[Immunoglobulin A nephropathy]. / Immunglobulin-A-Nephropathie.

Immunoglobulin A nephropathy (IgAN) is the most prevalent primary form of glomerulopathy in the western world. The pathogenetic relevance of autoimmune mechanisms, genetics and environmental or nutritional factors is not fully established. The majority of IgAN patients present with mild symptoms; however, the exact prognosis of the individual IgAN course is often difficult to predict. In approximately one third of the patients the disease remains on a stable benign course, whereas approximately 30% may develop end-stage renal disease. Risk factors for disease progression are a persistent microhematuria and proteinuria >1 g/day, arterial hypertension and the extent of tubulointerstitial fibrosis at the time of diagnosis. Recent genome-wide association studies (GWAS) identified numerous risk alleles, which can contribute to the pathophysiology of IgAN. The so-called gut-kidney axis as well as the complement system and genes that are linked to mucosal immunity appear to be important for the manifestation of the disease. Intensive supportive care should be initiated as first-line treatment and only rare cases with progressive features require treatment with corticosteroids. Other immunosuppressive treatment strategies have currently no indications for IgAN. Future approaches might be the use of local budesonide or the inhibition of lymphocyte activation.

A novel isoform of the orphan receptor RORγt suppresses IL-17 production in human T cells.

T helper (Th)17 cells constitute a distinct subset of CD4(+) helper T cells that is mainly characterized by abundant interleukin (IL)-17 production and is involved in the host defence against bacteria and fungi as well as in the pathogenesis of autoimmune diseases. The retinoic orphan receptor (ROR)γt directs the transcriptional activation of the IL17 gene. Here, we report the presence of a novel RORγt isoform, RORγt-Δ(5-8), which lacks the hinge-encoding exons 5-8 and represses potently IL17 and IL21 gene transcription. It thereby reduces the expression of multiple Th17-assigned cytokines. We propose that RORγt-Δ(5-8) acts as a dominant-negative regulator of RORγt-mediated gene regulation and the balance between the full-length RORγt and the novel repressor isoform may arbitrate IL-17 production in human T cells.

Dynamic CpG-DNA methylation of Il10 and Il19 in CD4+ T lymphocytes and macrophages: effects on tissue-specific gene expression.

The IL-10 family of cytokines consists of 9 members, including the immune-regulatory IL-10; Il19 is in close physical relationship with Il10 in the so-called IL-10 cytokine cluster on chromosome 1q32. While IL-10 is ubiquitously expressed, IL-19 expression is restricted to myeloid and epithelial cells. Little is known about molecular mechanisms that control tissue-specific expression of IL-10, and IL-19. Modifications in CpG-DNA methylation are a key mechanism in controlling transcription. Using bisulfite sequencing, we demonstrate that murine Il19 is methylated in CD4+ T lymphocytes. Macrophages display site-specific demethylation of Il19. The ubiquitously expressed Il10 gene is methylated to a lower degree and exhibits tissue-specific methylation patterns. DNA demethylation with 5-azacytidine resulted in an induction of IL-10, and IL-19 expression in CD4+ T cells, and CpG-DNA methylation through DNMT3a resulted in transcriptional silencing in macrophages. Thus, our findings suggest a role of CpG-DNA methylation in the regulation of Il10 and Il19.

Case series of idiopathic membranous nephropathy with long-term beneficial effects of ACTH peptide 1-24.

