The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells

We have previously demonstrated that apical Na⁺ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971–6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na⁺ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882–C888); however, it is unknown if the Na⁺ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for modulation of Na⁺ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for regulation of Na⁺ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na⁺ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na⁺ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na⁺ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 ± 5.42%) when compared to control (unstimulated) (f = 14.2 ± 0.77%) and vasopressin-treated (20 min) (f = 12.7 ± 2.4%) monolayers. The twofold increase in mobile fraction of Na⁺ channels corresponds to the average increase in Na⁺ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na⁺ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na⁺ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na⁺ channels.Supported by NIH grants DK37206 (DJB), NS26733 and NS28072 (KJA), DK46705 (PRS) and AHA New York Affiliate grant 91007G (LCS).     

Amiloride-sensitive apical membrane sodium channels of everted Ambystoma collecting tubule

Patch clamp methods were used to characterize sodium channels on the apical membrane of Ambystoma distal nephron. The apical membranes were exposed by everting and perfusing initial collecting tubules in vitro. In cell-attached patches, we observed channels whose mean inward unitary current averaged 0.39±0.05 pA (9 patches). The conductance of these channels was 4.3±0.2 pS. The unitary current approached zero at a pipette voltage of –92 mV. When clamped at the membrane potential the channel expressed a relatively high open probability (0.46). These characteristics, together with observation that doses of 0.5 to 2 m amiloride reversibly inhibited the channel activity, are consistent with the presence of the high amiloride affinity, high sodium selectivity channel reported for rat cortical collecting tubule and cultured epithelial cell lines.We used antisodium channel antibodies to identify biochemically the epithelial sodium channels in the distal nephron of Ambystoma. Polyclonal antisodium channel antibodies generated against purified bovine renal, high amiloride affinity epithelial sodium channel specifically recognized 110, 57, and 55 kDa polypeptides in Ambystoma and localized the channels to the apical membrane of the distal nephron. A polyclonal antibody generated against a synthetic peptide corresponding to the C-terminus of Apx, a protein associated with the high amiloride affinity epithelial sodium channel expressed in A6 cells, specifically recognized a 170 kDa polypeptide. These data corroborate that the apically restricted sodium channels in Ambystoma are similar to the high amiloride affinity, sodium selective channels expressed in both A6 cells and the mammalian kidney.This work was supported by American Heart Association, New York Affiliate Grant 91007G (LCS) and National Institute of Diabetes and Digestive and Kidney Disease Grants DK-37206 (DJB) and DK46705 (PRS).     

Analysis of molecular differentiation in a hybrid zone between chromosomally distinct races of the common shrew Sorex araneus (Insectivora: Soricidae) suggests their common ancestry

During postglacial colonization, populations that diverged in different refugia produced a patchwork of genomes, often delimited with sharp hybrid zones. The outcome of hybridization following the secondary contact of two genetically distinct populations is hard to predict. In this context, the present study investigated the genetic structure of the hybrid zone between the Drnholec and Białowieża chromosome races of the common shrew ( Sorex araneus ) in Poland using biparentally inherited (seven autosomal microsatellites) and uniparentally inherited (Y-linked microsatellite and mtDNA) molecular markers. On the basis of diagnostic chromosomes, the Drnholec and Białowieża races were classified to different karyotypic groups, which were believed to have independent glacial histories. It was found that genetic differentiation between the Drnholec and Białowieża races was weak and nonsignificant with respect to all molecular markers. However, these results are in contrast with the chromosomal structure of this hybrid zone. The very sharp frequency clines of the diagnostic chromosomes strongly suggest that gene flow between the Drnholec and Białowieża races was reduced. Nonsignificant correlations between genetic differentiation and both the presence of an environmental barrier and geographical distance reveal that only differences in karyotypes might be a reason for limited gene exchange between the races. It is assumed that a lack of molecular differences between the Drnholec and Białowieża races results from a shared ancestral variation.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 79–90.     

