Ureteric Epithelial Cells Research Articles

Targeted disruption of the histone lysine 79 methyltransferase Dot1L in nephron progenitors causes congenital renal dysplasia

ABSTRACT The epigenetic regulator Dot1, the only known histone H3K79 methyltransferase, has a conserved role in organismal development and homoeostasis. In yeast, Dot1 is required for telomeric silencing and genomic integrity. In Drosophila, Dot1 (Grappa) regulates homoeotic gene expression. Dysregulation of DOT1L (human homologue of Dot1) causes leukaemia and is implicated in dilated cardiomyopathy. In mice, germline disruption of Dot1L and loss of H3K79me2 disrupt vascular and haematopoietic development. Targeted inactivation of Dot1L in principal cells of the mature collecting duct affects terminal differentiation and cell type patterning. However, the role of H3K79 methylation in mammalian tissue development has been questioned, as it is dispensable in the intestinal epithelium, a rapidly proliferating tissue. Here, we used lineage-specific Cre recombinase to delineate the role of Dot1L methyltransferase activity in the mouse metanephric kidney, an organ that develops via interactions between ureteric epithelial (Hoxb7) and mesenchymal (Six2) cell lineages. The results demonstrate that Dot1LHoxb7 is dispensable for ureteric bud branching morphogenesis. In contrast, Dot1LSix2 is critical for the maintenance and differentiation of Six2+ progenitors into epithelial nephrons. Dot1LSix2 mutant kidneys exhibit congenital nephron deficit and cystic dysplastic kidney disease. Molecular analysis implicates defects in key renal developmental regulators, such as Lhx1, Pax2 and Notch. We conclude that the developmental functions of Dot1L-H3K79 methylation in the kidney are lineage-restricted. The link between H3K79me and renal developmental pathways reaffirms the importance of chromatin-based mechanisms in organogenesis.

Cu-bearing stainless steel reduces cytotoxicity and crystals adhesion after ureteral epithelial cells exposing to calcium oxalate monohydrate

Calcium oxalate monohydrate (COM), which is the main component of encrustation, may result in cell membrane injury. In addition, cellular damage is suggested to be the primary event attributing to COM crystal binding. To study the interaction between cells and crystals after incubating with a Cu-bearing stainless steel (316L-Cu SS), MTS and flow cytometric analyses were used to assess the cellular responses. The results confirmed that 316L-Cu SS could inhibit cytotoxicity and cellular apoptosis of ureteral epithelial cells (UECs) after COM treatment. Furthermore, molecular expressions of Cu/Zn superoxide dismutase (CuZnSOD), which were evaluated by western blot analysis and real-time quantitative PCR (qPCR), indicated that 316L-Cu SS could inhibit the oxidative stress attributing to up-regulating of CuZnSOD. Moreover, the crystal adhesion cytokine CD44 was examined with western blot and qPCR, and the corresponding hyaluronic (HA) secreted into the medium was measured by enzyme-linked immunosorbent assay (ELISA). All results were confirmed that the expressions of cells cultured with 316L-Cu SS were down-regulated, demonstrating the inhibitory performance of 316L-Cu SS against crystal adhesion.

Ketamine induces reactive oxygen species and enhances autophagy in SV-HUC-1 human uroepithelial cells.

This study was aimed at exploring the underlying mechanisms of ketamine in the SV-40 immortalized human ureteral epithelial (SV-HUC-1) cells. The viability and apoptosis of SV-HUC-1 cells treated with 0.01, 0.1, and 1 mM ketamine were respectively detected via cell counting kit-8(CCK-8) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Reactive oxygen species (ROS) level was measured through ROS probe staining. Apoptosis-related proteins (B-cell lymphoma 2 [Bcl-2] and Bax) and autophagy-associated proteins (light chain 3-I [LC3-I] and LC3-II) were determined by western blot or immunofluorescent assay. Additionally, transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes. After cotreatment of 3-methyladenine (3-MA) or N-acetyl-l-cysteine (NAC), the biological functions of SV-HUC-1 cells were analyzed to determine the association of ROS with cell viability and autophagy. CCK-8 assay and TUNEL staining indicated that ketamine effectively decreased the viability of SV-HUC-1 cells and accelerated apoptosis of SV-HUC-1 cells through regulating the expression level of IKBα (phospho), nuclear factorкB (P65), Bcl-2, and Bax proteins. Enhanced ROS production was also confirmed in ketamine-treated SV-HUC-1 cells treated with ketamine. Ketamine-induced autophagosomes in SV-HUC-1 cells were observed by means of TEM, and increased levels of LC3 II/I ratio and Beclin 1 were examined through western blot and immunofluorescent assay. Furthermore, ketamine exerted effects on SV-HUC-1 cells in a dose-dependent and time-dependent manner. Additionally, cotreatment of NAC with 3-MA significantly attenuated the ROS level and suppressed the cell autophagy. Ketamine promoted SV-HUC-1 cell autophagy and impaired the cell viability of SV-HUC-1 cells by inducing ROS.