BACKGROUND: In patients with idiopathic membranous nephropathy (IMN), immunosuppressive therapy is usually considered when severe nephrotic syndrome or risk for progressive renal failure exist. Recently, several studies showing beneficial effects of synthetic adrenocorticotropic hormone (ACTH) under such circumstances have been published. The objective of the present case series was to evaluate long-term ACTH effects on proteinuria and renal function. METHODS: Four patients with biopsy-proven membranous nephropathy and nephrotic syndrome were enrolled (median age 50 years (range 38 - 61), median GFR 39.5 ml/min (range 20 - 62), median proteinuria 9.6 g/d (range 6.0 - 20.0). Prior immunosuppressive treatment regimens included steroids, cyclosporine A, cyclophosphamide, mycophenolate mofetil or azathioprine. The patients received a synthetic ACTH analogue intramuscularly for a median duration of 8 months (range 3 - 24). ACTH dosage was adjusted according to side effects between 0.25 and 2.25 mg/week. Follow-up lasted between 24 and 82 months after therapy initiation. RESULTS: All 4 patients exhibited partial (n = 2) or complete (n = 2) remissions of their nephrotic syndrome within the first year. After discontinuation of ACTH therapy, proteinuria remained low in 3 of 4 cases, whereas 1 patient exhibited undulating proteinuria. Glomerular function (as assessed by glomerular filtration rate, GFR) was maintained in all patients. Side effects were minor and included weight gain, elevated blood pressure and hyperglycemia. CONCLUSION: In all 4 cases with IMN, ACTH treatment induced a lasting disease remission with relatively few side effects.

Evidence from surface tension and fluorescence data of a pyrene-assisted micelle-like assemblage of humic substances.

Surface activity and fluorescence of humic substances (HS) and HS/pyrene solutions were monitored under various pH conditions. For HS alone the surface tension of the solutions decreased with increasing acidity, with a minimum at around pH 4. This effect, which is a consequence of an increase in the amphiphilic character of structures, is much more pronounced in humic (HA) than in fulvic acids (FA). The addition of pyrene (0.1 micromolL(-1)) results, for HA, in a marked reduction in the migration of amphiphilic species to the solution surface. FA profiles are not modified in presence of pyrene at that concentration. A decrease in the pyrene I1/I3 ratio in HS solutions shows that below pH 9 pyrene molecules react progressively to the change to a more hydrophobic environment, the greatest effect being observed at around pH 6 to 7. These signals are followed by a significant increase in the pyrene excimer fluorescence (lambda(exc)/lambda(em)=334 nm/450 nm), which is a consequence of the proximity of pyrene molecules. For FA, the I1/I3 decrease is less significant and no excimers develop. This set of effects is explained in view of conformational adjustments of HS, mainly HA, which become arranged in micelle-like domains in aqueous solution, the aromatic moieties being assembled around the pyrene molecules.

Electrogenic glutamate transporters in the CNS: molecular mechanism, pre-steady-state kinetics, and their impact on synaptic signaling.

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. The spatiotemporal profile of the glutamate concentration in the synapse is critical for excitatory synaptic signalling. The control of this spatiotemporal concentration profile requires the presence of large numbers of synaptically localized glutamate transporters that remove pre-synaptically released glutamate by uptake into neurons and adjacent glia cells. These glutamate transporters are electrogenic and utilize energy stored in the transmembrane potential and the Na+/K+-ion concentration gradients to accumulate glutamate in the cell. This review focuses on the kinetic and electrogenic properties of glutamate transporters, as well as on the molecular mechanism of transport. Recent results are discussed that demonstrate the multistep nature of the transporter reaction cycle. Results from pre-steady-state kinetic experiments suggest that at least four of the individual transporter reaction steps are electrogenic, including reactions associated with the glutamate-dependent transporter halfcycle. Furthermore, the kinetic similarities and differences between some of the glutamate transporter subtypes and splice variants are discussed. A molecular mechanism of glutamate transport is presented that accounts for most of the available kinetic data. Finally, we discuss how synaptic glutamate transporters impact on glutamate receptor activity and how transporters may shape excitatory synaptic transmission.

Substrate translocation kinetics of excitatory amino acid carrier 1 probed with laser-pulse photolysis of a new photolabile precursor of D-aspartic acid.

