Renal Involvement in Primary Sj?gren's Syndrome: A Clinicopathologic Study

Background & objectives: Renal pathology and clinical outcomes in patients with primary Sjögren''s syndrome (pSS) who underwent kidney biopsy (KB) because of renal impairment are reported.Design, setting, participants, & measurements: Twenty-four of 7276 patients with pSS underwent KB over 40 years. Patient cases were reviewed by a renal pathologist, nephrologist, and rheumatologist. Presentation, laboratory findings, renal pathology, initial treatment, and therapeutic response were noted.Results: Seventeen patients (17 of 24; 71%) had acute or chronic tubulointerstitial nephritis (TIN) as the primary lesion, with chronic TIN (11 of 17; 65%) the most common presentation. Two had cryoglobulinemic GN. Two had focal segmental glomerulosclerosis. Twenty patients (83%) were initially treated with corticosteroids. In addition, three received rituximab during follow-up. Sixteen were followed after biopsy for more than 12 mo (median 76 mo; range 17 to 192), and 14 of 16 maintained or improved renal function through follow-up. Of the seven patients presenting in stage IV chronic kidney disease, none progressed to stage V with treatment.Conclusions: This case series supports chronic TIN as the predominant KB finding in patients with renal involvement from pSS and illustrates diverse glomerular lesions. KB should be considered in the clinical evaluation of kidney dysfunction in pSS. Treatment with glucocorticoids or other immunosuppressive agents appears to slow progression of renal disease. Screening for renal involvement in pSS should include urinalysis, serum creatinine, and KB where indicated. KB with characteristic findings (TIN) should be considered as an additional supportive criterion to the classification criteria for pSS because it may affect management and renal outcome.Primary Sjögren''s syndrome (pSS) is a progressive autoimmune disorder involving the exocrine glands (1), typically presenting with keratoconjunctivitis and xerostomia (2). It is characterized pathologically by a predominant lymphocytic infiltrate around epithelial ducts of exocrine glands on salivary gland biopsy (3). Extraglandular manifestations of pSS, once thought to be uncommon, occur in up to 25% of patients. Patients can be afflicted by severe interstitial lung disease (4), cutaneous vasculitis (5), peripheral neuropathy (6), and hematologic complications such as lymphoma (7). They are also at increased risk for celiac sprue (8) and complications from Helicobacter pylori infection (9) such as mucosa-associated lymphatic tissue (MALT)-type lymphoma.Much of our understanding of the clinical presentation of renal involvement in pSS is based on case reports (10–26) and small retrospective cohorts (27–29). Tubulointerstitial nephritis (TIN) remains the most common presentation of renal involvement in pSS and CD4/CD8 T cell subsets are reported to predominate (27,30). This is often characterized by a distal (type I) renal tubular acidosis (RTA) and less commonly proximal (type II) RTA (Fanconi syndrome) (11,31–33). GN is thought to be a rare occurrence, with only case reports available in the literature (10,12–23), and tends to be a late development (34) in the course of the disease.We examined the renal pathologic findings and clinical trends of all patients with pSS who underwent kidney biopsy (KB) at Mayo Clinic since 1967 and assembled a case series of patients with pSS with renal pathologic disease evaluated by renal biopsy at a single center in the United States. This case series aimed to describe the common clinical presentations of renal disease in pSS, the array of pathologic findings of renal involvement in pSS, and trends during follow-up and treatment.     

Recent assembly of the Cerrado,a neotropical plant diversity hotspot,by in situ evolution of adaptations to fire

