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A major quantitative trait locus (QTL) on rat chromosome (RNO)6 was linked to albuminuria in Munich Wistar Frömter rats (MWF). We tested whether transfer of MWF RNO6 into the background of albuminuria-resistant spontaneously hypertensive rats (SHR) induces albuminuria in consomic SHR-6(MWF) animals. Male MWF, SHR, and SHR-6(MWF) were sham operated and treated between 6 and 24 wk of age with normal water (Sham) or with water containing 20 mg/l N(G)-nitro-L-arginine methyl ester (L-NAME) or unilaterally nephrectomized (Nx). Compared with SHR albuminuria was not increased in SHR-6(MWF) in both Sham and Nx groups. All animals survived the observation period in Sham and Nx groups, while premature mortality occurred from 12-14 wk on in L-NAME-treated SHR and SHR-6(MWF) compared with MWF L-NAME animals, in which survival was not affected (P < 0.005, respectively). Subsequent further analysis of L-NAME-treated animals at 12 wk of age showed significantly increased arterial blood pressures in both SHR and SHR-6(MWF) compared with control (P < 0.05), with higher levels in SHR compared with consomics (P < 0.05). However, L-NAME-treated consomic animals demonstrated increased albuminuria compared with SHR (12.7 +/- 3.5 vs. 0.8 +/- 0.2 mg/24 h; P < 0.05) and an induction of tubulointerstitial structural injury and expression of neutrophil gelatinase-associated lipocalin mRNA (P < 0.05 vs. other strains). Our study demonstrates that isolation of the RNO6 albuminuria QTL from the MWF background and transfer into SHR fails to induce an albuminuria phenotype during normal conditions or after nephron reduction. Moreover, our data indicate that genes on RNO6 contribute to the development of L-NAME-induced renal damage in the SHR strain.

Intense neuronal activity in the sensory retina is associated with a volume increase of neuronal cells (Uckermann et al., J. Neurosci. 2004, 24:10149) and a decrease in the osmolarity of the extracellular space fluid (Dmitriev et al., Vis. Neurosci. 1999, 16:1157). Here, we show the existence of an endogenous purinergic mechanism that prevents hypoosmotic swelling of retinal glial (Müller) cells in mice. In contrast to the cells from wild-type mice, hypoosmotic stress induced rapid swelling of glial cell somata in retinal slices from mice deficient in P2Y(1), adenosine A(1) receptors, or ecto-5'-nucleotidase (CD73). Consistently, glial cell bodies in retinal slices from wild-type mice displayed osmotic swelling when P2Y(1) or A(1) receptors, or CD73, were pharmacologically blocked. Exogenous ATP, UTP, and UDP inhibited glial swelling in retinal slices, while the swelling of isolated glial cells was prevented by ATP but not by UTP or UDP, suggesting that uracil nucleotides indirectly regulate the glial cell volume via activation of neuronal P2Y(4/6) and neuron-to-glia signaling. It is suggested that autocrine/paracrine activation of purinergic receptors and enzymes is crucially involved in the regulation of the glial cell volume.

Background: We investigated whether alterations of transient receptor potential canonical (TRPC) channel expression may be observed in tissues from Munich Wistar Frömter (MWF) rats showing proteinuria compared to control Wistar rats., Methods: TRPC expression was investigated in tissue from MWF and Wistar rats using quantitative real time PCR, immunoblotting, and immunohistochemistry., Results: Compared to Wistar rats MWF rats showed significantly increased systolic blood pressure and significantly higher left ventricle weight (each p < 0.01). Quantitative real time PCR revealed that TRPC3 transcripts were significantly higher in kidney cortex from MWF rats compared to Wistar rats (p < 0.01). TRPC3 transcripts were not significantly different in kidney medulla nor in aorta from both groups (p = n.s.). Furthermore, TRPC6 transcripts were significantly lower in kidney cortex from MWF rats compared to Wistar rats (p < 0.001). Immunoblotting showed that TRPC3 channel protein expression was also significantly higher in kidney cortex from MWF rats compared to Wistar rats (p < 0.01). There was a significant correlation of TRPC3 mRNA and a specific marker for endothelium, von Willebrand factor (vWF; Spearman r = 0.564; p < 0.01). We observed a significant correlation between the TRPC3 transcripts to TRPC6 transcripts ratio in kidney cortex and urinary albumin excretion (Spearman r = 0.785, p < 0.001)., Conclusion: Altered TRPC expression pattern in kidney cortex is associated with kidney damage in MWF rats showing hypertension and albuminuria., (Copyright 2009 S. Karger AG, Basel.)

Aims: In patients with renal disease the cardiovascular risk is greatly increased, and endothelial dysfunction is assumed to play a pivotal role in this process. Therefore we compared treatment effects of a beta-blocker with additional vasodilatory capacities (nebivolol) and a beta-blocker lacking these actions (metoprolol) on intrarenal and coronary vascular function in a rat model of renal failure with hypertension., Main Methods: Renal failure was induced by 5/6-nephrectomy (Nx) and analyzed after 4 weeks in Wistar rats. Untreated Nx, Nx/nebivolol 6 mg/d (Nx-Nebi); Nx/metoprolol 60 mg/d (Nx-Meto) and sham-operated (Sham) animals were studied. Isolated small renal and coronary arteries were investigated for endothelium-dependent relaxation to acetylcholine (ACh) and for the contribution of the endothelial mediators NO and endothelium-derived hyperpolarizing factor (EDHF)., Key Findings: Systolic blood pressure (SBP) was significantly increased in Nx, Nx-Nebi, and Nx-Meto (168+/-5, 153+/-3, and 162+/-6 mmHg) compared to Sham (138+/-3 mmHg, p<0.05, respectively). The increase in albuminuria of Nx (120-fold vs. Sham, p<0.0001) was almost (-85%) normalized by nebivolol compared to Sham (p<0.05), whereas metoprolol induced no significant effect. Renal arteries showed significantly increased Ach-relaxation in Nx and Nx-Nebi (Emax 86+/-4% and 76+/-7%, p<0.05) due to an increase in EDHF-mediated dilation (Emax&#95;EDHF 78+/-7% and 73+/-6%) compared to Sham (Emax 54+/-4% and Emax&#95;EDHF 44+/-6%) and Nx-Meto (Emax 42+/-12% and Emax&#95;EDHF 18+/-5%). ACh-relaxation in coronary arteries was similar between groups but the contribution of NO (relative to EDHF) was strongly increased by nebivolol., Significance: The present findings offer an explanation of the nephroprotective effect of intrarenal endothelial function in renal failure.