Here we report the synthesis and photochemical and biological characterization of a new photolabile precursor of D-aspartic acid, alpha-carboxynitrobenzyl-caged D-aspartate (alpha-CNB-caged D-aspartate), and its application for studying the molecular mechanism of the neuronal excitatory amino acid carrier 1 (EAAC1). Investigation of the photochemical properties of alpha-CNB-caged D-aspartate by transient absorption spectroscopy of the aci-nitro intermediate revealed that it photolyzes with a quantum yield of 0. 19 at pH 7.0. The major component of the aci-nitro intermediate (77% of the total absorbance) decays with a time constant of 26 s. This decay is slowed by only a factor of 2 when increasing the pH to 10. A minor component (21%) decays with a time constant of 410 s and is pH insensitive. The compound was tested with respect to its biological activity with the glutamate transporter EAAC1 expressed in HEK293 cells. Whole-cell current recordings from these cells in the presence and absence of alpha-CNB-caged D-aspartate demonstrated that the compound neither activates nor inhibits EAAC1. Upon photolysis, D-aspartate-mediated whole-cell currents were generated. In contrast to laser-pulse photolysis experiments with alpha-CNB-caged L-glutamate, only a minor and much slower transient current component was observed. These results indicate that the substrate translocation step, which is not rate-limiting for the overall turnover of the transporter with L-glutamate, becomes rate-limiting when D-aspartate is translocated. The results demonstrate that the new caged D-aspartate derivative is a useful tool for the investigation of the molecular mechanism of glutamate transporters and probably other aspartate translocating systems using rapid chemical kinetic techniques.

Reduced synaptic clustering of GABA and glycine receptors in the retina of the gephyrin null mutant mouse.

Clustering of neurotransmitter receptors in postsynaptic densities involves proteins that aggregate the receptors and link them to the cytoskeleton. In the case of glycine and GABA(A) receptors, gephyrin has been shown to serve this function. However, it is unknown whether gephyrin is involved in the clustering of all glycine and GABA(A) receptors or whether it interacts only with specific isoforms. This was studied in the retinae of mice, whose gephyrin gene was disrupted, with immunocytochemistry and antibodies that recognize specific subunits of glycine and GABA(A) receptors. Because homozygous (geph -/-) mutants die around birth, an organotypic culture system of the mouse retina was established to study the clustering of gephyrin and the receptors in vitro. We found that all gephyrin and all glycine receptor clusters (hot spots) were abolished in the geph (-/-) mouse retina. In the case of GABA(A) receptors, there was a significant reduction of clusters incorporating the gamma2, alpha2, and alpha3 subunits; however, a substantial number of hot spots was still present in geph (-/-) mutant retinae. This shows that gephyrin interacts with all glycine receptor isoforms but with only certain forms of GABA(A) receptors. In heterozygous geph (+/-) mutants, no reduction of hot spots was observed in the retina in vivo, but a significant reduction was found in the organotypic cultures. This suggests that mechanisms may exist in vivo that allow for the compensation of a partial gephyrin deficit.

Diversity of glutamate transporter expression and function in the mammalian retina.

Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. Between photoreceptors and second order neurons, increases in light intensity are signaled by decreases in the concentration of glutamate within the synaptic cleft. In such a system the precise control of glutamate in the synaptic cleft is thus essential and glutamate transporters are thought to contribute to this process. As demonstrated here, all neuronal and macroglial cells of the retina appear to express high-affinity glutamate transporters. GLAST1, GLT1, EAAC1 and EAAT5 are expressed in the retina and exhibit unique localisation and functional properties. In the present study we summarize retinal glutamate transporter expression, identify the major glutamate uptake site in the mammalian retina and discuss the possible functional roles of different glutamate transporter subtypes in glutamatergic neurotranmission in the retina.

On the mechanism of proton transport by the neuronal excitatory amino acid carrier 1.