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

Human pluripotent stem cells, after directed differentiation in vitro, can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation.

Generation of kidney organoids from human pluripotent stem cells.

The human kidney develops from four progenitor populations-nephron progenitors, ureteric epithelial progenitors, renal interstitial progenitors and endothelial progenitors-resulting in the formation of maximally 2 million nephrons. Until recently, the reported methods differentiated human pluripotent stem cells (hPSCs) into either nephron progenitor or ureteric epithelial progenitor cells, consequently forming only nephrons or collecting ducts, respectively. Here we detail a protocol that simultaneously induces all four progenitors to generate kidney organoids within which segmented nephrons are connected to collecting ducts and surrounded by renal interstitial cells and an endothelial network. As evidence of functional maturity, proximal tubules within organoids display megalin-mediated and cubilin-mediated endocytosis, and they respond to a nephrotoxicant to undergo apoptosis. This protocol consists of 7 d of monolayer culture for intermediate mesoderm induction, followed by 18 d of 3D culture to facilitate self-organizing renogenic events leading to organoid formation. Personnel experienced in culturing hPSCs are required to conduct this protocol.

Ablation of the epithelial-specific splicing factor Esrp1 results in ureteric branching defects and reduced nephron number.

Background:Abnormalities in ureteric bud (UB) branching morphogenesis lead to congenital anomalies of the kidney and reduced nephron numbers associated with chronic kidney disease (CKD) and hypertension. Previous studies showed that the epithelial fibroblast growth factor receptor 2 (Fgfr2) IIIb splice variant supports ureteric morphogenesis in response to ligands from the metanephric mesenchyme during renal organogenesis. The epithelial‐specific splicing regulator Esrp1 is required for expression of Fgfr2‐IIIb and other epithelial‐specific splice variants. Our objective was to determine whether Esrp1 is required for normal kidney development.Results:Ablation of Esrp1 in mice, alone or together with its paralog Esrp2, was associated with reduced kidney size and increased incidence of renal aplasia. Three‐dimensional imaging showed that embryonic Esrp1 knockout (KO) kidneys had fewer ureteric tips and reduced nephron numbers. Analysis of alternative splicing in Esrp‐null ureteric epithelial cells by RNA‐Seq confirmed a splicing switch in Fgfr2 as well as numerous other transcripts.Conclusions:Our findings reveal that Esrp1‐regulated splicing in ureteric epithelial cells plays an important role in renal development. Defects in Esrp1 KO kidneys likely reflect reduced and/or absent ureteric branching, leading to decreased nephron induction secondary to incorrect Fgfr2 splicing and other splicing alterations. Developmental Dynamics 245:991–1000, 2016. © 2016 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Stromally expressed β-catenin modulates Wnt9b signaling in the ureteric epithelium.

