X-linked Alport syndrome presenting in mother and son with the same unique histopathological features.

Alport syndrome has been linked to three different genes, that is, COL4A3, COL4A4 and COL4A5. It is characterized by progressive and non-specific glomerulosclerosis with irregular thickening of the glomerular basement membrane (GBM). At times, the histopathologic picture is dominated by lesions that are consistent with focal and segmental glomerulosclerosis or IgA nephropathy. Here, we report the cases of two related individuals (mother and son) who were diagnosed with COL4A5-related Alport syndrome due to a missense variant (p.Gly1170Ser) in a G-X-Y repeat and found to present the same highly unusual histopathological abnormalities on their kidney biopsies. One of the abnormalities shared, which does not appear to have been reported, was reduced COL4A5 immunolabeling that was limited to Bowman's capsule even though the ultrastructure of the GBM was distorted. The other abnormality was superimposed segmental IgA deposition in both individuals, accompanied by mesangial changes in the mother. We feel that these findings provide novel insight into the mechanisms of disease manifestation in Alport syndrome. They suggest, in particular, that collagen expression and/or assemblies in Bowman's capsule is more vulnerable to missense mutations in COL4A5 than elsewhere in the kidney. Our findings also suggest that certain coinherited gene polymorphisms act as unexpectedly important phenotypic determinants in COL4A-related disorders.

The ever wider clinical spectrum of RMND1-related disorders and limitedness of phenotype-based classifications.

RMND1 has been identified as a mitochondriopathy-associated gene less than 12 years ago. The most common phenotype related to this gene is an early onset, severe form of encephalomyopathy that leads to death in a medium time of three years after birth. However, milder and later onset presentations have been reported in some individuals, including two in whom the mitochondriopathy was identified at ~ 40 years of age, and the early onset presentations have been the object of no reports in those who survived beyond age 10. It is thus unclear how lethal RMND1-related conditions really are. We herein describe the oldest case to have been identified hitherto with this condition, i.e., that of a white female who was 61 at the time of diagnosis but was still active in her everyday life. The gene defect identified was nonetheless associated with many manifestations including ovarian insufficiency and sensorineural hearing loss (two features of what is currently designated as Perrault syndrome) as well as chronic renal failure, asymptomatic myopathy, leukopenia, and a few others. In our opinion, this case is of great translational interest for at least three reasons. First, it hints towards the possibility of near-normal life expectancies in some if not many individuals with RMND1 insufficiency. Second, it underlines the wide clinical spectrum associated with this gene. Third, it brings us to question the use of eponyms and syndromic features to identify the true etiology of multisystemic phenotypes. KEY MESSAGES: RMND1-related conditions typically manifest at an early age with a progressive and lethal form of encephalomyopathy. More benign presentations have been described with some being categorized as Perrault syndrome but none have been diagnosed after the age of 45. The clinical spectrum and presenting age of RMND1-related mitochondriopathies are probably much more varied than implied in the current literature. The case reported in this manuscript illustrates the limitedness of phenotype-based classifications of genetic disorders to identify the defect at cause.

Genetic abnormalities in biopsy-proven, adult-onset hemolytic uremic syndrome and C3 glomerulopathy.

Atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) have been linked to mutations in many of the proteins that are involved in alternative complement pathway activation. Age and etiology confounded, the prevalence of such mutations has been reported to be over 30 to 50% in these diseases. However, the cohorts studied included many children or individuals with a familial history of complement-related disorders and genetic tests were usually limited to exome sequencing of known causative or risk-associated genes. In this study, a retrospective adult cohort of 35 patients with biopsy-proven thrombotic microangiopathy (the largest in Canada) and 10 patients with C3 glomerulopathy was tested through an extended exome panel to identify causative defects in associated or candidate genes including those of the alternative and terminal complement pathways. A variant of unknown significance was also analyzed for pathogenicity through in vitro studies. To our surprise, the prevalence of known causative or risk-associated variants in either of these cohorts was found to be less than ~ 15% overall. However, the panel used and analyses carried out allowed to identify novel variants of potential clinical significance and a number of candidate genes. The prevalence of known genetic defects in adult-onset aHUS and C3G is thus probably much lower than 30 to 50%. Our results also point towards the importance of investigating diseases of the alternative complement pathway through extended exome panels and in vitro analyses. KEY MESSAGES: The alternative complement pathway plays a major role in the pathogenesis of hemolytic uremic syndrome and C3 glomerulopathy. Based on previous studies, both disorders have been commonly linked to variants in the various intermediates that sustain or regulate this pathway. The prevalence of such mutations in the adult-onset and sporadic forms of these diseases is probably much lower than expected based on larger series. The sporadic forms of complementopathies are likely to involve additional genes that are yet to be uncovered.

