Differential expression of Na+-Cl- cotransporter and Na+-K+-Cl- cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes

The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na(+)-K(+)-Cl(-) cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na(+)-Cl(-) cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7× decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater.     

Transplantation of rat metanephroi into mice

To determine whether transplanted metanephroi grow and differentiate after implantation into the omentum in hosts of a different species, we implanted metanephroi from embryonic day 15 (E15) rat embryos into uninephrectomized mice (hosts). Some host mice received human CTLA4Ig (hCTLA4Ig), anti-CD45RB, and anti-CD154 (tolerance-inducing agents). E15 metanephroi contained only metanephric blastema, segments of ureteric bud, and primitive nephrons with no glomeruli. Rat metanephroi did not grow or differentiate in mice that received no tolerance-inducing agents. However, by 2 wk posttransplantation in mice that received hCTLA4Ig, anti-CD45RB, and anti-CD154, metanephroi from E15 rats had enlarged, become vascularized, and formed mature tubules and glomeruli. Rat metanephroi contained cells that stained specifically for mouse CD31, a marker for sprouting endothelial cells. Some rat glomerular capillary loops stained positively for mouse CD31. Here, we show that chimeric kidneys develop from metanephroi transplanted rat-->mouse and that glomeruli are vascularized, at least in part, by host vessels.     

Developmental expression of two CXC chemokines, MIP-2 and KC, and their receptors.

CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and KC, (a cloning designation based on ordinate and abscissa position) as well as the CXC chemokine receptor, CXCR2, are expressed in a variety of cells and tissues in adult mice. Targeted deletion of the gene encoding murine CXCR2 does not result in obvious changes in the development of the organ system of the mouse, though the CXCR2-/- mouse is compromised with regard to its ability to resist infection, heal wounds, and maintain homeostasis when challenged with microbes and/or chemicals. In an attempt to develop insight into additional possible subtle roles of CXCR2 and its ligands in the development of the mouse, we examined the expression of MIP-2, KC, CXCR2, as well as the Duffy antigen binding protein for chemokines during embryonic (p.c.) days 11.5 through 14.5 in the mouse. We observed strong correlation between the expression of MIP-2 and CXCR2 in the developing brain, cardiovascular system and condensing mesenchyme between 11.5 and 13.5 days. Moreover, the expression of KC was parallel to the expression of the Duffy antigen binding protein for chemokines with regard to temporal pattern and tissue localization. MIP-2 and CXCR2 are highly expressed in the brain, first in the cerebellum and in the head mesenchyme, the meninges and the floor plate, and by 14.5 days are also present in the telencephalon, thalamus and hypothalamus. In the developing brain KC and Duffy were prominently expressed in the neuronal tracts, the forebrain, sympathetic ganglia, and along the periphery of the neural tube. However, KC and Duffy were less prevalent in the developing cardiovascular system, lung and other organs, muscle and bone, than are CXCR2 and MIP-2. These data suggest that the roles for these chemokines and their receptors during development may be more significant than was initially thought based upon the phenotype of the mice with targeted deletion of CXCR2 and Duffy.     

Standardized,systemic phenotypic analysis of Slc12a1I299F mutant mice

Background

Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na⁺-K⁺-2Cl⁻ cotransporter NKCC2. We recently established the mutant mouse line Slc12a1^I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye.

Results

To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1^I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1^I299F in the German Mouse Clinic. Slc12a1^I299F homozygous mutant mice and Slc12a1^I299F heterozygous mutant littermates as controls were tested at the age of 4–6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1^I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed.

Conclusions

In this systemic analysis no clear primary effects of the Slc12a1^I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.     

