Wnt signaling mediates new nephron formation during zebrafish kidney regeneration.

Zebrafish kidneys use resident kidney stem cells to replace damaged tubules with new nephrons: the filtration units of the kidney. What stimulates kidney progenitor cells to form new nephrons is not known. Here, we show that wnt9a and wnt9b are induced in the injured kidney at sites where frizzled9b- and lef1-expressing progenitor cells form new nephrons. New nephron aggregates are patterned by Wnt signaling, with high canonical Wnt-signaling cells forming a single cell thick rosette that demarcates: domains of cell proliferation in the elongating nephron; and tubule fusion where the new nephron plumbs into the distal tubule and establishes blood filtrate drainage. Pharmacological blockade of canonical Wnt signaling inhibited new nephron formation after injury by inhibiting cell proliferation, and resulted in loss of polarized rosette structures in the aggregates. Mutation in frizzled9b reduced total kidney nephron number, caused defects in tubule morphology and reduced regeneration of new nephrons after injury. Our results demonstrate an essential role for Wnt/frizzled signaling in adult zebrafish kidney development and regeneration, highlighting conserved mechanisms underlying both mammalian kidney development and kidney stem cell-directed neonephrogenesis in zebrafish.

Fibroblast growth factor signaling mediates progenitor cell aggregation and nephron regeneration in the adult zebrafish kidney.

The zebrafish kidney regenerates after injury by development of new nephrons from resident adult kidney stem cells. Although adult kidney progenitor cells have been characterized by transplantation and single cell RNA seq, signals that stimulate new nephron formation are not known. Here we demonstrate that fibroblast growth factors and FGF signaling is rapidly induced after kidney injury and that FGF signaling is required for recruitment of progenitor cells to sites of new nephron formation. Chemical or dominant negative blockade of Fgfr1 prevented formation of nephron progenitor cell aggregates after injury and during kidney development. Implantation of FGF soaked beads induced local aggregation of lhx1a:EGFP  â€‹+ â€‹kidney progenitor cells. Our results reveal a previously unexplored role for FGF signaling in recruitment of renal progenitors to sites of new nephron formation and suggest a role for FGF signaling in maintaining cell adhesion and cell polarity in newly forming kidney epithelia.

A protein kinase A and Wnt-dependent network regulating an intermediate stage in epithelial tubulogenesis during kidney development.

Genetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.

Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate.

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).

Neuropeptide Y functions as a facilitator of GDNF-induced budding of the Wolffian duct.

Ureteric bud (UB) emergence from the Wolffian duct (WD), the initiating step in metanephric kidney morphogenesis, is dependent on GDNF; however, GDNF by itself is generally insufficient to induce robust budding of the isolated WD in culture. Thus, additional factors, presumably peptides or polypeptide growth factors, might be involved. Microarray data from in vivo budding and non-budding conditions were analyzed using non-negative matrix factorization followed by gene ontology filtering and network analysis to identify sets of genes that are highly regulated during budding. These included the GDNF co-receptors GFRalpha1 and RET, as well as neuropeptide Y (NPY). By using ANOVA with pattern matching, NPY was also found to correlate most significantly to the budded condition with a high degree of connectedness to genes with developmental roles. Exogenous NPY [as well as its homolog, peptide YY (PYY)] augmented GDNF-dependent budding in the isolated WD culture; conversely, inhibition of NPY signaling or perturbation of NPY expression inhibited budding, confirming that NPY facilitates this process. NPY was also found to reverse the decreased budding, the downregulation of RET expression, the mislocalization of GFRalpha1, and the inhibition of AKT phosphorylation that resulted from the addition of BMP4 to the isolated WD cultures, suggesting that NPY acts through the budding pathway and is reciprocally regulated by GDNF and BMP4. Thus, the outgrowth of the UB from the WD might result from a combination of the upregulation of the GDNF receptors together with genes that support GDNF signaling in a feed-forward loop and/or counteraction of the inhibitory pathway regulated by BMP4.

Functional maturation of drug transporters in the developing, neonatal, and postnatal kidney.

