Daily compared with alternate-day levamisole in pediatric nephrotic syndrome: an open-label randomized controlled study.

BACKGROUND: Levamisole is less expensive and has a better toxicity profile compared to other steroid sparing agents used in nephrotic syndrome. It has a plasma half-life of 2.0 to 5.6 hours, but is conventionally administered on alternate days. We aimed to assess whether daily levamisole is safe and more effective than standard alternate-day therapy in maintaining remission in children with frequently relapsing or steroid-dependent nephrotic syndrome (FR/SDNS). METHODS: An open-label randomized controlled trial was conducted in children with FR/SDNS. Group A received daily while Group B received alternate-day levamisole (2-3 mg/kg/dose) for 12 months. Prednisolone was tapered off by 3 months. Patients were monitored for relapses, further steroid requirement, and adverse effects. RESULTS: A total of 190 children with FR/SDNS (94 in Group A and 96 in Group B) were analyzed. Sustained remission for 12 months was observed in 36% of Group A and 27% of Group B patients (p = 0.18). Numbers completing 12 months in the study were 67% in Group A and 56% in Group B (p = 0.13). Time to first relapse, persistent FR/SDNS, and withdrawal due to poor compliance were statistically similar in both groups, while relapse rate and cumulative steroid dosage were significantly lower in Group A compared to Group B (p = 0.03 and p = 0.02, respectively). The incidence of adverse effects was comparable in both groups, with reversible leucopenia and hepatic transaminitis being the commonest. CONCLUSIONS: Daily levamisole therapy was not superior to alternate-day therapy in maintaining sustained remission over 12 months. Nevertheless, relapse rate and cumulative steroid dosage were significantly lower without increased adverse effects.

Antineutrophil cytoplasmic antibody in children with nephrotic syndrome treated with levamisole: a cross-sectional cohort study.

BACKGROUND: Levamisole is a commonly used steroid-sparing agent (SSA), but the reported incidence of antineutrophil cytoplasmic antibody (ANCA) positivity has been concerning. METHODS: Observational cross-sectional study wherein children aged 2 to 18 years with frequently relapsing/steroid dependent nephrotic syndrome (FRNS/SDNS) on levamisole for ≥ 12 months were tested for ANCA. RESULTS: A total of 210 children (33% female), median age of 7.3 (IQR: 5.6-9.6) years, and a median duration of levamisole exposure of 21 (IQR: 15-30) months were tested. ANCA was positive in 18% (n = 37): 89% (n = 33) perinuclear ANCA (pANCA), 3% (n = 1) cytoplasmic ANCA (cANCA), and 8% (n = 3) both. Of ANCA-positive children, none had reduced eGFR or abnormal urinalysis. The majority of these children were asymptomatic (81%, n = 30). Rash was more common among ANCA-positive children [6/37 (16%) vs. 3/173 (2%), p = 0.0001]. On multivariate analysis, higher age (OR = 1.02, [95th CI: 1.01 to 1.03], p = 0.007) and longer duration of levamisole exposure (OR = 1.05, [95th CI: 1.02 to 1.08], p = 0.0007) were associated with ANCA positivity. Levamisole was stopped in ANCA-positive children with the resolution of any clinical manifestations if present. Repeat ANCA testing was performed in 54% (20/37), and all were ANCA negative by 18 months. CONCLUSIONS: Children with FRNS/SDNS on longer duration of levamisole were associated with increasing prevalence of ANCA positivity, but most of these children were clinically asymptomatic. Prospective studies are required to determine the chronology of ANCA positivity and its clinical implication.

Cryo-EM captures a unique conformational rearrangement in 23S rRNA helices of the Mycobacterium 50S subunit.

Structural investigations of the ribosomes isolated from pathogenic and non-pathogenic Mycobacterium species have identified several mycobacteria-specific structural features of ribosomal RNA and proteins. Here, we report structural evidence of a hitherto unknown conformational switch of mycobacterium 23S rRNA helices (H54a and H67-H71). Cryo-electron microscopy (cryo-EM) structures (~3-4 Å) of the M. smegmatis (Msm) log-phase 50S ribosomal subunit revealed conformational variability in H67-H71 region of the 23S rRNA, and manifested that, while H68 possesses the usual stretched conformation in one class of the maps, another one exhibits a bulge-out, fused density of H68-H69 at the inter-subunit surface, indicating an intrinsic dynamics of these rRNA helices. Remarkably, altered conformation of H68 forming a more prominent bulge-out structure at the inter-subunit surface of the 50S subunit due to the conformational rearrangements of 23S rRNA H67-H71 region was clearly visualized in a 3 Å cryo-EM map of the 50S subunit obtained from the stationary phase ribosome dataset. The Msm50S subunit having such bulge-out conformation at the intersubunit surface would be incompatible for associating with the 30S subunit due to its inability to form major inter-subunit bridges. Evidently, availability of active 70S ribosome pool can be modulated by stabilizing either one of the H68 conformation.