The mammalian kidney undergoes cell interactions between the epithelium and mesenchyme to form the essential filtration unit of the kidney, termed the nephron. A third cell type, the kidney stroma, is a population of fibroblasts located in the kidney capsule, cortex and medulla and is ideally located to affect kidney formation. We found β-catenin, a transcriptional co-activator, is strongly expressed in distinctive intracellular patterns in the capsular, cortical, and medullary renal stroma. We investigated β-catenin function in the renal stroma using a conditional knockout strategy that genetically deleted β-catenin specifically in the renal stroma cell lineage (β-cats-/-). β-cats-/- mutant mice demonstrate marked kidney abnormalities, and surprisingly we show β-catenin in the renal stroma is essential for regulating the condensing mesenchyme cell population. We show that the population of induced mesenchyme cells is significantly reduced in β-cats-/- mutants and exhibited decreased cell proliferation and a specific loss of Cited 1, while maintaining the expression of other essential nephron progenitor proteins. Wnt9b, the key signal for the induction of nephron progenitors, was markedly reduced in adjacent ureteric epithelial cells in β-cats-/-. Analysis of Wnt9b-dependent genes in the neighboring nephron progenitors was significantly reduced while Wnt9b-independent genes remained unchanged. In contrast mice overexpressing β-catenin exclusively in the renal stroma demonstrated massive increases in the condensing mesenchyme population and Wnt9b was markedly elevated. We propose that β-catenin in the renal stroma modulates a genetic program in ureteric epithelium that is required for the induction of nephron progenitors.

Kif3a Controls Murine Nephron Number Via GLI3 Repressor, Cell Survival, and Gene Expression in a Lineage-Specific Manner

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms.

Fstl1 Antagonizes BMP Signaling and Regulates Ureter Development

Bone morphogenetic protein (BMP) signaling pathway plays important roles in urinary tract development although the detailed regulation of its activity in this process remains unclear. Here we report that follistatin-like 1 (Fstl1), encoding a secreted extracellular glycoprotein, is expressed in developing ureter and antagonizes BMP signaling activity. Mouse embryos carrying disrupted Fstl1 gene displayed prominent hydroureter arising from proximal segment and ureterovesical junction defects. These defects were associated with significant reduction in ureteric epithelial cell proliferation at E15.5 and E16.5 as well as absence of subepithelial ureteral mesenchymal cells in the urinary tract at E16.5 and E18.5. At the molecular level, increased BMP signaling was found in Fstl1 deficient ureters, indicated by elevated pSmad1/5/8 activity. In vitro study also indicated that Fstl1 can directly bind to ALK6 which is specifically expressed in ureteric epithelial cells in developing ureter. Furthermore, Sonic hedgehog (SHH) signaling, which is crucial for differentiation of ureteral subepithelial cell proliferation, was also impaired in Fstl1-/- ureter. Altogether, our data suggest that Fstl1 is essential in maintaining normal ureter development by antagonizing BMP signaling.

New ureteral scaffold constructed with composite poly(L‐lactic acid)–collagen and urothelial cells by new centrifugal seeding system

A tissue-engineered ureteral scaffold was constructed with composited poly L-lactic acid (PLLA)-collagen endoluminal stent and uroepithelial cells (UECs) using a new seeding system. The electrospun PLLA-collagen nanofibrous mesh was seeded efficiently with human ureteral epithelial cells using a modified centrifugal seeding device. The cellular nanofibrous mesh was then wound around a spiral endoluminal stent to form a cellular composited PLLA-collagen ureteral scaffold. The cellular ureteral scaffold was subcutaneously implanted into nude mice. Cell attachment, distribution, and viability in vitro were investigated along with the cell fate in vivo. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that scaffolds seeded with centrifugal method had higher cellular activity than scaffolds seeded with static method (p < 0.05), and the metabolic activity per cell had no significant differences between the two methods (p > 0.05). Histologic analysis showed that the entrapped UECs remained in the scaffolds after 2 wk of implantation. The results of the study indicated that the composited PLLA-collagen endoluminal stent could serve as alternative cell carrier for tissue engineering ureter. In addition, the new modified centrifugal seeding system allowed rapid homogeneous distribution of cells onto the nanofibrous mesh, which will be useful to ureteral reconstruction.

Disruption of polycystin-1 function interferes with branching morphogenesis of the ureteric bud in developing mouse kidneys

The polycystic kidney disease ( PKD1) gene-encoded protein, polycystin-1, is developmentally regulated, with highest expression levels seen in normal developing kidneys, where it is distributed in a punctate pattern at the basal surface of ureteric bud epithelia. Overexpression in ureteric epithelial cell membranes of an inhibitory pMyr-GFP-PKD1 fusion protein via a retroviral (VVC) delivery system and microinjection into the ureteric bud lumen of embryonic day 11 mouse metanephric kidneys resulted in disrupted branching morphogenesis. Using confocal quantitative analysis, significant reductions were measured in the numbers of ureteric bud branch points and tips, as well as in the total ureteric bud length, volume and area, while significant increases were seen as dilations of the terminal branches, where significant increases in outer diameter and volumes were measured. Microinjection of an activating 5TM-GFP-PKD1 fusion protein had an opposite effect and showed significant increases in ureteric bud length and area. These are the first studies to experimentally manipulate polycystin-1 expression by transduction in the embryonic mouse kidney and suggest that polycystin-1 plays a critical role in the regulation of epithelial morphogenesis during renal development.

Effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on fetal mouse urinary tract epithelium in vitro

2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), produces hydronephrosis by altering the differentiation and proliferation of ureteric epithelial cells in the fetal C57BL/6N mouse urinary tract. This study tests the hypothesis that the late fetal urinary tract epithelial cells respond to TCDD with increased proliferation and that the responses do not require contributions from other maternal or fetal tissues. This was achieved by exposing late gestation fetal urinary tract cells to TCDD in an in vitro model. Isolated ureteric cells from gestation day (GD) 18 fetal ureters were plated in medium supplemented with trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors. Both epithelial and mesenchymal cells remain viable under these conditions. The cultures were exposed to 0.1% dimethylsulfoxide (DMSO), 1×10 −8, 1×10 −9 or 1×10 −10 M TCDD. Exposure to 1×10 −10 M TCDD did not affect the cultures, while 1×10 −8 and 1×10 −9 M TCDD supported epithelial, but not mesenchymal, cell survival and stimulated epithelial cell proliferation and differentiation. The TCDD-exposed cells expressed high levels of keratin and little or no vimentin, confirming that the cells, which survive and differentiate are epithelial. However, after continuous exposure to epidermal growth factor (EGF), the TCDD-induced stimulation of ureteric epithelial growth could not be detected. In conclusion, this study demonstrates that late gestational ureteric cells respond to TCDD in vitro with the stimulation of epithelial cell growth and differentiation.

URETERAL GENE TRANSFER TO PORCINE INDUCED STRICTURES USING ENDOUROLOGIC DELIVERY OF AN ADENOVIRAL VECTOR

Direct gene transfer to the ureter is an attractive approach to prevent restenosis after endourologic management of ureteral strictures. We therefore assessed the rationale for adenovirus-mediated gene transfer in the ureter in vitro and in vivo using a porcine model. Primary cultures of porcine ureteral epithelial and stromal cells were infected with an adenoviral solution carrying a nucleus-targeted beta-Galactosidase (beta-Gal) reporter gene (6.5 10(8) pfu/ml.). In addition, in order to mimic the human clinical situation, we have devised a model of thermally-induced stricture in porcine ureter which produced tight fibrotic stenosis within 8 days. Using a purposely designed channelled balloon catheter prototype, these strictures were endoscopically dilated and then instilled with the same beta-Gal adenoviral construction. Application of recombinant adenovirus harboring a nucleus-targeted beta-Gal reporter gene to cultured porcine urothelial and stromal cells resulted in high transduction efficiency of up to 99% and 84% respectively. Seven days after infection, X-Gal staining of the strictured ureters demonstrated transfection up to 2 mm. depth within the fibrosis, confirmed by polymerase chain reaction (PCR) analysis. Adjacent and distal spread of the virus was excluded by histochemistry (X-Gal staining) and PCR. This data represents the first report of adenovirus-mediated gene transfer to the ureter. It remained site specific by endourologic retrograde clinically applicable techniques.

Developmental regulation of CFTR expression during human nephrogenesis.

Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein are expressed in proximal and distal tubules of the human kidney, but CFTR expression pattern during human nephrogenesis is unknown. We have now studied CFTR expression in fetal kidneys by immunohistochemistry and Western blot analysis, using six antibodies against human CFTR. CFTR was expressed in 12-wk human fetal kidneys, mostly in the apical membrane region of the ureteric bud epithelial cells. By 15 wk, CFTR was also diffusely expressed throughout the cytoplasm of proximal tubules and loops of Henle. No glomerular staining was seen at any state. From 15 to 24 wk of gestation this staining pattern remained constant and also included immunoreactivity of the transitional epithelium. Western blot for CFTR was performed on membrane extracts of human fetal kidneys, using T84 cells as a positive control. A 165-kDa protein corresponding to the predicted size of CFTR was seen at 13 wk and throughout development. We also observed a 75-kDa protein that was distinctly regulated during development. This protein was detected with several antibodies against the first half of CFTR (including the regulatory "R" domain) but not with a COOH-terminal-specific antibody and had the predicted size of a functional splice variant of CFTR identified in the human kidney. These results show the complex regulation of CFTR during nephrogenesis and raise the question of the respective roles of the full-length and the splice variant CFTR proteins in the human kidney.