The relative importance of local ecological and larger-scale historical processes in causing differences in species richness across the globe remains keenly debated. To gain insight into these questions, we investigated the assembly of plant diversity in the Cerrado in South America, the world''s most species-rich tropical savanna. Time-calibrated phylogenies suggest that Cerrado lineages started to diversify less than 10 Mya, with most lineages diversifying at 4 Mya or less, coinciding with the rise to dominance of flammable C4 grasses and expansion of the savanna biome worldwide. These plant phylogenies show that Cerrado lineages are strongly associated with adaptations to fire and have sister groups in largely fire-free nearby wet forest, seasonally dry forest, subtropical grassland, or wetland vegetation. These findings imply that the Cerrado formed in situ via recent and frequent adaptive shifts to resist fire, rather than via dispersal of lineages already adapted to fire. The location of the Cerrado surrounded by a diverse array of species-rich biomes, and the apparently modest adaptive barrier posed by fire, are likely to have contributed to its striking species richness. These findings add to growing evidence that the origins and historical assembly of species-rich biomes have been idiosyncratic, driven in large part by unique features of regional- and continental-scale geohistory and that different historical processes can lead to similar levels of modern species richness.The uneven distribution of species diversity across the globe and the occurrence of biodiversity hotspots with high concentrations of species are well established (1–3). However, the underlying causes of differences in species diversity and composition among biomes and the processes that have prompted accumulation of high species diversity in some areas remain poorly understood (4–6). Correlations between species richness and annual energy input, water supply, and physiographic complexity suggest that climatic and other environmental factors are major determinants of species diversity (7). Indeed, it has been shown that these factors can accurately predict the locations of most global diversity hotspots and account for the latitudinal gradient of species richness (7). However, such insights contribute little to our understanding of the historical assembly of species-rich biomes and the larger-scale evolutionary processes that have generated global patterns of diversity (4, 8, 9). Little is known about the historical and geographical assembly of species-rich biomes (5), in terms of when, how quickly, and from where the species and lineages that make up different biomes have been recruited and how they subsequently evolved in situ. This lack of data on geographical and temporal patterns of species diversification, especially in the tropics where most diversity resides, makes it difficult to assess why there are so many species in these areas, to what extent variation in diversity can be attributed to regional and continental-scale historical contingencies (4, 10, 11), and to compare patterns among different species-rich biomes (9, 12).Recent discussion has highlighted the potential role of phylogenetic niche conservatism in shaping regional species pools (8, 13) and explaining diversity gradients (11). Prominent examples of large-scale niche or biome conservatism have been documented for the tropical–temperate biotas (11), mangroves (14), southern hemisphere extratropical biomes (13), Andean grasslands (8, 15), and seasonally dry tropical forests (16–18). However, the extent to which this tendency to retain ancestral ecology across lineages has influenced species composition in the most species-rich tropical biomes is unknown.Recent insights into the historical assembly of species diversity and biomes have come from time-calibrated phylogenies for biome-specific lineages (5, 9, 19–23). Phylogenies have potential for reconstructing transitions from precursor to modern biotas and identifying the underlying factors that drive these processes (4, 11, 24). However, the sparse sampling of lineages and species in such studies (5, 24) has limited these insights to a few well-studied areas such as the Cape Floristic Region (12, 20).     

Outcomes Associated with Serum Calcium Level in Men with Non-Dialysis-Dependent Chronic Kidney Disease

Background and objectives: Elevated serum calcium has been associated with increased mortality in dialysis patients, but it is unclear whether the same is true in non-dialysis-dependent (NDD) chronic kidney disease (CKD). Outcomes associated with low serum calcium are also not well-characterized.Design, setting, participants, & measurements: We examined associations of baseline, time-varying, and time-averaged serum calcium with all-cause mortality in a historic prospective cohort of 1243 men with moderate and advanced NDD CKD by using Cox models.Results: The association of serum calcium with mortality varied according to the applied statistical models. Higher baseline calcium and time-averaged calcium were associated with higher mortality (multivariable adjusted hazard ratio (95% confidence interval): 1.31 (1.13, 1.53); P < 0.001 for a baseline calcium 1 mg/dl higher). However, in time-varying analyses, lower calcium levels were associated with increased mortality.Conclusions: Higher serum calcium is associated with increased long-term mortality (as reflected by the baseline and time-averaged models), and lower serum calcium is associated with increased short-term mortality (as reflected by the time-varying models) in patients with NDD CKD. Clinical trials are warranted to determine whether maintaining normal serum calcium can improve outcomes in these patients.Mineral and bone disorders in chronic kidney disease (CKD) (1) have emerged as novel mortality risk factors in dialysis patients (2–8). Some of these abnormalities (such as serum phosphorus and parathyroid hormone (PTH) levels) have also been implicated in similar ways in patients with non-dialysis-dependent (NDD) CKD (9–12). Serum calcium''s effect on outcomes has been the focus of attention mainly in dialysis patients, where calcium metabolism is significantly distorted (13–19). The use of calcium-containing phosphate binders further complicates the picture because these medications could be involved in the etiology of vascular calcification (20,21), and their roles as therapeutic agents have been intensely debated (22). Supporting the potential role for calcium in cardiovascular disease were epidemiologic studies showing an association between higher calcium and increased mortality (2–8). Some of the same studies have also suggested that extremely low calcium levels may themselves be deleterious (2,3), which has ultimately resulted in recommendations to attain a low-normal serum calcium level in dialysis patients (23). Studies examining the role of calcium in NDD CKD patients are fewer and failed to unequivocally show an association between abnormal calcium levels and vascular calcification (24–27). No study has yet examined the association of calcium levels with mortality in NDD CKD.We examined the association of serum calcium levels with all-cause mortality in a large number of male US veterans with moderate and advanced NDD CKD at a single medical institution.     