The clinical relevance of native vitamin D in pediatric kidney disease.

Hypovitaminosis D has been reported to be common in chronic kidney disease (CKD) as well as in proteinuric disorders. We reviewed available evidence to assess clinically relevant effects of low vitamin D status and native vitamin D (NVD) therapy, in pediatric renal diseases. Online medical databases were searched for articles related to vitamin D status, associations of hypovitaminosis D and effects of NVD therapy in kidney disease. Hypovitaminosis D was associated with worse skeletal, cardiovascular, inflammatory, and renal survival outcomes in CKD. Low serum 25 hydroxy-vitamin D (25[OH]D) levels correlated positively with glomerular filtration rate and negatively with serum parathyroid (PTH) levels. However, to date, evidence of benefit of NVD supplementation is restricted mainly to improvements in serum PTH, and biochemical 25[OH]D targets form the basis of clinical practice recommendations for NVD therapy. In nephrotic syndrome (NS) relapse, studies indicate loss of 25[OH]D along with vitamin D binding protein in urine, and serum total 25[OH]D levels are low. Preliminary evidence indicates that free 25[OH]D may be a better guide to the biologically active fraction. NVD therapy in NS does not show consistent results in improving skeletal outcomes and hypercalciuria has been reported when total 25[OH]D levels were considered as indication for therapy. NVD formulations should be regularised, and therapy monitored adequately to avoid adverse effects.

Ligand-Dependent Modulation of the Dynamics of Intracellular Loops Dictates Functional Selectivity of 5-HT2AR.

The serotonin 2A receptor (5-HT2AR) subtype of the G protein-coupled receptor (GPCR) family is involved in a plethora of neuromodulatory functions (e.g., neurogenesis, sleep, and cognitive processes). 5-HT2AR is the target of pharmacologically distinct classes of ligands, binding of which either activate or inactivate the receptor. Although high-resolution structures of 5-HT2AR as well as several other 5-HT GPCRs provided snapshots of both active and inactive conformational states, these structures, representing a truncated form of the receptor, cannot fully explain the mechanism of conformational transitions during their function. Importantly, biochemical studies have suggested the importance of intracellular loops in receptor functions. In our previous study, a model of the ligand-free form of 5-HT2AR with the third intracellular loop (ICL3) has been meticulously built. Here, we have investigated the functional regulation of 5-HT2AR with intact intracellular loops in ligand-free and five distinct ligand-bound configurations using unbiased atomistic molecular dynamics (MD) simulations. The selected ligands belong to either of the full, partial, or inverse agonist classes, which exert distinct pharmacological responses. We have observed significant structural, dynamic, and thermodynamic differences within ligand-bound complexes. Our results revealed, for the first time, that either activation or inactivation of the receptor upon specific ligand binding is primarily achieved through conformational transitions of its second and third intracellular loops (ICL2 and ICL3). A remarkable allosteric cross-talk between the ligand-binding site and the distal intracellular parts of the receptor, where binding of a specific ligand thermodynamically controls (either stabilizes or destabilizes) the intracellular region, consisting of crucial dynamic elements ICL2 and ICL3, and differential conformational transitions of these loops determine ligand-dependent functional selectivity.

Ribosome-membrane crosstalk: Co-translational targeting pathways of proteins across membranes in prokaryotes and eukaryotes.