Uptake of glutamate from the synaptic cleft is mediated by high affinity transporters and is driven by Na(+), K(+), and H(+) concentration gradients across the membrane. Here, we characterize the molecular mechanism of the intracellular pH change associated with glutamate transport by combining current recordings from excitatory amino acid carrier 1 (EAAC1)-expressing HEK293 cells with a rapid kinetic technique with a 100-micros time resolution. Under conditions of steady state transport, the affinity of EAAC1 for glutamate in both the forward and reverse modes is strongly dependent on the pH on the cis-side of the membrane, whereas the currents at saturating glutamate concentrations are hardly affected by the pH. Consistent with this, the kinetics of the pre-steady state currents, measured after saturating glutamate concentration jumps, are not a function of the pH. In addition, we determined the deuterium isotope effect on EAAC1 kinetics, which is in agreement with proton cotransport but not OH(-) countertransport. The results can be quantitatively explained with an ordered binding model that includes a rapid proton binding step to the empty transporter followed by glutamate binding and translocation of the proton-glutamate-transporter complex. The apparent pK of the extracellular proton binding site is approximately 8. This value is shifted to approximately 6.5 when the substrate binding site is exposed to the cytoplasm.

Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds.

The activity of glutamate transporters is essential for the temporal and spatial regulation of the neurotransmitter concentration in the synaptic cleft, and thus, is crucial for proper excitatory signaling. Initial steps in the process of glutamate transport take place within a time scale of microseconds to milliseconds. Here we compare the steady-state and pre-steady-state kinetics of the neuronal heterologously expressed glutamate transporter EAAC1, cloned from the mammalian retina. Rapid transporter dynamics, as measured by using whole-cell current recordings, were resolved by applying the laser-pulse photolysis technique of caged glutamate with a time resolution of 100 micros. EAAC1-mediated pre-steady-state currents are composed of two components: A transport current generated by substrate-coupled charge translocation across the membrane and an anion current that is not stoichiometrically coupled to glutamate transport. The two currents were temporally resolved and studied independently. Our results indicate a rapid glutamate-binding step occurring on a submillisecond time scale that precedes subsequent slower electrogenic glutamate translocation across the membrane within a few milliseconds. The voltage-dependent steady-state turnover time constant of the transporter is about 1/10 as fast, indicating that glutamate translocation is not rate limiting. A third process, the transition to an anion-conducting state, is delayed with respect to the onset of glutamate transport. These rapid transporter reaction steps are summarized in a sequential shuttle model that quantitatively accounts for the results obtained here and are discussed regarding their functional importance for glutamatergic neurotransmission in the central nervous system.

Fine tuning of glutamate uptake and degradation in glial cells: common transcriptional regulation of GLAST1 and GS.

Glutamate is the major excitatory neurotransmitter in the mammalian brain and retina, and glutamate uptake is essential for the normal function of glutamatergic synapses in the retina. As summarized here, all neuronal and macroglial cells of the retina express high-affinity glutamate transporters. GLAST1 is expressed in glial cells, whereas GLT1 and EAAC1 are neuronal. Glutamate uptake studies in intact retina revealed that Müller glial cells dominate the total retinal glutamate transport and that this uptake is strongly influenced by the activity of glutamine synthetase. A prerequisite for an effective glutamate-glutamine cycle in glial cells would be the regulated coordination between glutamate uptake and glutamate degradation. Using cultured retinal Müller glial cells, we demonstrate that protein expression of both, GLAST1 and glutamine synthetase, are inducible by the glucocorticoid hormone cortisol. These results suggest a common transcriptional regulation of the key proteins in the glial portion of the glutamate-glutamine cycle and may impact on transmitter clearance, transmitter recycling and, as discussed, on the development of the cellular architecture in retina.

High-affinity glutamate transporters in the rat retina: a major role of the glial glutamate transporter GLAST-1 in transmitter clearance.

Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. The reverse transcriptase-polymerase chain reaction (RT-PCR) has revealed the presence of three different high-affinity glutamate transporters in the rat retina, viz. GLAST-1, GLT-1 and EAAC-1. No message has been found in the retina for EAAT-4, a transporter recently cloned from human brain. By using membrane vesicle preparations of total rat retina, we show that glutamate uptake in the retina is a high-affinity electrogenic sodium-dependent transport process driven by the transmembrane sodium ion gradient. Autoradiography of intact and dissociated rat retinae indicates that glutamate uptake by Müller glial cells dominates total retinal glutamate transport and that this uptake is strongly influenced by the activity of glutamine synthetase. RT-PCR, immunoblotting and immunohistochemistry have revealed that Müller cells express only GLAST-1. The Km for glutamate of GLAST-1 is 2.1+/-0.4 microM. This study suggests a major role for the Müller cell glutamate transporter GLAST-1 in retinal transmitter clearance. By regulating the extracellular glutamate concentration, the action of GLAST-1 in Müller cells may extend beyond the protection of neurons from excitotoxicity; we suggest a mechanism by which Müller cell glutamate transport might play an active role in shaping the time course of excitatory transmission in the retina.

Differential expression of three glutamate transporter subtypes in the rat retina.

The immunocytochemical distribution of the three excitatory amino acid transporter subtypes GLT-1, GLAST-1 and EAAC-1 was studied in the rat retina using antibodies raised against synthetic peptides corresponding to the C-terminus of each transporter subtype (Rothstein et al. 1994). A comparative immunoblot analysis of rat cortex, cerebellum and retina membrane proteins suggested the following rank order of glutamate transporter subtype expression in retina: GLAST-1>/=EAAC-1>GLT-1. GLAST-1 immunoreactivity was seen in Müller cells and astrocytes. EAAC-1 was found in horizontal cells, in amacrine and displaced amacrine cells, and in ganglion cells. A minority of bipolar cells also expressed EAAC-1. GLT-1 was preferentially expressed by different types of bipolar cells; however, it was also found in some amacrine cells. The functional role of this differential distribution of glutamate transporters in the retina is discussed.

Coincidence of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) immunoreactions in retinal glia: evidence for coupling of GLAST and GS in transmitter clearance.

Our aim was to identify proteins that mediate the uptake and degradation of synaptically released glutamate, focusing on the rat retina with its well-defined glutamatergic pathways. Immunoreactivity against the L-glutamate/L-aspartate transporter (GLAST) is present in Müller cells. Ultrastructurally, even the finest glial processes, particularly those ensheathing identified structures of glutamatergic transmission (rod spherules), are immunoreactive for GLAST. Further light and electron microscopic observations revealed that also retinal astrocytes and pigment epithelial cells are immunoreactive for GLAST. No neuronal or microglial staining was observed. This is in line with uptake of exogenous [3H]glutamate previously localized specifically in Müller cells and pigment epithelium (Ehinger and Falck: Brain Res 33:157-172, 1971). Since endogenous glutamate can only be demonstrated in Müller cells if glutamine synthetase (GS) is inhibited (Pow and Robinson: Neuroscience 60:355-366, 1994), the immunocytochemical localization of GS was determined. GS immunoreactivity was found in all but only those cell types immunoreactive for GLAST. The light and electron microscopic patterns of immunoreactivity were very similar, particularly in the outer plexiform layer. The three cell types containing both GS and GLAST (Müller cells, astrocytes, and retinal pigment epithelium) are related developmentally. In the light of the two references quoted the present data indicate that the proteins mediating retinal uptake and degradation of synaptically released glutamate may be GLAST and GS, respectively, and that they may operate in concert to terminate the neurotransmitter action of glutamate.

Localization of the glutamate transporter GLT-1 in rat and macaque monkey retinae.

Antibodies directed against a glutamate transporter (GLT-1) purified from rat brain were applied to cryostat sections of rat and macaque monkey retinae. In the brain, GLT-1 expression is found mainly in astrocytes, and therefore it has been suggested that GLT-1 may be a glutamate transporter specific to glial cells. However, in the rat retina, cones and two distinct cone bipolar cell types were strongly immunoreactive. In the monkey retina, flat midget bipolars and one diffuse bipolar cell type (DB2)), were found to be labelled. Müller cells or astrocytes, the neuroglial cells of rat and monkey retinae, were not GLT-1-immunoreactive.