6284, 1997), branching morphogenesis was inhibited by both chemical agents (ilomastat and 1,10-orthophenanthroline) and a physiological protein factor [tissue inhibitor of metalloproteinases (TIMP)-2], known to act as matrix metalloproteinase (MMP) inhibitors. In addition, UB branching was inhibited in isolated UB culture (Qiao J, Sakurai H, and Nigam SK. Proc Natl Acad Sci USA 96: 7330-7335, 1999) by TIMP-2 and ilomastat, suggesting a direct role for MMPs in UB branching. Gelatin zymography and enzymatic measurement of MMP activity revealed that MMPs could originate from at least three different sources: the conditioned medium, the ECM, and the UB cells themselves. In the UB cells, transcription of several MMPs [gelatinase A (MMP2) and B (MMP9), stromelysin (MMP3), MT1-MMP] and TIMPs was altered by BSN-CM and changed as more complex branching structures formed. The ECM appeared to serve as both a reservoir for MMPs and modulated their expression because different ECM compositions altered the total MMP activity as well as specific subsets of MMPs expressed by the UB cells (as determined by zymography and Northern analysis). In the context of UB branching morphogenesis during kidney development, our data suggest a complex model in which soluble factors produced by the MM, in the context of specific ECM components, modulate the expression of specific subsets of MMPs and TIMPs in the UB, which alter as structures develop and the matrix environment changes. This suggests distinct roles for different subsets of MMPs and their inhibitors during different phases of branching morphogenesis.

A role for Timeless in epithelial morphogenesis during kidney development.

Central to the process of epithelial organogenesis is branching morphogenesis into tubules and ducts. In the kidney, this can be modeled by a very simple system consisting of isolated ureteric bud (UB) cells, which undergo branching morphogenesis in response to soluble factors present in the conditioned medium of a metanephric mesenchyme cell line. By employing a targeted screen to identify transcription factors involved early in the morphogenetic program leading to UB branching, we identified the mammalian ortholog of Timeless (mTim) as a potential immediate early gene (IEG) important in this process. In the embryo, mTim was found to be expressed in patterns very suggestive of a role in epithelial organogenesis with high levels of expression in the developing lung, liver, and kidney, as well as neuroepithelium. In the embryonic kidney, the expression of mTim was maximal in regions of active UB branching, and a shift from the large isoform of mTim to a smaller isoform occurred as the kidney developed. Selective down-regulation of mTim resulted in profound inhibition of embryonic kidney growth and UB morphogenesis in organ culture. A direct effect on the branching UB was supported by the observation that down-regulation of mTim in the isolated UB (cultured in the absence of mesenchyme) resulted in marked inhibition of morphogenesis, suggesting a key role for Tim in the epithelial cell morphogenetic pathway leading to the formation of branching tubules.

Role of hyaluronan and CD44 in in vitro branching morphogenesis of ureteric bud cells.

Mutual interaction between the metanephric mesenchyme (MM) and the ureteric bud (UB) in the developing kidney leads to branching morphogenesis and the formation of the ureteric tree. A UB-derived cell line, stimulated by conditioned medium derived from an embryonic MM cell line (or, similarly, by 10% fetal calf serum), forms branching tubules under three-dimensional culture conditions (H. Sakurai et al., 1997, Proc. Natl. Acad. Sci. USA 94, 6279-6284). The formation of branching tubules in this simple in vitro system for early nephrogenesis is highly sensitive to the matrix environment, a key component of which is the glycosaminoglycan hyaluronan (HA). Consistent with this, we found that HA in the extracellular environment markedly stimulated the formation of cellular processes and multicellular cords (early steps in branching morphogenesis) and also acted as a cell survival factor. Inhibition of HA binding to the cells by addition of blocking antibodies to CD44, the principal cell surface receptor for HA, or degradation of HA by the addition of Streptomyces hyaluronidase resulted in decreased cell survival and diminished morphogenesis, indicating that the HA-CD44 axis plays a central role in in vitro branching morphogenesis. Analysis of the expression of a large number of genes displayed on a cDNA array revealed that significant changes in gene expression in cells undergoing morphogenesis in the presence of HA were limited to a small subset of genes regulating apoptosis, proliferation, and morphogenesis. This included upregulation by HA of its receptor, CD44, which was found to largely localize to the tips of branching cellular processes. In the embryonic kidney, HA was found near the developing ureteric tree and CD44 was expressed basolaterally in UB-derived structures. In addition, both UB and MM appear to express HA synthase, suggesting their ability to secrete HA. We propose that HA promotes branching morphogenesis by creating a positive feedback loop that results in (1) enhanced interaction of HA-CD44 at branching tips (possibly leading to localization of HA binding morphoregulatory factors at the tips) and (2) an activated transcriptional program favoring cell survival/proliferation and migration/morphogenesis of cells through matrix by the expression of key morphoregulatory molecules. Furthermore, since HA, hyaluronidase, and CD44 have been functionally implicated in branching morphogenesis in this model, and since HA, CD44, and HA synthase are all expressed in an appropriate spatiotemporal fashion in the developing kidney, we propose that these molecules may, together, constitute a morphoregulatory pathway that plays a key role in sequential cycles of branching morphogenesis in the UB.