The neuronal ceroid lipofuscinoses (NCLs) are a group of genetic progressive brain diseases of children and young adults, characterized by a decline of mental and other capacities, epilepsy, and visual loss through retinal degeneration. The common pathology of NCLs is that of a storage disorder with accumulation of an autofluorescent material, ceroid lipofuscin, in combination with the degeneration of neuronal cells. At least 10 genetically distinct NCLs, designated CLN1 to CLN10, are presently known. Several NCLs exhibit a widely variable clinical picture, depending on the severity of the individual mutation. Some NCLs are not particularly rare. With increasing awareness of these disorders and better diagnostic techniques available, the number of recognized patients is rising. This overview briefly summarizes recent developments (or quotes corresponding literature) that are important to understand, diagnose, and manage patients suffering from one of these incurable disorders.

The neuronal ceroid lipofuscinoses (NCLs) form a group of autosomal recessively inherited neurodegenerative disorders that mainly affect children. Ten NCL forms can be distinguished by age at onset, clinicopathologic features, and genetics. In eight of these forms, the underlying genes have been identified. At present, approximately 10% of all patients do not fall into one of the eight known genetic forms of NCL. We have identified two Asian families with two novel homozygous mutations in the CLN5 gene. In the first Pakistani family, two children developed symptoms of an early juvenile NCL. After exclusion of mutations in genes known to be associated with this age of onset in families from many different countries (CLN1, CLN2, CLN3, CLN6, CLN8 and CLN10) SNP array-based homozygosity mapping led to the identification of a novel homozygous mutation c.1072&#95;1073delTT (p.Leu358AlafsX4) in CLN5. In the second Afghan family, two children developed symptoms of a late infantile NCL. The mutation c.1137G>T (p.Trp379Cys) in CLN5 was identified. The affected children in these families represent the first reported CLN5 patients originating in Asian sibships. Expression analysis showed that mutant p.Leu358AlafsX4 CLN5 is truncated and lacks a used N-glycosylation site at Asn401. The missense mutation p.Trp379Cys affected neither the size nor glycosylation of the CLN5 protein. Double immunofluorescence microscopy showed that while the wild-type CLN5 protein is localized in lysosomes, both mutant CLN5 proteins are retained in the endoplasmic reticulum rather than reaching the lysosome., (Copyright 2009 Wiley-Liss, Inc.)

Obese melanocortin-4-receptor-deficient (MC4R-/-) male mice are reported to have erectile dysfunction, while homozygous MC4R-/- female mice are apparently fertile. A recently established obese mouse strain, carrying an inactivating mutation in the MC4R gene, revealed difficulties in breeding for the homozygous female mice. This prompted us to determine the presence of follicles and corpora lutea (CL) in ovaries of MC4R-/- mice aged 3-6 months in comparison to wild type (MC4R+/+) littermates. Serial sections of formaldehyde-fixed ovaries of mice with vaginal signs of estrus and metestrus were assessed for the number of healthy and regressing follicles and CL. The number of CL, as an estimate for the ovulation rate, decreased to zero during aging in MC4R-/- mice. The number of small- (diameter 100-200 micrometer) and large-sized follicles namely antral follicles (diameter >200 micrometer) were slightly increased in MC4R-/- compared to MC4R+/+ mice. Greater differences were found in very large to cystic follicles, which were more numerous in MC4R-/- mice. The number of regressing antral follicles was higher in the MC4R-/- group compared to the MC4R+/+ group. This was associated with a wide range in the number of collapsed zonae pellucidae as the last remnants of regressed follicles. A conspicuous hypertrophy of the interstitial cells was noted in 6-month-old MC4R-/- mice. In conclusion, cystic follicles and the reduction in CL number point to a decreased ovulation rate in obese MC4R-/- mice.

Aims: Previously we confirmed an important role of rat chromosome 19 (RNO19) for salt-sensitive hypertension and target organ damage in male Dahl salt-sensitive rats (SS rats). The aim of this study was to further analyse the basis of left ventricular (LV) fibrosis development in both male and female rats in this model. To this end we utilized a consomic SS-19(SHR) rat strain in which RNO19 was transferred from spontaneously hypertensive rats (SHR) into the susceptible background of SS., Methods and Results: We compared the effects of low- (0.2% NaCl) and high-salt (4% NaCl) diet on the development of hypertension, blood lipids and LV fibrosis in male and female SS, SHR, and SS-19(SHR) rats. Systolic blood pressure was significantly lower in male and female SS-19(SHR) compared with SS under both diets (P < 0.001). Relative LV weight was similarly reduced in SS-19(SHR) compared with SS in either sex. Plasma cholesterol concentrations were significantly elevated in high-salt fed male and female SS (141 +/- 6 and 110 +/- 7 mg/dL) compared with SHR (47 +/- 2 and 62 +/- 8 mg/dL, P < 0.001) and were significantly lowered in male and female consomic rats (100 +/- 7 and 87 +/- 3 mg/dL). Both LV interstitial fibrosis (LVIF) and perivascular fibrosis (LVPF) were significantly reduced in high-salt male and female SS-19(SHR). A significant correlation between cholesterol concentrations and LVPF (r = 0.464) and LVIF (r = 0.401, P < 0.0001, respectively) was detected. Fibrosis parameters demonstrated no correlation with blood pressure, LV weight or plasma triglycerides concentrations. LV immunohistochemistry analysis showed a significant higher number of ED-1 positive cells in SS compared with SS-19(SHR). Depositions of collagen I and fibronectin were also greater in LV tissue of SS compared with SS-19(SHR)., Conclusion: Our findings point to a link between hypercholesterolemia and LV fibrosis in salt-sensitive hypertension of SS rats which is genetically modulated by RNO19.