Hoxa-5 in mouse developing lung: cell-specific expression and retinoic acid regulation

Hoxa-5 is a homeobox gene that is highly expressed in the developing mouse lung. However, little is known about the molecular mechanisms controlling expression. We characterized the ontogeny of Hoxa-5 gene and protein expressions during lung development and then studied the cell-specific effects of retinoic acid (RA) on Hoxa-5 mRNA in fetal lung fibroblasts and MLE-12 mouse lung epithelial cells. Strong but constant Hoxa-5 gene and protein expressions were detected from mouse lung on embryonic day 13.5 to postnatal day 2. At baseline, the gene was strongly expressed in the fibroblasts of day 17.5 fetal mouse lungs. A very weak but reproducible expression was present in the MLE-12 cells. RA stimulated gene expression in both cell types in a time- and dose-dependent manner. Peak expression occurred much later in the MLE-12 cells compared with that in fibroblasts. Cycloheximide and actinomycin D treatment studies suggested that the differences in RA effect on each cell type may involve the presence of a repressor that can be overcome by RA.     

Novel expression and regulation of the renin-angiotensin system in metanephric organ culture

To evaluate the presence and regulation of the renin-angiotensin system (RAS) in metanephric organ culture, embryonic day 14 (E14) rat metanephroi were cultured for 6 days. mRNAs for renin and both ANG II receptors (AT(1) and AT(2)) are expressed at E14, and all three genes continue to be expressed in culture. Renin mRNA is localized to developing tubules and ureteral branches in the cultured explants. At E14, renin immunostaining is found in isolated cells scattered within the mesenchyme. As differentiation progresses, renin localizes to the ureteric epithelium, developing tubules and glomeruli. E14 metanephroi contain ANG II, and peptide production persists in culture. Renin activity is present at E14 (6.13 +/- 0.61 pg ANG I. kidney(-1). h(-1)) and in cultured explants (28.84 +/- 1. 13 pg ANG I. kidney(-1). h(-1)). Renin activity in explants is increased by ANG II treatment (70.1 +/- 6.36 vs. 40.97 +/- 1.94 pg ANG I. kidney(-1). h(-1) in control). This increase is prevented by AT(1) blockade, whereas AT(2) antagonism has no effect. These studies document an operational local RAS and a previously undescribed positive-feedback mechanism for renin generation in avascular, cultured developing metanephroi. This novel expression pattern and regulatory mechanism highlight the unique ability of developing renal cells to express an active RAS.     

Expression of the tudor-related gene Tdrd5 during development of the male germline in mice

Homologues of Drosophila germ cell determinant genes such as vasa, nanos and tudor have recently been implicated in development of the male germline in mice. In the present study, the mouse gene encoding Tudor domain containing protein 5 (TDRD5) was isolated from a 12.5-13.5 days post coitum (dpc) male-enriched subtracted cDNA library. Whole-mount in situ hybridization analysis of Tdrd5 expression in the mouse embryonic gonad indicated that this gene is upregulated in the developing testis from 12.5 dpc, with expression levels remaining higher in testis than ovary throughout embryogenesis. Expression of Tdrd5 was absent in testes isolated from We/We embryos, which lack germ cells. In situ hybridization (ISH) on cryosectioned 13.5 dpc testes suggests that expression of Tdrd5, like that of Oct4, is restricted to germ cells. Northern hybridization analysis of expression in adult tissues indicated that Tdrd5 is expressed in the testis only, implying that expression of this gene is restricted to the male germline throughout development to adulthood.     

Redirection of renal mesenchyme to stromal and chondrocytic fates in the presence of TGF-β2

Many members of the transforming growth factor-β (TGF-β) superfamily have been shown to be important regulators of metanephric development. In this study, we characterized the effect of TGF-β2 on metanephric development. Rat and mouse metanephroi cultured in the presence of exogenous TGF-β2 for up to 15 days were small, and contained rudimentary ureteric branches and few glomeruli. These metanephroi were mostly comprised of mesenchymal cells, with two cell populations (designated Type 1 and Type 2 cells) evident. Type 1 cells were only observed when TGF-β2 was added from the commencement of culture, they resembled chondroblasts and were Alcian Blue and Col IIB positive. Type 2 cells were observed whenever TGF-β2 was added to the media, formed a band at the periphery of the explants consisting of 5–10 layers of spindle-shaped cells, and were alpha-smooth muscle actin positive. Molecular and RNA in situ hybridization analysis of metanephroi cultured in the presence of TGF-β2 for 6 days demonstrated that Type 1 and 2 cells were negative for Pax2, WT1, GDNF and FoxD1. Gene expression profiling demonstrated an upregulation of chondrocyte, myogenic and stromal genes, some of which were identified as markers of Type 1 and Type 2 cells. In addition, TGF-β2 was capable of maintaining the survival of mouse isolated metanephric mesenchyme (iMM) in the absence of serum or inductive signals from the ureteric epithelium. TGF-β2 also induced the differentiation of iMM into Type 1 and 2 cells. The presence of chondrocytes and muscle in these cultures is reminiscent of the cell types found in some Wilms' tumors. These studies demonstrate that TGF-β2 is capable of differentiating metanephric mesenchyme away from a renal cell fate.     