Ribosomes are the molecular machine of living cells designed for decoding mRNA-encoded genetic information into protein. Being sophisticated machinery, both in design and function, the ribosome not only carries out protein synthesis, but also coordinates several other ribosome-associated cellular processes. One such process is the translocation of proteins across or into the membrane depending on their secretory or membrane-associated nature. These proteins comprise a large portion of a cell's proteome and act as key factors for cellular survival as well as several crucial functional pathways. Protein transport to extra- and intra-cytosolic compartments (across the eukaryotic endoplasmic reticulum (ER) or across the prokaryotic plasma membrane) or insertion into membranes majorly occurs through an evolutionarily conserved protein-conducting channel called translocon (eukaryotic Sec61 or prokaryotic SecYEG channels). Targeting proteins to the membrane-bound translocon may occur via post-translational or co-translational modes and it is often mediated by recognition of an N-terminal signal sequence in the newly synthesizes polypeptide chain. Co-translational translocation is coupled to protein synthesis where the ribosome-nascent chain complex (RNC) itself is targeted to the translocon. Here, in the light of recent advances in structural and functional studies, we discuss our current understanding of the mechanistic models of co-translational translocation, coordinated by the actively translating ribosomes, in prokaryotes and eukaryotes.

The role of membranes in function and dysfunction of intrinsically disordered amyloidogenic proteins.

Membrane-protein interactions play a major role in human physiology as well as in diseases pathology. Interaction of a protein with the membrane was previously thought to be dependent on well-defined three-dimensional structure of the protein. In recent decades, however, it has become evident that a large fraction of the proteome, particularly in eukaryotes, stays disordered in solution and these proteins are termed as intrinsically disordered proteins (IDPs). Also, a vast majority of human proteomes have been reported to contain substantially long disordered regions, called intrinsically disordered regions (IDRs), in addition to the structurally ordered regions. IDPs exist in an ensemble of conformations and the conformational flexibility enables IDPs to achieve functional diversity. IDPs (and IDRs) are found to be important players in cell signaling, where biological membranes act as anchors for signaling cascades. Therefore, IDPs modulate the membrane architectures, at the same time membrane composition also affects the binding of IDPs. Because of intrinsic disorders, misfolding of IDPs often leads to formation of oligomers, protofibrils and mature fibrils through progressive self-association. Accumulation of amyloid-like aggregates of some of the IDPs is a known causative agent for numerous diseases. In this chapter we highlight recent advances in understanding membrane interactions of some of the intrinsically disordered proteins involved in the pathogenesis of human diseases.

Hidden electrostatic energy contributions define dynamic allosteric communications within p53 during molecular recognition.

Molecular recognition is fundamental to transcription regulation. As a transcription factor, the tumor suppressor p53 has to recognize either specific DNA sequences or repressor protein partners. However, the molecular mechanism underlying the p53 conformational switch from the DNA-bound to repressor-bound states is not fully characterized. The highly charged nature of these interacting molecules prompted us to explore the nonbonded energy contributions behind molecular recognition of either a DNA or the repressor protein iASPP by p53 DNA binding domain (p53DBD), using molecular dynamics simulation followed by rigorous analyses of energy terms. Our results illuminate the allosteric pathway by which iASPP binding to p53 diminishes binding affinity between p53 and DNA. Even though the p53DBD uses a common framework of residues for recognizing both DNA and iASPP, a comparison of the electrostatics in the two p53DBD complexes revealed significant differences in residue-wise contributions to the electrostatic energy. We found that an electrostatic allosteric communication path exists in the presence of both substrates. It consists of evolutionarily conserved residues, from residue K120 of the binding loop L1 to a distal residue R213 of p53DBD. K120 is near the DNA in the p53DBD-DNA complex, whereas iASPP binding moves it away from its DNA binding position in the p53DBD-iASPP complex. The "energy hubs" (the residues show a higher degree of connectivity with other residues in the electrostatic networks) determined from the electrostatic network analysis established that this conformational change in K120 completely rewires the electrostatic network from K120 to R213, thereby impeding DNA binding. Furthermore, we found shifting populations of hydrogen bonds and salt bridges reduce pairwise electrostatic energies within p53DBD in its DNA-bound state.

Conformational distortion in a fibril-forming oligomer arrests alpha-Synuclein fibrillation and minimizes its toxic effects.

The fibrillation pathway of alpha-Synuclein, the causative protein of Parkinson's disease, encompasses transient, heterogeneous oligomeric forms whose structural understanding and link to toxicity are not yet understood. We report that the addition of the physiologically-available small molecule heme at a sub-stoichiometric ratio to either monomeric or aggregated α-Syn, targets a His50 residue critical for fibril-formation and stabilizes the structurally-heterogeneous populations of aggregates into a minimally-toxic oligomeric state. Cryo-EM 3D reconstruction revealed a 'mace'-shaped structure of this monodisperse population of oligomers, which is comparable to a solid-state NMR Greek key-like motif (where the core residues are arranged in parallel in-register sheets with a Greek key topology at the C terminus) that forms the fundamental unit/kernel of protofilaments. Further structural analyses suggest that heme binding induces a distortion in the Greek key-like architecture of the mace oligomers, which impairs their further appending into protofilaments and fibrils. Additionally, our study reports a novel mechanism of prevention as well as reclamation of amyloid fibril formation by blocking an inter-protofilament His50 residue using a small molecule.