Mammals adapted to a great variety of habitats with different accessibility to water. In addition to changes in kidney morphology, e.g. the length of the loops of Henle, several hormone systems are involved in adaptation to limited water supply, among them the renal-neurohypophysial vasopressin/vasopressin receptor system. Comparison of over 80 mammalian V2 vasopressin receptor (V2R) orthologs revealed high structural and functional conservation of this key component involved in renal water reabsorption. Although many mammalian species have unlimited access to water there is no evidence for complete loss of V2R function indicating an essential role of V2R activity for survival even of those species. In contrast, several marsupial V2R orthologs show a significant increase in basal receptor activity. An increased vasopressin-independent V2R activity can be interpreted as a shift in the set point of the renal-neurohypophysial hormone circuit to realize sufficient water reabsorption already at low hormone levels. As found in other desert mammals arid-adapted marsupials show high urine osmolalities. The gain of basal V2R function in several marsupials may contribute to the increased urine concentration abilities and, therefore, provide an advantage to maintain water and electrolyte homeostasis under limited water supply conditions.

Genetic factors influence renal disease progression, and several loci have been linked to the spontaneous development of proteinuria and glomerulosclerosis in animal models. However, the role of genetic susceptibility in glomerulonephritis-induced progressive glomerulosclerosis is unknown. In a rat model of mesangial proliferative glomerulonephritis, anti-Thy-1 glomerulonephritis (antiThy1GN), Lewis/Maastricht (Lew/Maa) rats exhibit progression to glomerulosclerosis, whereas in genetically related Lewis/Møllegard (Lew/Moll) rats, glomerular lesions are repaired within 3 wk. The genetic factors underlying this strain-related difference are not known. To identify novel quantitative trait loci (QTL) involved in progression or repair in Lewis rats, 145 female backcross rats [F1(Lew/Maa x Lew/Moll) x Lew/Maa] were studied. After induction of antiThy1GN proteinuria, we determined mesangial activation, the percentage of microaneurysms, and the glomerular damage score for each animal; a genome scan using 187 microsatellite markers was performed. QTL mapping revealed a significant QTL for glomerular damage score on chromosome 1 with a logarithm of odds (LOD) score of 3.9. Homozygosity for Lew/Maa DNA in this region was associated with a higher percentage of damaged glomeruli on day 21. Furthermore, suggestive linkage was found for the percentage of glomeruli with microaneurysms on day 3 on chromosome 1, 6, and 11; for mesangial activation on day 7 on chromosome 18, while proteinuria was suggestively linked to chromosome 5 (day 0), 4 (day 3), and 6 (day 7). This study identifies a QTL on rat chromosome 1 that is significantly linked to progressive glomerulosclerosis after acute glomerulonephritis.

The Munich Wistar Frömter (MWF) rat represents a genetic model with an inherited nephron deficit and exhibits mild hypertension and progressive albuminuria, which is more pronounced in males than females. Previously, we demonstrated in a consomic strain that replacement of a quantitative trait locus on chromosome 6 normalized the nephron deficit and suppressed albuminuria development, suggesting a link between the two findings. Here we tested the role of a second major locus linked to albuminuria in MWF on chromosome 8 and generated the consomic strain MWF-8(SHR) by transfer of chromosome 8 from spontaneously hypertensive rats (SHR) into MWF. The early onset of albuminuria at 8 wk of age in MWF (>50-fold increase compared with SHR) was significantly suppressed in consomic animals, and the development of marked proteinuria at 32 wk significantly diminished. Total nephron number in consomic rats (23,771 +/- 1,352) and MWF (27,028 +/- 1,322) were similar and significantly lower (-36%) compared with SHR (36,979 +/- 1,352, P < 0.0001). The development of mild albuminuria in female MWF was also significantly diminished in MWF-8(SHR). Thus, the development of overt and mild albuminuria in male and female MWF rats is not a mandatory consequence of the inherited nephron deficit. The locus on chromosome 8 appears of interest, because its exchange between MWF and SHR protects against the development of albuminuria in MWF-8(SHR) animals despite their inherited nephron deficit and higher systolic blood pressure.

Background: Gene knock down by RNAi is a highly effective approach to silence gene expression in experimental as well as therapeutic settings. However, this widely used methodology entails serious pitfalls, especially concerning specificity of the RNAi molecules., Results: We tested the most widely used control siRNA directed against GFP for off-target effects and found that it deregulates in addition to GFP a set of endogenous target genes. The off-target effects were dependent on the amount of GFP siRNA transfected and were detected in a variety of cell lines. Since the respective siRNA molecule specific for GFP is widely used as negative control for RNAi experiments, we studied the complete set of off-target genes of this molecule by genome-wide expression profiling. The detected modulated mRNAs had target sequences homologous to the siRNA as small as 8 basepairs in size. However, we found no restriction of sequence homology to 3'UTR of target genes., Conclusion: We can show that even siRNAs without a physiological target have sequence-specific off-target effects in mammalian cells. Furthermore, our analysis defines the off-target genes affected by the siRNA that is commonly used as negative control and directed against GFP. Since off-target effects can hardly be avoided, the best strategy is to identify false positives and exclude them from the results. To this end, we provide the set of false positive genes deregulated by the commonly used GFP siRNA as a reference resource for future siRNA experiments.