Treatment with IFN-α, -β, or -γ Is Associated with Collapsing Focal Segmental Glomerulosclerosis

Background and objectives: Treatment with IFN is rarely associated with nephrotic syndrome and renal biopsy findings of minimal-change disease or FSGS.Design, setting, participants, & measurements: We report 11 cases of collapsing FSGS that developed during treatment with IFN and improved after discontinuation of therapy.Results: The cohort consists of seven women and four men with a mean age of 48.2 yr. Ten of the 11 patients were black. Six patients were receiving IFN-α for hepatitis C virus infection (n = 5) or malignant melanoma (n = 1), three were receiving IFN-β for multiple sclerosis, and two were treated with IFN-γ for idiopathic pulmonary fibrosis. After a mean and median duration of therapy of 4.0 and 12.6 months, respectively, patients presented with acute renal failure (mean creatinine 3.5 mg/dl) and nephrotic-range proteinuria (mean 24-hour urine protein 9.7 g). Renal biopsy revealed collapsing FSGS with extensive foot process effacement and many endothelial tubuloreticular inclusions. Follow-up was available for 10 patients, all of whom discontinued IFN. At a mean of 23.6 months, nine of 10 patients had improvement in renal function, including one with complete remission and two with partial remission. Among the seven patients with available data, mean proteinuria declined from 9.9 to 3.0 g/d. Four of the seven patients were treated with immunosuppression, and there was no detectable benefit.Conclusions: Collapsing FSGS may occur after treatment with IFN-α, -β, or -γ and is typically accompanied by the ultrastructural finding of endothelial tubuloreticular inclusions. Optimal therapy includes discontinuation of IFN.FSGS is the most common cause of idiopathic nephrotic syndrome in black patients and may be the most frequent cause of nephrotic syndrome in the general population (1–6). The spectrum of FSGS includes primary forms mediated by a putative circulating or permeability factor and a number of secondary forms caused by such diverse insults as hereditable mutations in podocyte genes, drugs, viral infections, and adaptive responses to reduced renal mass or other hemodynamic stress (1). A variety of histologic variants of FSGS have been identified and can be applied to both primary and secondary forms (7–9). Many secondary forms tend to manifest as particular morphologic subtypes (1).The collapsing variant of FSGS is defined by implosive wrinkling and “collapse” of the glomerular basement membrane associated with hypertrophy and hyperplasia of overlying podocytes (10–12). Collapsing FSGS was mainly described in patients with HIV-associated nephropathy (HIVAN) (13) but also was recognized as a variant of idiopathic FSGS (11,12). Both idiopathic collapsing FSGS and HIVAN are most commonly seen in young black patients (8–12,14). Compared with the usual, most common form of FSGS with discrete segmental scars (FSGS not otherwise specified [FSGS NOS]), collapsing FSGS is distinguished by more severe nephrotic syndrome and renal insufficiency at presentation and a more rapid course to renal failure (8–12,14). Central to the morphogenesis of the collapsing variant is podocyte injury that leads to podocyte dedifferentiation, apoptosis, and proliferation, in part through dysregulation of cell cycle–related proteins (15–19). Podocyte precursor cells from the parietal cell layer may contribute to the glomerular epithelial cell proliferation (20).HIVAN is not the only established secondary cause of collapsing FSGS. Collapsing FSGS has been reported in the setting of Parvovirus B19 infection (21) and in patients with hemophagocytic syndrome (with or without underlying lymphoma) (22). Collapsing FSGS also may follow treatment with pamidronate (23), with 15 cases reported in the medical literature (23,24). In contrast, FSGS NOS has been reported to result from treatment with lithium (25), sirolimus (26), and more recently anabolic steroids (27). Although rare cases of collapsing FSGS also have been reported after treatment with IFN-α (28–30), this therapeutic agent is more commonly associated with minimal-change disease (MCD) (31–38) and FSGS NOS (39–47). We report 11 additional cases of collapsing FSGS that developed during treatment with IFN, including six IFN-α (for hepatitis C virus [HCV] infection or melanoma), three IFN-β (for multiple sclerosis [MS]), and two IFN-γ (for idiopathic pulmonary fibrosis).     