Shedding a new light on Huntington's disease: how blood can both propagate and ameliorate disease pathology.

Huntington's disease (HD) is a monogenic neurodegenerative disorder resulting from a mutation in the huntingtin gene. This leads to the expression of the mutant huntingtin protein (mHTT) which provokes pathological changes in both the central nervous system (CNS) and periphery. Accumulating evidence suggests that mHTT can spread between cells of the CNS but here, we explored the possibility that mHTT could also propagate and cause pathology via the bloodstream. For this, we used a parabiosis approach to join the circulatory systems of wild-type (WT) and zQ175 mice. After surgery, we observed mHTT in the plasma and circulating blood cells of WT mice and post-mortem analyses revealed the presence of mHTT aggregates in several organs including the liver, kidney, muscle and brain. The presence of mHTT in the brain was accompanied by vascular abnormalities, such as a reduction of Collagen IV signal intensity and altered vessel diameter in the striatum, and changes in expression of Glutamic acid decarboxylase 65/67 (GAD65-67) in the cortex. Conversely, we measured reduced pathology in zQ175 mice by decreased mitochondrial impairments in peripheral organs, restored vessel diameter in the cortex and improved expression of Dopamine- and cAMP-regulated phosphoprotein 32 (DARPP32) in striatal neurons. Collectively, these results demonstrate that circulating mHTT can disseminate disease, but importantly, that healthy blood can dilute pathology. These findings have significant implications for the development of therapies in HD.

The grapevine NIP2;1 aquaporin is a silicon channel.

Silicon (Si) supplementation has been shown to improve plant tolerance to different stresses, and its accumulation in the aerial organs is mediated by NIP2;1 aquaporins (Lsi channels) and Lsi2-type exporters in roots. In the present study, we tested the hypothesis that grapevine expresses a functional NIP2;1 that accounts for root Si uptake and, eventually, Si accumulation in leaves. Own-rooted grapevine cuttings of the cultivar Vinhão accumulated >0.2% Si (DW) in leaves when irrigated with 1.5 mM Si for 1 month, while Si was undetected in control leaves. Real-time PCR showed that VvNIP2;1 was highly expressed in roots and in green berries. The transient transformation of tobacco leaf epidermal cells mediated by Agrobacterium tumefaciens confirmed VvNIP2;1 localization at the plasma membrane. Transport experiments in oocytes showed that VvNIP2;1 mediates Si and arsenite uptake, whereas permeability studies revealed that VvNIP2;1 expressed in yeast is unable to transport water and glycerol. Si supplementation to pigmented grape cultured cells (cv. Gamay Freáux) had no impact on the total phenolic and anthocyanin content, or on the growth rate and VvNIP2;1 expression. Long-term experiments should help determine the extent of Si uptake over time and whether grapevine can benefit from Si fertilization.

Si permeability of a deficient Lsi1 aquaporin in tobacco can be enhanced through a conserved residue substitution.

Silicon (Si) is a beneficial substrate for many plants, conferring heightened resilience to environmental stress. A plant's ability to absorb Si is primarily dependent on the presence of a Si-permeable Lsi1 (NIP2-1) aquaporin in its roots. Structure-function analyses of Lsi1 channels from higher plants have thus far revealed two key molecular determinants of Si permeability: (a) the amino acid motif GSGR in the aromatic/arginine selectivity filter and (b) 108 amino acids between two highly conserved NPA domains. Curiously, tobacco (Nicotiana sylvestris) stands as a rare exception as it possesses an Lsi1 (NsLsi1) with these molecular signatures but is reported as a low Si accumulator. The aim of this study was therefore to identify whether additional determinants influence Si permeability via Lsi1 channels, focusing on the role of residues that differ uniquely in NsLsi1 relative to functional Lsi1 homologs. We observed tobacco indeed absorbed Si poorly (0.1% dw), despite NsLsi1 being expressed constitutively in planta. Si influx measured in NsLsi1-expressing Xenopus oocytes was very low (<13% that of OsLsi1 from rice (Oryza sativa) over a 3-hr time course), which likely explains why tobacco is a low Si accumulator. Interestingly, NsLsi1P125F displayed a significant gain of function (threefold increase in Si influx relative to NsLsi1WT), which coincided with a threefold increase in plasma membrane localization in planta, as measured by transient expression of GFP constructs in Nicotiana benthamiana leaves. These findings thus reveal a novel molecular determinant of Si transport in plants and inform breeding, biotechnological, and agricultural practices to effectively utilize Si in the context of plant resilience to environmental stress.

Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer.

Maintenance of ion balance requires that ionoregulatory epithelia modulate ion flux in response to internal or environmental osmotic challenges. We have explored the basis of this functional plasticity in the gills of the euryhaline killifish Fundulus heteroclitus. The expression patterns of several genes encoding ion transport proteins were quantified after transfer from near-isosmotic brackish water [10 parts/thousand (ppt)] to either freshwater (FW) or seawater (SW). Many changes in response to SW transfer were transient. Increased mRNA expression occurred 1 day after transfer for Na(+)-K(+)-ATPase-alpha(1a) (3-fold), Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) (3-fold), and glucocorticoid receptor (1.3-fold) and was paralleled by elevated Na(+)-K(+)-ATPase activity (2-fold). The transient increase in NKCC1 mRNA expression was followed by a later 2-fold rise in NKCC protein abundance. In contrast to the other genes studied in the present work, mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel generally remained elevated (2-fold) in SW. No change in protein abundance was detected, however, suggesting posttranscriptional regulation. The responses to FW transfer were quite different from those to SW transfer. In particular, FW transfer increased Na(+)-K(+)-ATPase-alpha(1a) mRNA expression and Na(+)-K(+)-ATPase activity to a greater extent than did SW transfer but had no effect on V-type H(+)-ATPase expression, supporting the current suggestion that killifish gills transport Na(+) via Na(+)/H(+) exchange. These findings demonstrate unique patterns of ion transporter expression in killifish gills after salinity transfer and illustrate important mechanisms of functional plasticity in ion-transporting epithelia.

Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter.

Purinergic inhibition of Na-K-Cl cotransport has been noted in various renal epithelial cells derived from the collecting tubule, including Madin-Darby canine kidney (MDCK) cells. In recent studies, we have observed purinergic inhibition of Na-K-Cl cotransport in C11-MDCK subclones (alpha-intercalated-like cells). Interestingly, Na-K-Cl cotransport activity was also detected in C7-MDCK subclones (principal-like cells) but was not affected by ATP. In this investigation, we have transfected the human Na-K-Cl cotransporter (huNKCC1) in both C11 and C7 cells to determine whether these differences in NKCC regulation by ATP were due to cell-specific purinoceptor signaling pathways or to cell-specific isoforms/splice variants of the transporter. In both cell lines, we found that endogenous as well as huNKCC1-derived cotransport activity was restricted to the basolateral side. In addition, we were able to show that extracellular application of 100 microM ATP or 100 microM UTP abolished NKCC activity in both mock- and huNKCC1-transfected C11 cells but not in mock- and huNKCC1-transfected C7 cells; in C11 cells, intriguingly, this inhibition was not affected by inhibitors of RNA and protein synthesis and occurred even though expression levels of UTP-sensitive P2Y2-, P2Y4-, and P2Y6-purinoceptors were not different from those observed in C7 cells. These results suggest that C11 cells express an undetermined type of UTP-sensitive P2-purinoceptors or a unique P2Y-purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1.

Functional comparison of renal Na-K-Cl cotransporters between distant species.

In the shark (sa), two variants of the renal Na-K-Cl cotransporter (saNKCC2A and saNKCC2F) are produced by alternative splicing of the second transmembrane domain (tm(2)). In mammals, these splice variants, as well as a third variant (NKCC2B), are spatially distributed along the thick ascending limb of Henle and exhibit divergent kinetic behaviors. To test whether different tm(2) in saNKCC2 are also associated with different kinetic phenotypes, we examined the ion dependence of (86)Rb influx for shark and rabbit splice variants expressed in Xenopus laevis oocytes. We found that, in both species, A forms have higher cation affinities than F forms. In regard to Cl affinity, however, the A-F difference was more pronounced in rabbit, and the relationship between transport activity and Cl concentration was not always sigmoidal. These results show that the tm(2) of saNKCC2 is, as in rabbit, important for Cl transport, and they suggest that the ability of the distal NKCC2-expressing segment to extract Cl from the luminal fluid differs among species. We have also found that the renal NKCC2 of distant vertebrates share similar affinities for cations. This finding points to the existence of highly conserved residues that mediate the kinetic behavior of the NKCC2 splice variants.