GPCRs (G-protein-coupled receptors) exist in a spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and inverse agonists respectively. Because ligand binding of agonists and inverse agonists often occurs in a competitive manner, one can assume an overlap between both binding sites. Only a few studies report mutations in GPCRs that convert receptor blockers into agonists by unknown mechanisms. Taking advantage of a genetically modified yeast strain, we screened libraries of mutant M(3)Rs {M(3) mAChRs [muscarinic ACh (acetylcholine) receptors)]} and identified 13 mutants which could be activated by atropine (EC50 0.3-10 microM), an inverse agonist on wild-type M(3)R. Many of the mutations sensitizing M(3)R to atropine activation were located at the junction of intracellular loop 3 and helix 6, a region known to be involved in G-protein coupling. In addition to atropine, the pharmacological switch was found for other M(3)R blockers such as scopolamine, pirenzepine and oxybutynine. However, atropine functions as an agonist on the mutant M(3)R only when expressed in yeast, but not in mammalian COS-7 cells, although high-affinity ligand binding was comparable in both expression systems. Interestingly, we found that atropine still blocks carbachol-induced activation of the M(3)R mutants in the yeast expression system by binding at the high-affinity-binding site (Ki approximately 10 nM). Our results indicate that blocker-to-agonist converting mutations enable atropine to function as both agonist and antagonist by interaction with two functionally distinct binding sites.

Clinical and experimental studies indicate that the progression of renal disease is faster in males than females. These observations are corroborated by a sexual dimorphism observed in the polygenetic MWF (Munich Wistar Frömter) rat model. The age-dependent spontaneous progression of increased UAE (urinary albumin excretion) in male MWF rats is influenced by multiple QTLs (quantitative trait loci). In contrast, female MWF rats only develop a slight increase in UAE, while the role of genetic factors for this phenotype is unknown. In the present study, we show that, compared with resistant SHRs (spontaneously hypertensive rats), both male and female MWF rats develop a significant increase in UAE at 24 weeks of age (P<0.0001), although blood pressures were lower compared with SHRs (P<0.0001). UAE was significantly higher in male (7-fold) compared with female MWF rats (162.6+/-15.9 compared with 24.0+/-5.5 mg/24 h respectively; P<0.0001), and only male MWF rats developed significant glomerulosclerosis and tubulointerstitial damage in the kidney (P<0.0001). To test the role of genetic factors in the development of low grade albuminuria in female MWF rats, we analysed the role of a major UAE QTL on rat chromosome 6. To this end, we analysed a consomic MWF-6(SHR) strain in which chromosome 6 from SHRs was introgressed into the MWF rat background. Time course analysis of UAE in females indicated that the small increase in UAE in MWF rats was fully suppressed by exchange of rat chromosome 6. Thus, taken together with previous studies in males, we show that RNO6 protects against the increase in albuminuria with age in both female and male MWF rats.

Background: To study the regulation of a naturally occurring genetic variant of high angiotensin-converting enzyme (ACE) gene (Ace in rat) expression, i.e., the Ace allele of the normotensive Wistar-Kyoto (WKY) rat, in the hypertensive background of stroke-prone spontaneously hypertensive (SHRSP) rats., Methods: We analyzed a congenic strain termed SHRSP.WKY-Ace derived from SHRSP in which a chromosomal fragment of rat chromosome 10 including Ace was replaced by the WKY locus. We compared blood pressures by radiotelemetry, measured plasma ACE activity, tissue ACE messenger RNA (mRNA) and enzyme activities in lung, kidney, and left ventricle (LV) of the heart in adult animals., Results: Congenic animals demonstrated a twofold increase in plasma ACE activity in comparison to SHRSP (P < 0.05) and thus similar levels to WKY. The increased tissue expression of ACE mRNA and enzyme activities in lung, kidney, and LV observed in WKY were similarly found in congenic animals when compared to SHRSP (P < 0.05, respectively). Systolic and diastolic blood pressures were not different between congenic and SHRSP animals. Analysis of renin in plasma and angiotensin peptides in LV tissues indicated the induction of compensatory mechanisms by downregulation of renin and angiotensin I (Ang I) concentrations in congenic animals., Conclusions: We demonstrated that genetically determined high ACE expression linked to WKY Ace remains unchanged in the hypertensive background of SHRSP.WKY-Ace. Our data indicate that buffering mechanisms in the renin-angiotensin system contribute to the finding that the development of spontaneous hypertension is not affected, despite an average twofold higher expression of ACE in congenic animals.

Objective: Increased urinary albumin excretion (UAE) represents an independent cardiovascular risk factor in the general population and particularly in patients with diabetes or arterial hypertension. It has been suggested that increased UAE may be related to a generalized endothelial dysfunction. We set out to identify candidate genes for increased UAE by glomerular transcriptome analysis in the Munich Wistar Frömter (MWF) genetic rat model with spontaneous hypertension and albuminuria., Methods: First, we performed microarray expression analysis in isolated glomerular tissue in MWF with established albuminuria and normal Wistar rats. Second, in validation experiments and follow-up studies we focused on the identified upregulation of glomerular complement component C1q expression in MWF., Results: Overall, 38 genes with a regulation score > 2 were differentially expressed in glomerular RNA. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), in-situ hybridization and immunohistochemistry analysis revealed that C1q is indeed significantly upregulated in the glomerulus of MWF. Additionally, CD24, although not detected by the microarray experiment, was found to be differentially expressed in MWF glomeruli using quantitative real-time RT-PCR and immunohistochemstry. Interestingly, we could show for the first time that the glomerular endothelium represents the site of increased C1q and CD24 expression in MWF. In contrast, endothelial expression of this gene is low or absent in normotensive Wistar and in spontaneously hypertensive rats (SHR) without albuminuria., Conclusions: The induction of C1q and CD24 expression confined to the glomerular endothelium might represent a possible repair mechanism of the capillary wall damage.

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y(12)-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y(12), have been deorphanized. The P2Y(12)-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y(12) in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y(12)-like receptor members.