Structural insights into the interplay of protein biogenesis factors with the 70S ribosome.

Bacterial co-translational N-terminal methionine excision, an early event of nascent polypeptide chain processing, is mediated by two enzymes: peptide deformylase (PDF) and methionine aminopeptidase (MetAP). Trigger factor (TF), the only ribosome-associated bacterial chaperone, offers co-translational chaperoning assistance. Here, we present two high-resolution cryoelectron microscopy structures of tRNA-bound E. coli ribosome complexes showing simultaneous binding of PDF and TF, in the absence (3.4 Å) and presence of MetAP (4.1 Å). These structures establish molecular details of the interactions of the factors with the ribosome, and thereby reveal the structural basis of nascent chain processing. Our results suggest that simultaneous binding of all three factors is not a functionally favorable mechanism of nascent chain processing. Strikingly, an unusual structural distortion of the 70S ribosome, potentially driven by binding of multiple copies of MetAP, is observed when MetAP is incubated with a pre-formed PDF-TF-bound ribosome complex.

Integrative approaches in cryogenic electron microscopy: Recent advances in structural biology and future perspectives.

Cellular factories engage numerous highly complex "molecular machines" to perform pivotal biological functions. 3D structural visualization is an effective way to understand the functional mechanisms of these biomacromolecules. The "resolution revolution" has established cryogenic electron microscopy (cryo-EM) as a preferred structural biology tool. In parallel with the advances in cryo-EM methodologies aiming at atomic resolution, several innovative approaches have started emerging where other techniques are sensibly integrated with cryo-EM to obtain additional insights into the biological processes. For example, combining the time-resolved technique with high-resolution cryo-EM enables discerning structures of short-lived intermediates in the functional pathway of a biomolecule. Likewise, integrating mass spectrometry (MS) techniques with cryo-EM allows deciphering structural organizations of large molecular assemblies. Here, we discuss how the data generated upon combining either time resolve or MS techniques with cryo-EM supplement structural elucidations with in-depth understanding of the function of cellular macromolecules when they participate in fundamental biological processes.

Dynamics and electrostatics define an allosteric druggable site within the receptor-binding domain of SARS-CoV-2 spike protein.

The pathogenesis of the SARS-CoV-2 virus initiates through recognition of the angiotensin-converting enzyme 2 (ACE2) receptor of the host cells by the receptor-binding domain (RBD) located at the spikes of the virus. Here, using molecular dynamics simulations, we have demonstrated the allosteric crosstalk within the RBD in the apo- and the ACE2 receptor-bound states, revealing the contribution of the dynamics-based correlated motions and the electrostatic energy perturbations to this crosstalk. While allostery, based on correlated motions, dominates inherent distal communication in the apo-RBD, the electrostatic energy perturbations determine favorable pairwise crosstalk within the RBD residues upon binding to ACE2. Interestingly, the allosteric path is composed of residues which are evolutionarily conserved within closely related coronaviruses, pointing toward the biological relevance of the communication and its potential as a target for drug development.

Hibernating ribosomes exhibit chaperoning activity but can resist unfolded protein-mediated subunit dissociation.

Ribosome hibernation is a prominent cellular strategy to modulate protein synthesis during starvation and the stationary phase of bacterial cell growth. Translational suppression involves the formation of either factor-bound inactive 70S monomers or dimeric 100S hibernating ribosomal complexes, the biological significance of which is poorly understood. Here, we demonstrate that the Escherichia coli 70S ribosome associated with stationary phase factors hibernation promoting factor or protein Y or ribosome-associated inhibitor A and the 100S ribosome isolated from both Gram-negative and Gram-positive bacteria are resistant to unfolded protein-mediated subunit dissociation and subsequent degradation by cellular ribonucleases. Considering that the increase in cellular stress is accompanied by accumulation of unfolded proteins, such resistance of hibernating ribosomes towards dissociation might contribute to their maintenance during the stationary phase. Analysis of existing structures provided clues on the mechanism of inhibition of the unfolded protein-mediated disassembly in case of hibernating factor-bound ribosome. Further, the factor-bound 70S and 100S ribosomes can suppress protein aggregation and assist in protein folding. The chaperoning activity of these ribosomes is the first evidence of a potential biological activity of the hibernating ribosome that might be crucial for cell survival under stress conditions.