Because renal function in newborns is immature, the pharmacokinetics of drugs administered to neonates vary significantly from adult patients. The establishment of drug transport systems is a key process in the functional maturation of the nephron. However, a thorough examination of the expression of the main drug transporters in the kidney throughout all stages of development (embryonic, postnatal, and mature) has yet to be carried out, and the functional (physiological) impact is not well understood. Using time-series microarray data, we analyzed the temporal behavior of mRNA levels for a wide range of SLC and ABC transporters in the rodent kidney throughout a developmental time series. We find dynamic increases between the postnatal and mature stages of development for a number of transporters, including the proximal tubule-specific drug and organic anion transporters (OATs) OAT1 (SLC22a6) and OAT3 (SLC22a8). The OATs are the major multispecific basolateral drug, toxin, and metabolite transporters in the proximal tubule responsible for handling of many drugs, as well as the prototypical OAT substrate para-aminohippurate (PAH). We therefore performed specific in vivo pharmacokinetic analysis of the transport of PAH in postnatal and maturing rodent kidney. We show that there is a 4-fold increase in PAH clearance during this period. Clearance studies in Oat1 and Oat3 knockouts confirm that, as in the adult, Oat1 is the principle transporter of PAH in the postnatal kidney. The substantial differences observed supports the need for better understanding of pharmacokinetics in the newborn and juvenile kidney compared with the adult kidney at the basic and clinical level.

Hs2st mediated kidney mesenchyme induction regulates early ureteric bud branching.

Heparan sulfate proteoglycans (HSPGs) are central modulators of developmental processes likely through their interaction with growth factors, such as GDNF, members of the FGF and TGFbeta superfamilies, EGF receptor ligands and HGF. Absence of the biosynthetic enzyme, heparan sulfate 2-O-sulfotransferase (Hs2st) leads to kidney agenesis. Using a novel combination of in vivo and in vitro approaches, we have reanalyzed the defect in morphogenesis of the Hs2st(-)(/)(-) kidney. Utilizing assays that separately model distinct stages of kidney branching morphogenesis, we found that the Hs2st(-/-) UB is able to undergo branching and induce mesenchymal-to-epithelial transformation when recombined with control MM, and the isolated Hs2st null UB is able to undergo branching morphogenesis in the presence of exogenous soluble pro-branching growth factors when embedded in an extracellular matrix, indicating that the UB is intrinsically competent. This is in contrast to the prevailing view that the defect underlying the renal agenesis phenotype is due to a primary role for 2-O sulfated HS in UB branching. Unexpectedly, the mutant MM was also fully capable of being induced in recombination experiments with wild-type tissue. Thus, both the mutant UB and mutant MM tissue appear competent in and of themselves, but the combination of mutant tissues fails in vivo and, as we show, in organ culture. We hypothesized a 2OS-dependent defect in the mutual inductive process, which could be on either the UB or MM side, since both progenitor tissues express Hs2st. In light of these observations, we specifically examined the role of the HS 2-O sulfation modification on the morphogenetic capacity of the UB and MM individually. We demonstrate that early UB branching morphogenesis is not primarily modulated by factors that depend on the HS 2-O sulfate modification; however, factors that contribute to MM induction are markedly sensitive to the 2-O sulfation modification. These data suggest that key defect in Hs2st null kidneys is the inability of MM to undergo induction either through a failure of mutual induction or a primary failure of MM morphogenesis. This results in normal UB formation but affects either T-shaped UB formation or iterative branching of the T-shaped UB (possibly two separate stages in collecting system development dependent upon HS). We discuss the possibility that a disruption in the interaction between HS and Wnts (e.g. Wnt 9b) may be an important aspect of the observed phenotype. This appears to be the first example of a defect in the MM preventing advancement of early UB branching past the first bifurcation stage, one of the limiting steps in early kidney development.

The instructive role of metanephric mesenchyme in ureteric bud patterning, sculpting, and maturation and its potential ability to buffer ureteric bud branching defects.