Classical methods have been recruited to determine the molecular function and the physiological relevance of G-protein-coupled receptors (GPCRs), including ligand-binding and signal transduction studies, pharmacological receptor profiling in tissues and the characterization of transgenic mouse models. Evolutionary data from both sequenced genomes and targeted retrieved orthologs are increasingly used as a source of structural information. Recent success in sequencing and functionally expressing GPCRs from fossils opens the possibility of studying signaling pathways even in extinct species. Therefore, mining evolutionary data provides an additional source for understanding the functional relevance of individual GPCRs, for interpreting naturally occurring receptor mutations in patients and for guiding structural modeling and mutagenesis studies of GPCRs.

The common seven-transmembrane-domain (TMD) architecture of G protein-coupled receptors (GPCRs) has been preserved over a vast period of time, and highly conserved amino acid motifs and residues have evolved to establish ligand and signal transduction specificities. The mining of evolutionary data from sequenced genomes and targeted retrieved orthologs has proven helpful for understanding the physiological relevance of individual GPCRs and for interpreting the clinical significance of GPCR mutations in structural terms. Sequence analysis of GPCR pseudogenes, which are considered as genomic traces of past functions, as well as recent success in sequence analysis of GPCR genes from extinct species, provide further information. This review discusses recent advances and approaches aimed at developing a better understanding of GPCR biology based on evolutionary data.

In a cross between the Munich Wistar Frömter (MWF) rat and spontaneously hypertensive rats (SHR), a major quantitative trait locus (QTL) was identified on rat chromosome 6 (RNO6) that demonstrated the strongest linkage to albuminuria among several QTL identified. The QTL represented the only locus that is linked to both early-onset albuminuria and increased renal interstitial fibrosis in adult animals. A consomic MWF-6(SHR) strain in which chromosome 6 from SHR was introgressed into the MWF background therefore was generated to test the relevance of this QTL. Phenotype analysis at 8 wk of age revealed that early onset of albuminuria in MWF with a 55-fold elevation of urinary albumin excretion compared with SHR (P < 0.0001) was completely abolished in MWF-6(SHR). Time-course analysis until week 24 demonstrated only a moderate increase of urinary albumin excretion in MWF-6(SHR), whereas MWF reached levels in the nephrotic range (16.6 +/- 3.5 versus 162.6 +/- 16.0 mg/24 h; P < 0.0001). At this age, analysis of glomerulosclerosis, tubulointerstitial damage, renal interstitial fibrosis, and renal collagen III mRNA expression revealed a significant improvement of all parameters in MWF-6(SHR) compared with MWF (P < 0.05). At 32 wk, MWF but not MWF-6(SHR) demonstrated overt proteinuria (354.6 +/- 37.6 versus 48.8 +/- 13.2; P < 0.0001), whereas serum urea, cholesterol, and triglyceride concentrations were lower and creatinine clearance was higher in MWF-6(SHR) compared with MWF (P < 0.05). Therefore, although albuminuria in MWF is determined by a complex interplay of several QTL, our data demonstrate that genetic exchange of one locus on RNO6 leads to marked suppression of early-onset albuminuria and renal damage in MWF.

Objectives: Unlike Dahl salt-sensitive (SS) rats, some strains of spontaneously hypertensive (SHR) rats develop only minor organ damage even when exposed to high-salt diet. In previous linkage studies, we identified quantitative trait loci on rat chromosome 19 (RNO19) linked to the SHR allele suggesting a protective effect against salt-induced hypertensive organ damage in SS., Methods: To test the relevance of this finding, we generated and characterized a consomic strain SS-19SHR in which RNO19 from SHR was introgressed into the susceptible background of SS. We compared the effects of low-salt (0.2% NaCl) and high-salt (4% NaCl) diet exposure for 8 weeks on the development of hypertension and target organ damage in male consomic and SS animals (n=14-20, each)., Results: Systolic blood pressure, relative left ventricular weight and urinary protein excretion were significantly lower in SS-19SHR compared to SS under both low-salt and high-salt diet (P < 0.05, respectively). Left ventricular atrial natriuretic peptide mRNA expression showed a more pronounced 4.5-fold increase in SS compared to SS-19 (two-fold) after high-salt (P < 0.05). In comparison to low diet, high-salt exposure induced a significant increase in vascular aortic hypertrophy index, left ventricular interstitial fibrosis (+210%) and perivascular fibrosis (+195%) in SS but not in consomic SS-19SHR (P < 0.05, respectively)., Conclusions: These results demonstrate a strong protective effect of RNO19 from SHR on the development of hypertension, salt-sensitivity, cardiovascular and renal organ damage in SS. In particular, we demonstrate a genetic effect protecting against the development of cardiac fibrosis in salt-sensitive hypertension.

Maintenance of water and electrolyte homeostasis is central to mammalian survival and, therefore, under stringent hormonal control. Water homeostasis is achieved by balancing fluid intake with water excretion, governed by the antidiuretic action of arginine vasopressin. Arginine vasopressin stimulation of renal V 2 vasopressin receptors in the basolateral membrane of principal cells induces aquaporin-2-mediated water reabsorption in the kidney. The importance of this system is apparent when mutations inactivate V 2 vasopressin receptors and aquaporin-2 and cause the clinical phenotype of nephrogenic diabetes insipidus. To date, over 190 mutations in the V 2 vasopressin receptors gene (AVPR2) and approximately 38 mutations in the aquaporin-2 gene have been identified in patients with inherited nephrogenic diabetes insipidus. Extensive in vitro expression and mutagenesis studies of V 2 vasopressin receptors and aquaporin-2 have provided detailed insights into the molecular mechanisms of G-protein-coupled receptor and water channel dysfunction per se. Targeted deletions of AVPR2 and AQP2 in mice have extended the knowledge of nephrogenic diabetes insipidus pathophysiology and have stimulated testing of old and new ideas to therapeutically restore normal kidney function in animal models and patients with this disease. In this review, we summarize the current knowledge relevant to understand the molecular basis of inherited nephrogenic diabetes insipidus forms and the rationales for the current pharmacological treatment of patients with this illness.