Structure, dynamics and lipid interactions of serotonin receptors: excitements and challenges.

Serotonin (5-hydroxytryptamine, 5-HT) is an intrinsically fluorescent neurotransmitter found in organisms spanning a wide evolutionary range. Serotonin exerts its diverse actions by binding to distinct cell membrane receptors which are classified into many groups. Serotonin receptors are involved in regulating a diverse array of physiological signaling pathways and belong to the family of either G protein-coupled receptors (GPCRs) or ligand-gated ion channels. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions such as sleep, mood, pain, anxiety, depression, aggression, and learning. Serotonin receptors act as drug targets for a number of diseases, particularly neuropsychiatric disorders. The signaling mechanism and efficiency of serotonin receptors depend on their amazing ability to rapidly access multiple conformational states. This conformational plasticity, necessary for the wide variety of functions displayed by serotonin receptors, is regulated by binding to various ligands. In this review, we provide a succinct overview of recent developments in generating and analyzing high-resolution structures of serotonin receptors obtained using crystallography and cryo-electron microscopy. Capturing structures of distinct conformational states is crucial for understanding the mechanism of action of these receptors, which could provide important insight for rational drug design targeting serotonin receptors. We further provide emerging information and insight from studies on interactions of membrane lipids (such as cholesterol) with serotonin receptors. We envision that a judicious combination of analysis of high-resolution structures and receptor-lipid interaction would allow a comprehensive understanding of GPCR structure, function and dynamics, thereby leading to efficient drug discovery.

Heptameric Peptide Interferes with Amyloid-ß Aggregation by Structural Reorganization of the Toxic Oligomers.

Pathogenesis of Alzheimer's disease (AD), the most common type of dementia, involves misfolding and aggregation of the extracellular amyloid-ß (Aß) protein where the intermediate oligomers, formed during the aggregation progression cascade, are considered the prime toxic species. Here, we identify an active peptide fragment from a medicinal plant-derived (Aristolochia indica) fibrinolytic enzyme having anti-amyloidogenic effects against Aß fibrillation and toxicity. Liquid chromatography with tandem mass spectrometry (LC-MS/MS), followed by computational analysis of the peptide pool generated by proteolytic digestion of the enzyme, identifies two peptide sequences with predictive high-propensity binding to Aß42. Microscopic visualizations in conjunction with biochemical and biophysical assessments suggest that the synthetic version of one of the peptides (termed here Pactive, GFLLHQK) arrests Aß molecules in off-pathway oligomers that can no longer participate in the cytotoxic fibrillation pathway. In contrast, the other peptide (termed P1) aggravates the fibrillation process. Further investigations confirm the strong binding affinity of Pactive with both Aß42 monomers and toxic oligomers by biolayer interferometric assays. We have also shown that, mechanistically, Pactive binding induces conformational alterations in the Aß molecule along with modification of Aß hydrophobicity, one of the key players in aggregation. Importantly, the biostability of Pactive in human blood serum and its nontoxic nature make it a promising therapeutic candidate against Alzheimer's, for which no disease-modifying treatments are available to date.

Resveratrol as a nontoxic excipient stabilizes insulin in a bioactive hexameric form.

Insulin aggregation is the leading cause of considerable reduction in the amount of active drug molecules in liquid formulations manufactured for diabetes management. Phenolic compounds, such as phenol and m-cresol, are routinely used to stabilize insulin in a hexameric form during its commercial preparation. However, long term usage of commercial insulin results in various adverse secondary responses, for which toxicity of the phenolic excipients is primarily responsible. In this study we aimed to find out a nontoxic insulin stabilizer. To that end, we have selected resveratrol, a natural polyphenol, as a prospective nontoxic insulin stabilizer because of its structural similarity with commercially used phenolic compounds. Atomic force microscopy visualization of resveratrol-treated human insulin revealed that resveratrol has a unique ability to arrest hINS in a soluble oligomeric form having discrete spherical morphology. Most importantly, resveratrol-treated insulin is nontoxic for HepG2 cells and it effectively maintains low blood glucose in a mouse model. Cryo-electron microscopy revealed 3D morphology of resveratrol-stabilized insulin that strikingly resembles crystal structures of insulin hexamer formulated with m-cresol. Significantly, we found that, in a condition inductive to amyloid fibrillation at physiological pH, resveratrol is capable of stabilizing insulin more efficiently than m-cresol. Thus, this study describes resveratrol as an effective nontoxic natural molecule that can be used for stabilizing insulin in a bioactive oligomeric form during its commercial formulation.