Kidney organogenesis depends on reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) to form the UB-derived collecting system and MM-derived nephron. With the advent of in vitro systems, it is clear that UB branching can occur independently of MM contact; however, little has been done to detail the role of MM cellular contact in this process. Here, a model system in which the cultured isolated UB is recombined with uninduced MM is used to isolate the effects of the MM progenitor tissue on the development and maturation of the collecting system. By morphometrics, we demonstrate that cellular contact with the MM is required for vectorial elongation of stalks and tapering of luminal caliber of UB-derived tubules. Expression analysis of developmentally significant genes indicates the cocultured tissue is most similar to an embryonic day 19 (E19) kidney. The likely major contributor to this is the functional maturation of the collecting duct and proximal nephron segments in the UB-induced MM, as measured by quantitative PCR, of the collecting duct-specific arginine vasopressin receptor and the nephron tubule segment-specific organic anion transporter OAT1, Na-P(i) type 2 cotransporter, and Tamm-Horsfall protein gene expressions. However, expression of aquaporin-2 is upregulated similarly in isolated UB and cocultured tissue, suggesting that some aspects of functional maturation can occur independently of MM cellular contact. In addition to its sculpting effects, the MM normalized a "branchless" UB morphology induced by FGF7 or heregulin in isolated UB culture. The morphological changes induced by the MM were accompanied by a reassignment of GFRalpha1 (a receptor for GDNF) to tips. Such "quality control" by the MM of UB morphology may provide resiliency to the branching program. This may help to explain a number of knockout phenotypes in which branching and/or cystic defects are less impressive than expected. A second hit in the MM may thus be necessary to make these defects fully apparent.

Advances in cellular reprogramming: moving toward a reprieve from immunogenicity.

Somatic cell nuclear reprogramming is opening new doors for the modeling of human disease phenotypes in vitro, the identification of novel therapeutic compounds and diagnostic factors as well as future autologous cell replacement therapies. Despite the potential that reprogramming technologies bring, there are remaining concerns preventing their broad application in the short-term. One of them is the safety concern associated with the use of stem cell derivatives, those generated by reprogramming or even when embryonic stem cells are employed. Here we summarize the current knowledge in the field of stem cells and reprogramming with a particular focus on the pitfalls preventing rapid translation of stem cell technologies into the clinic. We discuss the most recent findings on immunogenicity and tumorigenicity of reprogrammed cells. We additionally provide an overview on the potential applications that reprogramming approaches might bring to the immunological field and elaborate on the use of induced pluripotent stem cells (iPSCs) with pre-arranged immune receptors for the development of future immunotherapeutic approaches. The use of reprogramming approaches can represent and provide groundbreaking strategies previously unachievable for stem cell engineering aimed at modulating immune responses. In summary, we provide an overview on the different topics related to the use of stem cells and highlight the most provocative, yet perhaps currently underappreciated, aspect of combining immunological and reprogramming strategies for the treatment of human disease.

Organic anion and cation SLC22 "drug" transporter (Oat1, Oat3, and Oct1) regulation during development and maturation of the kidney proximal tubule.

Proper physiological function in the pre- and post-natal proximal tubule of the kidney depends upon the acquisition of selective permeability, apical-basolateral epithelial polarity and the expression of key transporters, including those involved in metabolite, toxin and drug handling. Particularly important are the SLC22 family of transporters, including the organic anion transporters Oat1 (originally identified as NKT) and Oat3 as well as the organic cation transporter Oct1. In ex vivo cultures of metanephric mesenchyme (MM; the embryonic progenitor tissue of the nephron) Oat function was evident before completion of nephron segmentation and corresponded with the maturation of tight junctions as measured biochemically by detergent extractability of the tight junction protein, ZO-1. Examination of available time series microarray data sets in the context of development and differentiation of the proximal tubule (derived from both in vivo and in vitro/ex vivo developing nephrons) allowed for correlation of gene expression data to biochemically and functionally defined states of development. This bioinformatic analysis yielded a network of genes with connectivity biased toward Hnf4α (but including Hnf1α, hyaluronic acid-CD44, and notch pathways). Intriguingly, the Oat1 and Oat3 genes were found to have strong temporal co-expression with Hnf4α in the cultured MM supporting the notion of some connection between the transporters and this transcription factor. Taken together with the ChIP-qPCR finding that Hnf4α occupies Oat1, Oat3, and Oct1 proximal promoters in the in vivo differentiating rat kidney, the data suggest a network of genes with Hnf4α at its center plays a role in regulating the terminal differentiation and capacity for drug and toxin handling by the nascent proximal tubule of the kidney.