Phase 1 Study of Anti-CTGF Monoclonal Antibody in Patients with Diabetes and Microalbuminuria

Background and objectives: This report summarizes the first phase 1 trial treating patients with microalbuminuric diabetic kidney disease (DKD) using FG-3019, a human monoclonal antibody to connective tissue growth factor (CTGF). CTGF is critically involved in processes of progressive fibrosis, including DKD. This phase 1, open-label, dose-escalation trial evaluated safety, pharmacokinetics, and possible therapeutic effects of FG-3019 on albuminuria, proteinuria, and tubular proteins.Design, setting, participants, and measurements: Microalbuminuric subjects (n = 24) with type 2 (79%) or type 1 (21%) diabetes received 3 or 10 mg/kg FG-3019 dosed intravenously every 14 days for four doses. Albuminuria and safety follow-up were to days 62 and 365, respectively.Results: No infusion was interrupted for symptoms, although 5 of 24 subjects had mild infusion-day adverse events thought to be possibly drug-related. No subject developed anti-FG-3019 antibodies. FG-3019 clearance was lower at 10 mg/kg than at 3 mg/kg, suggesting a saturable elimination pathway. Although this study was not designed for efficacy testing, it was notable that urinary albumin/creatinine ratio (ACR) decreased significantly from mean pretreatment ACR of 48 mg/g to mean post-treatment (day 56) ACR of 20 mg/g (P = 0.027) without evidence for a dose-response relationship.Conclusions: Treatment of microalbuminuric DKD subjects using FG-3019 was well tolerated and associated with a decrease in albuminuria. The data demonstrate a saturable pathway for drug elimination, minimal infusion adverse events, and no significant drug-attributable adverse effects over the year of follow-up. Changes in albuminuria were promising but require validation in a prospective, randomized, blinded study.Patients with diabetic kidney disease (DKD) are at increased risk for cardiovascular complications and early mortality. Those who survive long enough tend to progress to ESRD requiring dialysis or transplantation. Although advances in therapy with angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor type II blockers (ARBs) have attenuated the incident rate of ESRD (1), disease progression remains common (2–4) and diabetes continues to be the leading cause for initiation of dialysis in the United States (1).Connective tissue growth factor (CTGF) is a 349-amino-acid secreted pleiotropic protein belonging to the cysteine-rich CCN (CTGF/Cyr61/Cef10/NOVH) family. Numerous glomerular, tubulointerstitial, and vascular cells types can produce CTGF, and many factors associated with the diabetic condition can stimulate CTGF expression, including hypertension, hyperglycemia, and hyperlipidemia (5–24).CTGF is a critical mediator of extracellular matrix accumulation and coordinates a final common pathway of fibrosis (5,25,26). CTGF has been shown to amplify the fibrogenic activity of TGFβ (27) and IGF-1 (17) and to inhibit the action of antifibrotic and regenerative factors bone morphogenic protein-7 (27,28) and vascular endothelial growth factor (29,30).In type 1 diabetes, plasma and urine CTGF levels correlate with the level of albuminuria and the stage of progressive renal insufficiency (31–34), and the plasma CTGF level is an independent predictor of vascular disease as assessed by intimal medial thickness (35) and of mortality and progression to ESRD (36). In renal biopsy specimens from patients with diabetes, elevated levels of CTGF mRNA are associated with chronic tubulointerstitial damage, albuminuria, and progression of renal insufficiency (37–39).FG-3019 is a recombinant human anti-CTGF monoclonal IgG₁ antibody that has shown activity in rodent models of kidney dysfunction associated with type 1 and 2 diabetes (40–42). Here, we report results of an open-label dose-escalation trial of FG-3019 infusions administered biweekly over 56 days in patients with DKD, the first study designed to evaluate safety and potential therapeutic effect of FG-3019 in this patient population.     