A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients.

Directed cloning approaches and large-scale sequencing of several vertebrate genomes unveiled many new members of the G-protein-coupled receptor (GPCR) superfamily, among them GPR34. Initial studies showed that GPR34 is an evolutionarily old GPCR structurally related to a group of ADP-like receptors. To gain insight into the genomic organization, regulation of expression, and supragenomic diversification of GPR34 several vertebrate species were analyzed. In contrast to the obviously intronless coding region GPR34 displays an evolutionary preserved 5' noncoding intron-exon structure. Further, an alternatively used cryptic intron was identified within the coding region, which shortens the N terminus by 47 amino acids. Ubiquitous expression of GPR34 is driven by genomic sequences upstream of at least two transcriptional start regions in mouse and rat but only one region in human. In rodents, both promoters are active in all tissues investigated, but the level of activity is tissue-specific. At the translational level, several conserved in-frame AUGs within the first 150 bp of the coding region may serve as start points for translation in human and other mammals. Combinatory mutagenesis and expression of reporter constructs confirmed these multiple translational start points and revealed a preference for the second in-frame AUG in human GPR34. Our data show that multiple translation initiation starts and alternative splicing contribute to the supragenomic diversification of GPR34.

Chemokine and chemoattractant receptors are members of the large superfamily of G protein-coupled receptors (GPCR), which control leukocyte chemotaxis. In addition to their physiological role, several chemokine and chemoattractant receptors, such as CCR5 and Duffy, have been directly associated with pathogen entry. GPR33 is an orphan chemoattractant GPCR that was previously identified as a pseudogene in humans. GPR33 evolved in mammals about 125-190 million years ago. The cloning and analysis of more than 120 mammalian GPR33 orthologs from 16 of 18 eutherian orders revealed an inactivation of this chemoattractant GPCR not only in humans, but also in several great ape and rodent species. Intriguingly, in all ape and some rodent species where the inactivation occurred, samples harbored both pseudogene and intact gene variants. The analysis of over 1200 human individuals representing all major linguistic groups revealed that the intact allele of GPR33 is still present in the human population. Estimates of the age of the human alleles suggest inactivation in the past 1 million years. Similarly, analysis of more than 120 wild-caught gray rats (Rattus norvegicus), revealed that inactivation of gpr33 is worldwide fixed and occurred in less than 0.7 million years ago. The coincidental inactivation and its fixation in several species of distantly related mammalian orders suggest a selective pressure on this chemoattractant receptor gene.

G-protein-coupled receptors (GPCR) are involved in directly and indirectly controlling an extraordinary variety of physiological functions. Their key roles in cellular communication have made them the target for more than 60% of all currently prescribed drugs. Mutations in GPCR can cause acquired and inherited diseases such as retinitis pigmentosa (RP), hypo- and hyperthyroidism, nephrogenic diabetes insipidus, several fertility disorders, and even carcinomas. To date, over 600 inactivating and almost 100 activating mutations in GPCR have been identified which are responsible for more than 30 different human diseases. The number of human disorders is expected to increase given the fact that over 160 GPCR have been targeted in mice. Herein, we summarize the current knowledge relevant to understanding the molecular basis of GPCR function, with primary emphasis on the mechanisms underlying GPCR malfunction responsible for different human diseases.

We describe the ninth variant of neuronal ceroid lipofuscinosis (NCL) or Batten disease, due to defects in a putative new gene, CLN9. We therefore refer to the new variant as CLN9-deficient. Two Serbian sisters and two German brothers are described. Their clinical history is characteristic for juvenile NCL. They show similar gene expression patterns. The existence of this variant is supported by the presence of curvilinear inclusions, fingerprint profiles, and granular osmiophilic deposits in neurons, lymphocytes, and conjunctival cells. Enzyme screening and sequencing of the coding regions of other NCL genes was negative. CLN9-deficient cells have a distinctive phenotype. They have rounded cell bodies, have prominent nucleoli, attach poorly to the culture dish, and are sensitive to apoptosis but have increased growth rates. Gene expression of proteins involved in cell adhesion and apoptosis is altered in these cells. Sphingolipid metabolism is also perturbed. They have decreased levels of ceramide, sphingomyelin, lactosylceramide, ceramide trihexoside, and globoside and increased activity of serine palmitoyl transferase.

Our aim was to study the effects of high-salt diet on the genetics of albuminuria and renal injury in the Dahl salt-sensitive (SS) rat. We compared SS with salt-resistant spontaneously hypertensive rats (SHR) and with genetically related salt-sensitive stroke-prone SHR (SHRSP). Moreover, we performed genome-wide linkage analysis to identify quantitative trait loci (QTL) contributing to salt-induced renal injury in an F2 population derived from SS and SHR (n = 230). In response to high-salt diet SS and SHRSP developed a striking increase in systolic blood pressure, urinary albumin excretion (UAE), and renal damage indices compared with SHR. Both SHRSP and SS developed severe glomerulosclerosis, whereas microangiopathy, tubulointerstitial fibrosis, and inflammation were more pronounced in SHRSP. We detected two QTL with significant linkage to UAE on rat chromosomes (RNO) 6 and 19. Comparison with the recently identified salt-independent UAE QTL in young animals revealed that the UAE QTL on RNO6 is unique to high-salt conditions, whereas RNO19 plays a significant role during both low- and high-salt conditions. Some F2 animals demonstrated severe microangiopathy and tubulointerstitial injury, which exceeded the degree observed in the parental SS strain. Three loci demonstrated suggestive linkage to these phenotypes on RNO3, RNO5, and RNO20, whereas no linkage to glomerular damage was found. Further analyses at these loci indicated that the severity of renal injury was attributable to the SHR allele. Our data suggest that the SHR genetic background confers greater susceptibility for the development of microangiopathy and tubulointerstitial injury in salt-sensitive hypertension than the SS background.