Subtractive hybridization unravels a role for the ion cotransporter NKCC1 in the murine intestinal pacemaker

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. Our aim was to identify ICC-specific genes and their function in the mouse jejunum. Suppression subtractive hybridization using two independent ICC-deficient mouse models identified 56 genes putatively downregulated in the muscularis propria compared with wild-type littermates. Differential expression was confirmed by real-time quantitative PCR for the tyrosine kinase receptor KIT, the established marker for ICC, and for the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Immunoreactivity for NKCC1 was detected in myenteric ICC but not in the ICC population located at the deep muscular plexus. NKCC1 was also expressed in enteric neurons and mucosal crypts. Bumetanide, an NKCC1 inhibitor, reversibly affected the shape, amplitude, and frequency of the slow waves. Similar alterations were observed in NKCC1 knockout mice. These data support the hypothesis that NKCC1 expressed in myenteric ICC is involved in the mechanism of slow waves in the murine jejunum.     

Expression of the Na+-K+-2Cl- cotransporter in the rat endolymphatic sac

The endolymphatic sac (ES) is a part of the membranous labyrinth that contains the cochlea, vestibular organs, and semicircular canals, and is believed to absorb endolymphatic fluid. Na⁺–K⁺–2Cl⁻ (NKCC) is a cotransporter that occurs as two isoforms (NKCC-1 and NKCC-2). Especially, NKCC-2 is suggested to participate in ES endolymph absorption. In the present study, the expression and cellular localization of NKCC-1 and NKCC-2 in the rat ES were examined by RT-PCR and in situ hybridization, respectively. The findings indicate that both NKCC-1 and NKCC-2 are expressed in the rat ES and suggest that NKCC is involved in ES homeostasis. NKCC-2 may be particularly involved in endolymph absorption. This is the first report confirming NKCC expression in the ES.     

Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin

Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4(-/-), are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO(3) (-) transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4(-/-) mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4(+/-) and Slc26a4(-/-) mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4(-/-) mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4(-/-) mice, and possibly in humans, lacking functional pendrin expression.     

Transplantation of metanephroi across the major histocompatibility complex in rats

To determine whether transplanted metanephroi grow, differentiate, and function in hosts that differ in major histocompatibility complex loci (RT1 loci in rats) from donors in a defined way, we implanted metanephroi from embryonic day (E) 15 PVG (RT1(c)) rat embryos into the omentum of nonimmunosupressed uninephrectomized PVG-RT1(avl) (host) rats. By 4 wk posttransplantation, metanephroi had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts had normal structure and ultrastructure. At 12 wk posttransplantation, weights of metanephroi were 54 +/- 8 mg. Inulin clearances were 0.9 +/- 0.3 microl. min(-1). 100 g rat wt(-1). In vitro, splenocytes from PVG rats stimulated the proliferation of cells originating from both PVG-RT1(avl) rats in which a transplant had been performed and PVG-RT1(avl) rats with no transplant. Full-thickness PVG-RT1(avl) skin engrafted normally on PVG-RT1(avl) rats in which PVG metanephroi had been previously implanted and metanephroi retained a normal appearance. In contrast, skin from PVG rats sloughed, and the tubular architecture of metanephroi was obliterated by a mononuclear cell infiltrate consistent with acute rejection. Here we show for the first time that functional chimeric kidneys develop from metanephroi transplanted across the MHC into nonimmunosupressed hosts and provide evidence that a state of peripheral immune tolerance secondary to T cell "ignorance" permits their survival.