Phosphoinositides and SNARE chaperones synergistically assemble and remodel SNARE complexes for membrane fusion

Yeast vacuole fusion requires 4 SNAREs, 2 SNARE chaperone systems (Sec17p/Sec18p/ATP and the HOPS complex), and 2 phosphoinositides, phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. By reconstituting proteoliposomal fusion with purified components, we now show that phosphoinositides have 4 distinct roles: PI(3)P is recognized by the PX domain of the SNARE Vam7p; PI(3)P enhances the capacity of membrane-bound SNAREs to drive fusion in the absence of SNARE chaperones; either PI(3)P or PI(4,5)P₂ can activate SNARE chaperones for the recruitment of Vam7p into fusion-competent SNARE complexes; and either PI(3)P or PI(4,5)P₂ strikingly promotes synergistic SNARE complex remodeling by Sec17p/Sec18p/ATP and HOPS. This ternary synergy of phosphoinositides and 2 SNARE chaperone systems is required for rapid fusion.Intracellular membrane fusion is a conserved reaction, vital for vesicle trafficking, hormone secretion, and neurotransmission. Fusion is regulated by NSF (N-ethylmaleimide-sensitive factor)/Sec18p, αSNAP (soluble NSF attachment protein)/Sec17p, SNAREs (SNAP receptors), Sec1p/Munc18–1p family (SM) proteins, Rab GTPases, and Rab:GTP-binding proteins, termed “Rab effectors” (1–3). Lipids, including phosphoinositides, sterols, diacylglycerol (DAG), and phosphatidic acid (PA), have specific roles in fusion (4–14). Proteins and lipids cooperate for their enrichment in membrane fusion microdomains (6, 8, 15, 16).SNARE proteins are integral or peripheral membrane proteins required for membrane fusion. SNAREs have either a Q or R residue at the center of their SNARE domain and associate in 4-helical QabcR complexes in cis (anchored to one membrane) or in trans (anchored to apposed membranes), where a, b, and c are families of related Q-SNAREs (2, 17, 18). Reconstituted proteoliposomes (RPLs) bearing Q-SNAREs fuse with RPLs bearing an R-SNARE through trans-SNARE-complex assembly (19, 20). This fusion has slow kinetics, requires nonphysiologically high SNARE densities, and causes substantial leakage of luminal contents of the RPLs (21–24).We study membrane fusion with yeast vacuoles (lysosomes). Vacuole fusion (25) requires 3 Q-SNAREs (Vam3p, Vti1p, and Vam7p) and 1R-SNARE (Nyv1p) (26, 27), two SNARE chaperone systems, Sec17p/Sec18p/ATP (28), and the HOPS (homotypic fusion and vacuole protein sorting)/Vps Class C complex (29, 30), the Rab-GTPase Ypt7p (31), and chemically minor but functionally vital “regulatory lipids”: ergosterol (ERG), DAG, PI(3)P, and PI(4,5)P₂ (8). Inactive 4SNARE cis-complexes on isolated organelles are disassembled by Sec17p/Sec18p/ATP (27). The heterohexameric HOPS complex, containing the SM protein Vps33p as a subunit, promotes and proofreads SNARE-complex assembly (32–34). HOPS can physically interact with the Q-SNAREs [Vam7p (35) and Vam3p (36, 37)], 4SNARE cis-complexes (32), GTP-bound Ypt7p (29), and phosphoinositides (35). PI(3)P supports the membrane association of the Qc-SNARE Vam7p, which has no transmembrane domain, through binding its PX domain (38). SNAREs, HOPS, Ypt7p, and regulatory lipids assemble in an interdependent fashion to form a fusion-competent membrane microdomain, the “vertex ring” (8, 16, 39). Trans-SNARE complexes are essential for fusion (26), yet fusion can be accelerated by SNARE-associating factors such as HOPS (14, 35) and by cycles of SNARE complex disassembly and reassembly, termed “remodeling” (40).Membrane fusion has been reconstituted with all purified yeast vacuolar components, including 4SNAREs, vacuolar lipids, 2 SNARE chaperone systems, and phosphoinositides (14). We now show distinct functions of phosphoinositides in RPL fusion: the PX-domain of the SNARE Vam7p recognizes PI(3)P, as reported (38); PI(3)P activates the 3Q-SNAREs to be more fusogenic in the absence of SNARE chaperones; either PI(3)P or PI(4,5)P₂ accelerates fusion by promoting the synergy between Sec17p/Sec18p and HOPS, although this synergy is not a function of the membrane recruitments of these SNARE chaperones. This ternary synergy between phosphoinositides and SNARE chaperones is essential for the assembly and remodeling of SNARE complexes.     