Integrin-mediated adhesive properties of uroepithelial cells are inhibited by treatment with bacterial toxins

Gram-negative bacteria are a dominant cause of urinary tract infection, and their ability to produce toxins is an important virulence attribute. Cellular mechanisms triggered by the production of toxins in the lower urinary tract have not been completely defined. Ureteral epithelial cells (UT; A. Elgavish, Infect. Immun. 61: 3304-3312, 1993) have served as an in vitro model to explore the possibility that bacterial toxins act on UT by affecting integrin-mediated adhesive properties. The effect of treatment with lipopolysaccharides (LPS) from three strains of the gram-negative Escherichia coli [055:B5 (LPS-1), 0111:B4 (LPS-4), and 0127:B8 (LPS-5)] and lipoteichoic acids from two gram-positive bacteria, Streptococcus faecalis (LT-2) and Bacillus subtilis (LT-3), were examined. LPS-5 inhibited markedly UT attachment to collagen and fibronectin. LPS-4 had no effect, whereas LPS-1 inhibited UT attachment to collagen but not to fibronectin. The fact that LPS-5 and LT-2 inhibited an Arg-Gly-Asp sequence-sensitive component of UT attachment to fibronectin is consistent with the possibility that these toxins acted via a mechanism involving typical fibronectin receptors. UT spreading was inhibited markedly by LPS-1, LT-2, and LT-3, whereas LPS-4 and LPS-5 had no effect. Because clustering of integrins is a crucial step in integrin-mediated signal transduction, the possibility that toxins inhibited spreading by affecting clustering was tested. Treatment with LT-2, which inhibited spreading dramatically, abolished completely a UT cell population containing more than five to eight beta 1- or beta 4-subunit-containing integrin clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Escherichia coli and E. coli lipopolysaccharides on the function of human ureteral epithelial cells cultured in serum-free medium

Escherichia coli is the microorganism most commonly isolated from human urinary tract infections. Earlier studies by others have shown that bacterial attachment and production of toxins (e.g., lipopolysaccharides [LPS]) enhance recruitment of leukocytes to the infection site and mucosal inflammation. The mechanisms by which these changes occur have not been completely defined. In the present study, epithelial cell cultures isolated from the human ureter (UT cells) (A. Elgavish, J. J. Wille, F. Rahemtulla, and L. Debro, Am. J. Physiol. 261:C916-C926, 1991; J. J. Wille, J. Park, and A. Elgavish, J. Cell. Physiol. 150:52-58, 1992) served as a model system with which to explore the mechanisms of action of Escherichia coli and E. coli LPS in UT cells. E. coli adhered to UT cells and inhibited carrier-mediated sulfate uptake to half of that in untreated UT cells, suggesting that the intracellular pool of sulfate available for sulfation may be lower in infected cells and may lead to the production of undersulfated glycoconjugates. Incubation of UT cells with E. coli LPS inhibited carrier-mediated sulfate uptake to an extent similar to that caused by whole E. coli, indicating that the effect of E. coli on sulfate uptake may be mediated by LPS. LPS caused an increase in Na+ content in rapidly proliferating UT cells but not in quiescent cells. We postulated that this change in the intracellular ionic environment or changes coupled to it (e.g., pH or Ca2+ levels) may serve as a transducing signal. This possibility was supported by the fact that LPS stimulated clustering of ICAM-1 on the cell surface of rapidly proliferating but not quiescent UT cells. This study suggests that, in vivo, LPS stimulation of ICAM-1 clustering on the surface of the urothelium may allow more effective binding of leukocytes. This may be the mechanism underlying earlier findings in vivo indicating a role for LPS in the recruitment of leukocytes to the urinary tract as a host defense mechanism following urinary tract infection.