Many human diseases are caused by inactivating mutations in specific G-protein-coupled receptors (GPCRs). In about 10% of these cases, a premature stop codon leads to the generation of a truncated, functionally inactive receptor protein. In this study, we tested the hypothesis that such GPCR mutations can be functionally rescued in vitro and in vivo by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature termination codons. As a model system, we studied a mutant V2 vasopressin receptor (AVPR2) containing the inactivating E242X nonsense mutation which mimics human X-linked nephrogenic diabetes insipidus (XNDI) when introduced into mice via gene targeting techniques. Studies with cultured mammalian cells expressing the E242X mutant receptor showed that G418 (geneticin) was by far the most potent aminoglycoside antibiotic capable of suppressing the E242X nonsense codon. Strikingly, G418 treatment increased AVP-mediated cAMP responses in cultured kidney collecting duct cells prepared from E242X mutant mice in vitro, and significantly improved the urine-concentrating ability of E242X mutant mice in vivo. This is the first study demonstrating that G418 (aminoglycosides) can ameliorate the clinical symptoms of a disease-causing premature stop codon in a member of the GPCR superfamily.

The development of renal interstitial fibrosis (RIF) represents an important step in the progression of chronic proteinuric nephropathies. The Munich Wistar Frömter (MWF) rat represents a valuable model to study the progression in proteinuric renal disease. MWF animals demonstrate a significant increase of urinary albumin excretion (UAE) and RIF compared with the spontaneously hypertensive rat (SHR) with low UAE. The aim of this study was to analyze the genetic basis and the relation between UAE and RIF by genetic linkage and quantitative trait loci (QTL) mapping analysis. The authors generated a backcross population between MWF and SHR including 215 male animals. UAE was determined in young backcross animals at 8 wk, and at 14 and 24 wk of age, respectively. RIF was evaluated by Sirius red staining of kidney sections and quantified by computer-assisted image analysis at 24 wk. Total genome scan analysis identified in total eight QTL linked to UAE and a major locus on chromosome 6. At this locus, homozygosity for the MWF allele exhibited a strong effect on UAE levels (threefold elevation) and displayed significant linkage already at 8 wk (logarithm of odds [LOD] = 4.3) with increasing significance at 14 and 24 wk (LOD = 7.8 and 10.1, respectively). In addition, this was the only QTL that was linked to the amount of RIF (P = 0.0009, LOD = 2.4). These data establish a genetic link between early onset albuminuria and progression of RIF at the QTL on RNO6. This study demonstrates the power of genetic linkage analysis for the dissection of physiologic pathways involved in renal disease progression.

Based on the now available crystallographic data of the G-protein-coupled receptor (GPCR) prototype rhodopsin, many studies have been undertaken to build or verify models of other GPCRs. Here, we mined evolution as an additional source of structural information that may guide GPCR model generation as well as mutagenesis studies. The sequence information of 61 cloned orthologs of a P2Y-like receptor (GPR34) enabled us to identify motifs and residues that are important for maintaining the receptor function. The sequence data were compared with available sequences of 77 rhodopsin orthologs. Under a negative selection mode, only 17% of amino acid residues were preserved during 450 million years of GPR34 evolution. On the contrary, in rhodopsin evolution approximately 43% residues were absolutely conserved between fish and mammals. Despite major differences in their structural conservation, a comparison of structural data suggests that the global arrangement of the transmembrane core of GPR34 orthologs is similar to rhodopsin. The evolutionary approach was further applied to functionally analyze the relevance of common scaffold residues and motifs found in most of the rhodopsin-like GPCRs. Our analysis indicates that, in contrast to other GPCRs, maintaining the unique function of rhodopsin requires a more stringent network of relevant intramolecular constrains.

The aim of the study was to characterize the genetic basis for the early onset of increased urinary albumin excretion (UAE) observed in the salt-sensitive Dahl rat (SS). We first characterized blood pressures and UAE values in adult SS compared with the spontaneously hypertensive rat (SHR) strain. Blood pressure measurements by radiotelemetry at 14 wk demonstrated similar spontaneous hypertension in both strains on a low-sodium diet containing 0.2% NaCl by weight, whereas UAE was markedly increased in SS compared with SHR (253.07 +/- 68.39 vs. 1.65 +/- 1.09 mg/24 h, P < 0.0001). Analysis of UAE in young animals of both strains fed a low-sodium diet demonstrated that UAE is elevated in SS as early as 4 wk of age (P < 0.0001), when ultrastructural evaluation of glomeruli by electron microscopy appears still normal. At 8 wk SS demonstrated a 280-fold elevated UAE compared with SHR (P < 0.0001). Consequently, to identify quantitative trait loci (QTLs) contributing to salt-independent early manifestation of increased UAE in the SS rat, we performed genome-wide linkage and QTL mapping analysis in a young F(2) population derived from the two contrasting strains. UAE was determined in 539 F(2) animals at 8 wk. We identified seven suggestive or significant UAE QTLs on rat chromosomes (RNO) RNO2, RNO6, RNO8, RNO9, RNO10, RNO11, and RNO19, accounting together for 34% of the overall variance of UAE in this F(2) population. Thus early onset albuminuria in the SS rat is under polygenetic influence and independent from salt loading.

Different activation mechanisms of glycoprotein hormone receptors, which are members of the G protein-coupled receptor superfamily, have been proposed. For example, the large ectodomain of glycoprotein hormone receptors may function as an inverse agonist keeping the transmembrane domain in an inactive conformation. To provide support for this hypothesis, we have generated different lutropin/choriogonadotropin receptor (LHR) constructs lacking the ectodomain. Although some ectodomain-deficient LHR constructs were targeted to the cell surface, cAMP levels remained unchanged under basal conditions and agonist application but could be increased by a mutation within the transmembrane domain 6 (D578H). Taking advantage of a constitutive activating mutation (S277N) located in the extracellular domain, we showed that the intact leucine-rich repeat-containing ectodomain is essential for constitutive activation of the LHR by mutation of the hinge region. Our findings support an activation scenario in which agonist binding or mutational alterations expose a structure within the ectodomain, which then activates the transmembrane core.