Hypophosphatemic Effect of Niacin in Patients without Renal Failure: A Randomized Trial

Background and objectives: Niacin administration lowers the marked hyperphosphatemia that is characteristic of renal failure. We examined whether niacin administration also reduces serum phosphorus concentrations in patients who have dyslipidemia and are free of advanced renal disease.Design, setting, participants, & measurements: We performed a post hoc data analysis of serum phosphorus concentrations that had been determined serially (at baseline and weeks 4, 8, 12, 18, and 24) among 1547 patients who had dyslipidemia and were randomly assigned in a 3:2:1 ratio to treatment with extended release niacin (ERN; 1 g/d for 4 weeks and dose advanced to 2 g/d for 20 weeks) combined with the selective prostaglandin D2 receptor subtype 1 inhibitor laropiprant (L; n = 761), ERN alone (n = 518), or placebo (n = 268).Results: Repeated measures analysis revealed that ERN-L treatment resulted in a net mean (95% confidence interval) serum phosphorus change comparing ERN-L with placebo treatment of −0.13 mmol/L (−0.15 to −0.13 mmol/L; −0.41 mg/dl [−0.46 to −0.37 mg/dl]). These results were consistent across the subgroups defined by estimated GFR of <60 or ≥60 ml/min per 1.73 m², a serum phosphorus of >1.13 mmol/L (3.5 mg/dl) versus ≤1.13 mmol/L (3.5 mg/dl), the presence of clinical diabetes, or concomitant statin use.Conclusions: We have provided definitive evidence that once-daily ERN-L treatment causes a sustained 0.13-mmol/L (0.4-mg/dl) reduction in serum phosphorus concentrations, approximately 10% from baseline, which is unaffected by estimated GFR ranging from 30 to ≥90 ml/min per 1.73 m² (i.e., stages 1 through 3 chronic kidney disease).Abnormalities in calcium-phosphorus homeostasis, including significant elevations in serum phosphorus concentrations, are thought to contribute to arterial stiffening, hypertension, and cardiovascular disease (CVD) risk in patients with advanced chronic kidney disease and ESRD that requires maintenance dialysis (1–6). Observational data from population-based studies suggested that even serum phosphorus concentrations within the normative range are linearly associated with measures of subclinical arteriosclerosis and the development of incident CVD outcomes (7–12). Two cross-sectional studies from patients who underwent cardiac catheterization have further indicated that serum phosphorus concentrations, primarily within the normative range, were directly associated with both the presence and the severity of angiographic coronary artery disease (13,14). Moreover, a graded, independent association between serum phosphorus concentrations (again, within the normative range) and recurrent CVD events was reported among a large clinical trial cohort of patients with a previous myocardial infarction (15).Supplementation of calcium salts, despite their efficacy and tolerability as a phosphorus-lowering treatment in ESRD, may enhance coronary artery and aortic valve calcification (16,17). This observation highlights the need for hyperphosphatemia treatment protocols to balance potential benefits and adverse effects (18–22). Phosphorus-lowering drugs that target other cardiovascular risk factors in chronic kidney disease (CKD), simultaneously, including, for example, dyslipidemia (23), might have additive or synergistic benefits. These findings may also be relevant to populations with less advanced CKD or normal renal function.Preliminary studies suggested that niacin administration (as niacinamide, niceritrol, or nicotinic acid) could be a useful primary or adjunctive treatment for the marked hyperphosphatemia that is characteristic of ESRD (24–30). Several reports from clinical trials of extended-release niacin (ERN) that was given to patients who had dyslipidemia and were free of clinical renal disease and hyperphosphatemia have contained limited additional data noting up to 10% reductions in the serum phosphorus concentrations of actively treated patients (31–34). These repeated clinical observations (24–34) are most plausibly explained by the direct inhibitory effect of niacin compounds on active transport-mediated phosphorus absorption in the mammalian small intestine (35–39).Published studies of patient populations who had dyslipidemia and were receiving ERN that included phosphorus data may have failed to provide information on baseline phosphorus values (33,34), and none (31–34) performed repeated measures analyses to examine the potential effects of niacin treatment on serum phosphorus and calcium concentrations, as well as the calcium-phosphorus products.Focused reexamination of the large, placebo-controlled clinical trial data set assembled by Maccubbin et al. (34) afforded us a unique opportunity to elucidate these and other unresolved issues regarding the impact of niacin given as the fixed-dose combination of ERN and laropiprant (ERN-L), a selective prostaglandin D2 receptor subtype 1 inhibitor that reduces niacin-induced flushing (34) or ERN alone on serum phosphorus and calcium concentrations and calcium-phosphorus products. We further evaluated whether there was evidence for significant effect modification by estimated GFR (eGFR), baseline serum phosphorus concentration, the presence of diabetes, or concurrent hepatic hydroxymethyl glutaryl–CoA reductase inhibitor (statin) use when assessing the potential impact of niacin on these routine clinical measures of calcium-phosphorus homeostasis.     