The Role of Mammals in Creating and Modifying Seedshadows in Tropical Forests and Some Possible Consequences of Their Elimination

Mammal populations are increasingly hunted, yet the consequences of their disappearance from tropical forests have only recently been explored. Here, we summarize current research on the role of mammals in seed dispersal and postdispersal processes, such as seed predation and secondary dispersal, in different tropical regions. We evaluate how mammal features influence seedshadows and ultimately forest regeneration. Finally, we discuss the potential effect of changes in seedshadows caused by the elimination of many medium- and large-sized mammals. The complex role that mammals play in creating and modifying seedshadows in tropical forests cannot be easily quantified, and in this review we emphasize the variation that exists both within and among mammal taxa and across continents. To bridge this gap in information, we suggest that more studies should evaluate the relative importance of the disappearance of both seed dispersers and seed predators for particular plant species so that we may begin to understand the balance between these two influences. We also suggest that future studies identify ecological redundancy in nonhunted vertebrates within any particular community to evaluate compensatory behavior that may help ameliorate some of the negative effects of hunting of large and medium mammals.     

Associations between intensive diabetes therapy and NMR-determined lipoprotein subclass profiles in type 1 diabetes

Our objective is to define differences in circulating lipoprotein subclasses between intensive versus conventional management of type 1 diabetes during the randomization phase of the Diabetes Control and Complications Trial (DCCT). NMR-determined lipoprotein subclass profiles (NMR-LSPs), which estimate molar subclass concentrations and mean particle diameters, were determined in 1,294 DCCT subjects after a median of 5 years (interquartile range: 4–6 years) of randomization to intensive or conventional diabetes management. In cross-sectional analyses, we compared standard lipids and NMR-LSPs between treatment groups. Standard total, LDL, and HDL cholesterol levels were similar between randomization groups, while triglyceride levels were lower in the intensively treated group. NMR-LSPs showed that intensive therapy was associated with larger LDL diameter (20.7 vs. 20.6 nm, P = 0.01) and lower levels of small LDL (median: 465 vs. 552 nmol/l, P = 0.007), total IDL/LDL (mean: 1,000 vs. 1,053 nmol/l, P = 0.01), and small HDL (mean: 17.3 vs. 18.6 μmol/l, P < 0.0001), the latter accounting for reduced total HDL (mean: 33.8 vs. 34.8 μmol/l, P = 0.01). In conclusion, intensive diabetes therapy was associated with potentially favorable changes in LDL and HDL subclasses in sera. Further research will determine whether these changes contribute to the beneficial effects of intensive diabetes management on vascular complications.     

Conservation Throughout Vertebrate Evolution of the Predicted β-Strands in Myelin Basic Protein

To identify functionally important parts of the 18.5-kDa myelin basic protein (MBP), the amino acid sequences from 10 species ranging from shark to human were aligned using the SEQHP computer program. The residues that are invariant or very conservatively substituted (Arg/Lys, Ser/Thr, Ile/Leu, Asp/Glu) among all 10 proteins were scored. Of the 72 conserved residues in the 170-residue human protein (42% conserved), 32 are found within the five beta-strands previously predicted (45 residues, 71% conserved), 23 within the small-loops region (42 residues, 55% conserved), but only 17 within the large-loops region (83 residues, 20% conserved). Of the 22 hydrophobic residues within the predicted beta-sheet of human MBP, 20 hydrophobic residues remain in the shark protein, 19 of them in the same positions. In contrast, there are 10 hydrophobic residues elsewhere in the human protein, but only 7 remain in the shark protein and only 1 of them is in the same position. The triprolyl sequence found in all mammalian MBPs and in the chicken MBP is not conserved in the shark protein. The four alternately spliced forms of mouse MBP can be accommodated by the beta-structural model, but not the 17-kDa human MBP, which lacks exon 5. These findings confirm the crucial role of the hydrophobic residues in the predicted beta-sheet for the structure and function of the protein. It seems likely that the conserved portions of the protein make an important contribution to the highly ordered lamellar structure of myelin.