Retrospect and Prospect of Single Particle Cryo-Electron Microscopy: The Class of Integral Membrane Proteins as an Example.

A giant technological leap in the field of cryo-electron microscopy (cryo-EM) has assured the achievement of near-atomic resolution structures of biological macromolecules. As a recognition of this accomplishment, the Nobel Prize in Chemistry was awarded in 2017 to Jacques Dubochet, Joachim Frank, and Richard Henderson, the pioneers in the field of cryo-EM. Currently, the technique has become the method of choice for structural analysis of heterogeneous and intrinsically dynamic biological complexes. In the past few years, the most prolific branch of cryo-EM, single particle analysis, has revolutionized the structural biology field and structural investigation of membrane proteins, in particular. To achieve high-resolution structures of macromolecules in noncrystalline specimens, from sample and grid preparation to image acquisition, image data processing, and analysis of 3D maps, methodological advances in each of the steps play critical roles. In this Review, we discuss two areas in single particle cryo-EM, the remarkable developments in sample preparation strategies, particularly for membrane proteins, and breakthroughs in methodologies for molecular model building on the high-resolution 3D density maps that brought promises to resolve high-resolution (<4 Å) structures of biological macromolecules.

Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock.

The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell cultures. In this protocol, we describe a step-by-step method to express and purify serotonin 5-HT2AR using a baculoviral expression vector system in Sf9 cell cultures, derived from our work with the rat (matching Uniprot ID P14842) and human (matching Uniprot ID P28223) 5-HT2ARs. A unique feature of this method is the utilization of cell culture additives to infect cells at low multiplicity of infection, thereby using several fold less quantity of viral titer compared to prior methods without the additive. This protocol can be tweaked to selectively over-express glycosylated or non-glycosylated forms of the receptor by varying the post-infection harvest times.

Comprehensive structural modeling and preparation of human 5-HT2A G-protein coupled receptor in functionally active form.

The serotonin 2A receptor (5-HT2A R) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT2A R has marked distinction with ligand-bound conformations of 5-HT2 subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT2A R by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier.

Free vitamin D levels in steroid-sensitive nephrotic syndrome and healthy controls.

INTRODUCTION: Body stores of vitamin D are measured as "total" serum 25-hydroxy vitamin D (25(OH)D). Its largest component is protein bound and lost in urine in nephrotic syndrome (NS). Our study investigates whether "free" 25(OH)D levels are a better guide to bone health and need for vitamin D supplementation in patients with steroid-sensitive NS (SSNS). METHODS: A cross-sectional study was performed in children with SSNS and healthy controls. Blood was tested for albumin, creatinine, calcium, phosphate, ALP, total and free (by direct ELISA) 25(OH)D, iPTH, and urine for protein-creatinine ratio. RESULTS: Seventy-nine NS patients (48 in relapse, 31 in remission) and 60 healthy controls were included. The levels of total 25(OH)D were significantly different (lowest in NS relapse and highest in controls) (p < 0.001). Corrected calcium and phosphate levels were normal, and there were no differences in free 25(OH)D, ALP, or iPTH levels between groups. Only total and not free 25(OH)D correlated significantly and negatively with urinary protein creatinine ratios (rs = - 0.42 vs. 0.04). Free 25(OH)D values of 3.75 and 2.85 pg/ml corresponded to total 25(OH)D levels of 20 and 12 ng/ml, respectively, in healthy controls. CONCLUSION: These results confirm that total 25(OH)D levels are low in NS and related to degree of proteinuria. However levels of free 25(OH)D, ALP, and iPTH did not change in relapse or remission in comparison with healthy controls. Our results suggest that in proteinuric renal diseases, free 25(OH)D rather than total 25(OH)D levels should be used to diagnose vitamin D deficiency and guide therapy.