Training of Surgeons in Peritoneal Dialysis Catheter Placement in the United States: A National Survey

Background and objectives: Peritoneal dialysis (PD) depends on timely and skilled placement of a PD catheter (PDC). Most PDCs are placed surgically, but little is known about the residency training of surgeons in this procedure. Inadequate residency training could limit surgical expertise in PDCs, resulting in high complication rates that discourage PD use. This study assessed surgical PDC training in the United States to explore this issue.Design, setting, participants, & measurements: A survey was sent to program directors of 248 U.S. surgery residency programs regarding the amount of PDC training, attitudes toward PDCs, and barriers to PDC training. Results were compared between academic and private centers.Results: Ninety-three surgery programs (38%) responded: 82% provided training in PDC and 69% were academic centers. Most surgeons placed 2 to ≤5 catheters during residency. Forty-eight percent of program directors felt that PDC training was important, 61% felt PDC training affected outcomes and increased the likelihood surgeons would place PDCs in practice, and 62% of programs expressed willingness to provide more PDC training. Lack of referrals from nephrology was the most frequently cited barrier to PDC training.Conclusions: Although many U.S. surgery residency programs provide PDC training, this training appears inadequate. Low PD use and lack of referrals limits surgical training at most centers. Nephrologists need to develop initiatives with surgeons to improve PDC training and outcomes.The use of peritoneal dialysis (PD) in the United States is declining. Despite comparable efficacy, improving outcomes, and cost savings compared with hemodialysis (HD), only 6% of incident and 7.2% of prevalent dialysis patients are treated with PD (1–4). Although many factors determine success on PD, a well functioning PD catheter (PDC) is absolutely necessary. Placement of a PDC by an experienced operator is strongly recommended to reduce complications (5–9). Little attention has been given to the potential effect of surgical PDC training on PD use and outcomes (1–2,10). Conversely, considerable focus has been placed on improving surgical training and outcomes for HD access (11–15).Problems with PDC placement and malfunction can disrupt efforts to grow and develop a PD program (5,9,16–18). PDC problems frustrate patients, nurses, and nephrologists alike, leading to dissatisfaction with PD and an early switch to HD (18). PDC malfunction is second only to infection as the cause of technique failure in PD (19,20). Surgeons insert most PD catheters in the United States because most nephrologists are not trained in PDC placement (5,21–23). Unfortunately, there is a shortage of surgeons interested and skilled in performing this procedure (5).Surgical outcomes correlate strongly with training during residency (24). Reluctance by surgeons to place PDCs and suboptimal PDC outcomes might stem from inadequate residency training. Unfortunately, little is known about the training surgeons undergo in this outwardly simple, yet critical procedure. We sought to investigate PDC training in U.S. surgery residency programs and explore surgical program directors'' attitudes toward this procedure.     