Lewis Blood Group Antigen Expression by Cultured Normal Ureteral Epithelial Cells

The importance of Lewis blood group antigens in recurrent urinary tract infections has led to a more detailed study of the expression of these antigens in an in vitro culture system. Expression of A, B, H, and Lewis blood group antigens by primary cultures of ureter epithelial cells was analyzed by flow cytometry. Cells maintained blood group determinants for up to 7 weeks in culture over three passages. In several specimens, the cells that exhibited expression of Lewis A (Lea) and Lewis B (Leb) antigens increased dramatically over 2-3 passages in culture. Lewis Y (Ley) antigen expression was positive on all primary cultures tested. The effect of fetal bovine serum (FBS) on blood group antigen expression was evaluated. Increased concentrations of FBS in the growth media increased the expression of Lea and Leb antigens in certain specimens, but did not affect expression of the other blood group antigens. The effect of FBS on blood group antigen expression of cultured cells depended on the Lewis blood type of the individual donating the specimen. To evaluate the heterogeneity often seen in Lea and Leb antigen expression, dual staining of cells for Lea and Leb antigens was performed. The results of these studies show for the first time that Lewis blood group antigens are expressed by cultured normal ureteral epithelial cells and that the pattern of expression of Lea and Leb changes over time. Furthermore, an individual cell may simultaneously express more than one Lewis antigen on its surface.

Effects of growth factors, hormones, bacterial lipopolysaccharides, and lipotechoic acids on the clonal growth of normal ureteral epithelial cells in serum‐free culture

In vitro tissue culture techniques were employed to study the effects of bacterial endotoxins on the growth of normal epithelial cells from the human ureter (NHU). Primary cultures of NHU cells were initiated from explant outgrowth cultures of human ureteral tissue and cultured on collagen gel in F-12* medium containing 1% fetal calf serum (FCS). Optimal clonal growth of secondary cultures of NHU cells seeded at relatively low seeding cell densities, directly on plastic dishes, was achieved in F-12* medium containing bovine pituitary extract (0.5% BPE) and 0.05% BSA. Results indicated that insulin in the F-12* medium could be replaced by three orders of magnitude less IGF-1. Further clonal growth experiments demonstrated that PGE1 is growth stimulatory and can replace BPE as a growth factor requirement. This finding was in agreement with the fact that BPE growth requirement could be replaced by cholera toxin or dibutyryl cAMP. These results suggested that both BPE and cholera toxin operated by activation of a cAMP-dependent mitogenic pathway. Seven gram-negative bacterial lipopolysaccharides (LPS) and three gram-positive bacterial lipotechoic acids (LT) were tested for their effects on NHU clonal growth. Three out of the five LPS derived from Escherichia coli (strains 055:B5, 0128:B12, and 0127:B8), LPS from Klebsiella pneumoniae, and LPS from Pseudomonas aeruginosa all showed significant growth inhibitory effects at minimally effective doses ranging from 5 to 25 micrograms/ml. LPS derived from E. coli strain (0111:B4) had no growth effects at the highest concentration tested (100 micrograms/ml). In contrast, LT derived from Streptococcus pyogenes, S. faecalis, Staphylococcus aureas, and Bacillus subtilis all markedly enhanced clonal growth at concentrations ranging from 1 microgram/ml less than [LT] less than 50 micrograms/ml. LT from Strep. pyogenes was inhibitory to clonal growth at 100 micrograms/ml. The growth inhibitory effects of LPS were shown to be sensitive to the presence of hydrocortisone in the growth medium, indicating that LPS effects on growth are mediated via the arachidonic acid cascade. We speculate that these results indicate a link between the susceptibility of uroepithelial tissue to the pathogenic microflora seen in urinary tract diseases and the differential sensitivity of proliferation-competent uroepithelial cells to growth inhibition by LPS produced by gram-negative bacteria. However, further studies with uropathogenic serotypes will be necessary to corroborate this possibility. The growth-stimulating activity of LTs produced by gram-positive bacteria may be due to their ability to bind to cell-associated fibronectin and to activate the fibronectin receptor as part of ligand receptor-induced mitogenic transmembrane signalling pathway.