Background: Human chorionic gonadotropin/luteinizing hormone (hCG/LH) function in the male is mediated by the LH receptor (LHR) and is crucial for the normal development of internal and external genitalia. We report a 46, XY patient who presented at the age of 16 with a female phenotype and delayed puberty. Gonads were located bilaterally in the inguinal canal, removed surgically and showed hypoplastic Leydig cells. Immunostaining for the LHR revealed that some Leydig cell progenitors were positive, while others were negative, reflecting different developmental stages of Leydig cell maturation., Methods and Results: Molecular analysis of the LHR was performed on DNA extracted from blood samples of the patient, her parents and sister. The 11 exons of the LHR gene were amplified by PCR and subjected to further single stranded conformation polymorphism (SSCP) analysis. Aberrant migration patterns were observed in exon 7. Upon sequencing, a homozygous T to G transversion was identified, resulting in a F194V substitution located in the extracellular domain. The parents and sister were heterozygous carriers of this mutation. Functional studies in transiently transfected COS-7 cells with the F194V LHR mutation showed the lack of cAMP production upon hCG stimulation, indicating complete inactivation of the receptor due to impaired trafficking of the receptor to the membrane. The mutation is located within a stretch of five amino acids Ala (A)-Phe (F)-Asn (N)-Gly (G)-Thr (T), highly conserved in glycoprotein hormone receptors. For the follicle-stimulating hormone (FSH) receptor (FSHR) loss-of-function mutations have been allocated to this region, a homozygous A189V mutation resulting in a resistant ovary syndrome and impaired spermatogenesis and a heterozygous N191I mutation with no apparent phenotype. Further mutational and functional analysis of the AFN region in the LHR and FSHR revealed that the integrity of this amino acid sequence is crucial for receptor function.

By screening patients with X-linked nephrogenic diabetes insipidus (NDI) for mutations within the V(2) vasopressin receptor (AVPR2) gene, we have identified six novel and two recurrent mutations. Additionally, one patient revealed a genomic deletion of 3.2 kb encompassing most of the AVPR2 gene and the last exon/3'-region of C1 gene, which is in close proximity to the AVPR2 locus. In-depth characterization of the mutant AVPR2s by a combination of functional and immunological techniques allowed to gain further insight into molecular mechanisms leading to the receptor dysfunction. Aiming at the functional reconstitution of mutant G protein-coupled receptors, several strategies of potential therapeutic usefulness have been tested. Because the functional rescue of truncated receptors is most challenging, we addressed this issue by applying an aminoglycoside approach. Here, we demonstrate that the misreading capacity of the aminoglycoside antibiotic geneticin was sufficient to restore function of mutant AVPR2s harboring premature stop codons in an in vitro expression system.

An elevated urinary albumin excretion (UAE) is a risk factor for the development of chronic nephropathy and for cardiovascular mortality. The Munich Wistar Frömter (MWF) rat represents a genetic model that develops spontaneously mild hypertension and a 142-fold increased UAE compared with normal Lewis rats at 14 wk of age. The genetic basis of UAE in male MWF was analyzed in relation to BP by using a quantitative trait (QTL) mapping strategy. F1-hybrids generated from a cross between MWF and Lewis rats showed normal UAE rates similar to Lewis. A backcross strategy including 213 animals was therefore used. To account for age-of-onset effects, UAE was determined in young backcross animals at 8 wk and in adult animals at 14 and 24 wk of age, respectively. Total genome scan analysis identified three QTL with significant linkage to UAE and one QTL with suggestive linkage to UAE. These loci showed no linkage to BP, and BP explained only 2% of the total variance of UAE. Homozygosity for the MWF allele accounted for a similar mean increase in UAE in adult backcross animals at each UAE QTL. When single gene effects were analyzed, only one UAE QTL led to a significant increase in UAE. Early onset of albuminuria and a considerable increase of UAE in adult animals required homozygosity at three loci at least. This study demonstrates the polygenetic recessive determination of increased UAE by a set of genes that are unlinked to BP.

Background: Albuminuria is an independent risk factor of coronary artery disease and has been proposed to reflect a general endothelial disorder. The Munich Wistar Frömter (MWF) rat strain develops spontaneous albuminuria and, therefore, may be an interesting experimental model to study alterations of endothelial function under conditions of increased albuminuria. Our aim was to investigate if the MWF strain shows generalized endothelial dysfunction or endothelial dysfunction localized to the coronary vascular bed, and if so, determine which endothelial dilative mediators are involved., Methods: Coronary and mesenteric arteries were investigated for endothelium-dependent relaxation and the contribution of prostacyclin, nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) in MWF rats compared to normal Wistar rats. In addition, as MWF rats show increased blood pressure, spontaneously hypertensive rats (SHR) with similar hypertension but without increased albuminuria also were studied., Results: Maximal total endothelium-dependent relaxation of coronary arteries was strongly impaired in MWF rats (55 +/- 3%) compared to Wistar (89 +/- 5%) and SHR (89 +/- 2%) P < 0.05, respectively. The NO-mediated relaxation as well as the relaxation mediated by EDH were significantly lower in coronary arteries from MWF compared to Wistar. In mesenteric arteries of MWF the endothelium-dependent relaxation was intact., Conclusions: The strong impairment of coronary endothelium-dependent relaxation in the MWF model of spontaneous albuminuria may be due to defects in production or activity of NO and EDH. The intact mesenteric endothelium-dependent relaxation suggests that increased albuminuria may not be related to generalized endothelial vasodilator dysfunction in this model. Selective impairment of coronary endothelial function in a setting of spontaneous albuminuria may be a feature of the MWF that may be employed to further study cause-effect relations between albuminuria and coronary artery disease.