Carrier-mediated sulfate transport in human ureteral epithelial cells cultured in serum-free medium

Sulfate transport studies were carried out in secondary cultures of epithelial cells isolated from the human ureter. Results demonstrate the presence of carrier-mediated SO4(2-) transport as supported by three lines of evidence: 1) saturation kinetics, 2) substrate specificity, and 3) inhibition by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The DIDS-insensitive component of SO4(2-) transport was markedly lower than the DIDS-sensitive component and was not affected by changes in extracellular pH (pHo) or Cl- concentration. The mechanism of this DIDS-insensitive component is not clear. The DIDS-sensitive component of SO4(2-) uptake was a saturable function of the extracellular sulfate concentration ([SO4(2-)]o). Increasing the extracellular chloride concentration ([Cl-]o) inhibited DIDS-sensitive SO4(2-) uptake competitively. Taken together with the fact that increasing [Cl-]o stimulated SO4(2-) efflux, these results suggest that SO4(2-) uptake in uroepithelial cells occurs via SO4(2-)-Cl- anion exchange. Cis-inhibition studies with a variety of anions indicate that this anion-exchange system may be shared by S2O3(2-) and MoO4(2-) but not by NO3- and H2PO4-. SO4(2-) uptake was stimulated at decreasing pHo with a pK approximately 7.4. Decreasing pHo from 7 to 6 lowered the apparent Michaelis constant significantly but had no significant effect on kcat, suggesting that protons may increase the affinity of the SO4(2-) transporter for SO4(2-). SO4(2-) efflux was inhibited at low pHo and was stimulated by increasing [Cl-]o. This study is the first to demonstrate an ion transport process in epithelial cell cultures isolated from the human ureter. In contrast to epithelial cells from the upper urinary tract, no Na(+)-dependent SO4(2-) transport could be demonstrated in these lower urinary tract epithelial cells. In conclusion, the major mechanism for SO4(2-) transport in ureteral epithelial cells is a carrier-mediated, DIDS-sensitive, pHo-sensitive SO4(2-)/Cl- anion-exchange mechanism. These studies suggest that varying [SO4(2-)]o and [Cl-]o or pHo in the ureteral lumen will affect SO4(2-) influx and efflux and may influence the size of the intracellular pool of SO4(2-) available for macromolecular sulfation in these cells.

Effects of TCDD on embryonic ureteric epithelial EGF receptor expression and cell proliferation

The potent toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing hydronephrosis and cleft palate. Because of the long half-life of TCDD, the urinary tract is exposed throughout development after a single dose on gestation day (GD) 10 or earlier. TCDD-induced hydronephrosis is a consequence of occlusion of the ureter by epithelial cells. Since embryonic growth factors and the epidermal growth factor (EGF) receptor are probably involved in regulation of embryonic cell proliferation, this study examines the effects of TCDD on expression of EGF receptors and proliferation of ureteric epithelial cells in vivo and in culture. After exposure to TCDD by gavage (12, 24, or 30 micrograms/kg on GD 10; 6 or 24 micrograms/kg on GD 12) the mean cell depth of the ureteric and bladder epithelia was increased. EGF receptors were detected immunohistochemically in sectioned urinary tracts. The expression of receptors decreased with advancing development in control ureteric epithelia. However, after TCDD exposure the level of EGF receptors failed to decline. The incorporation of 3H-TdR was observed in sections by autoradiography, and after exposure to TCDD more epithelial cells showed incorporation than was apparent in controls. Transmission electron microscopy (TEM) of embryonic ureters from fetuses exposed to TCDD in vivo showed no cytotoxicity in basal cells and the cells remained undifferentiated, as in controls. Ureters taken from GD 12 embryos and cultured with 1 x 10(-10)M TCDD showed ureteric epithelial hyperplasia without cytotoxicity, but at 1 x 10(-8)M TCDD evidence of cytotoxicity was observed by TEM. The levels of TCDD found in fetuses after in vivo exposure (204-307 pg/fetus, with 1-2 pg in the urinary tract) compare well with the in vitro level (32 pg/ml), which was most effective in producing hyperplasia of the epithelial cells. The present study correlates a TCDD-induced increase in cell depth with altered regulation of EGF receptors and excessive proliferation, both in vivo and in cultured embryonic ureters.