A state-mutating genetic algorithm to design ion-channel models

Realistic computational models of single neurons require component ion channels that reproduce experimental findings. Here, a topology-mutating genetic algorithm that searches for the best state diagram and transition-rate parameters to model macroscopic ion-channel behavior is described. Important features of the algorithm include a topology-altering strategy, automatic satisfaction of equilibrium constraints (microscopic reversibility), and multiple-protocol fitting using sequential goal programming rather than explicit weighting. Application of this genetic algorithm to design a sodium-channel model exhibiting both fast and prolonged inactivation yields a six-state model that produces realistic activity-dependent attenuation of action-potential backpropagation in current-clamp simulations of a CA1 pyramidal neuron.The importance of modeling ion channels has been understood since Hodgkin and Huxley''s seminal work with the squid giant axon (1). Subsequently, the development of the patch-clamp method (2) enabled the characterization of the properties of a wide range of channels, and intensive efforts followed to produce quantitative models that could predict and explain specific ion-channel behavior (3–7). Such efforts have led to two broad classes of models: those describing single-channel and gating currents, and those describing macroscopic currents.Models of single-channel and gating currents can be used to analyze properties such as open-channel probabilities, dwell times, and activation kinetics; they therefore facilitate an improved understanding of channel biophysics (3–5, 7–9). By contrast, models of macroscopic currents are usually intended as empirical tools as part of larger compartmental models of neurons. Such a macroscopic model may not necessarily describe the actual molecular state changes of the channel; the goal is rather for it to function as a “black-box” that reproduces the mean behavior of a population of channels. Hodgkin and Huxley''s (1) original formulation of sodium- and potassium-channel models is a prime example of this case, and its basic formalism continues to be widely used to generate empirical ion-channel models.An alternative to the Hodgkin–Huxley formalism is the state-dependent modeling approach (3). In reality, state-dependent models subsume Hodgkin–Huxley models because the latter can be recast as the former (3). State-dependent models are more general, however, because they can describe certain behaviors more easily than Hodgkin–Huxley type models, such as having widely different transition rates into and out of a given state (10). In general, the gold standard for the use of state-dependent models is single-channel recording (3, 5, 11), but the state-dependent formalism is also often employed in models of macroscopic currents (12–15) because of the generality and flexibility it affords.Methods to make empirical state-dependent models conform to data have been studied extensively and have involved a multitude of techniques such as hand fitting (12–14, 16), principal-axis fitting (17), maximum-likelihood estimation (5, 7, 17, 18), and genetic algorithms (15), among others. Here, we present a new fitting technique based on a topology-mutating genetic algorithm. Genetic algorithms have a number of useful characteristics: First, they have been shown to explore a large area of parameter space with relatively quick convergence, especially for problems with many parameters (19). Second, they are easily parallelizable. Third, they have been successfully applied to neuronal modeling, both for Hodgkin–Huxley-type ion-channel parameters and for compartmental neuronal models with voltage-activated conductances (15). Here we show that if the ion-channel model is formulated properly, genetic algorithms provide a natural way to incorporate changes in model topology as mutations.The algorithm presented here has several key features. Most notably, whereas other published optimization algorithms fix the model topology and optimize the rate constants, our algorithm searches over the space of model topologies and the space of rate parameters simultaneously. In order to design such an algorithm appropriate for state-dependent ion-channel models, we formulated an automated, computationally efficient method to satisfy the principle of microscopic reversibility, an equilibrium condition that imposes constraints on topologies with loops (20, 21). Finally, our algorithm uses a sequential approach, also known as goal programming (22), to optimize multiple protocols without the need to assign weights to each of the objective functions. The ability of this genetic algorithm to select and examine topologies not previously explored demonstrates its flexibility in developing working empirical models.We applied this genetic algorithm to devise a sodium-channel model that exhibits both fast and slow inactivation. Fast inactivation refers to a nonconducting channel state that follows quickly after depolarization and activation (within milliseconds) and from which channels recover quickly when the voltage is restored to resting levels (1). In response to either sustained depolarization (23, 24) or a train of depolarizing pulses (16, 25), however, the fraction of sodium channels available for activation also decreases rapidly, but in this case recovery occurs much more slowly, on the order of seconds rather than milliseconds. This form of inactivation has therefore been called “prolonged” or “slow” (16, 25). The presence of such widely disparate time scales makes the creation of state-dependent models of these channels a challenge. At the same time, the effect of prolonged inactivation on processes such as action-potential backpropagation (26, 27), transitions from bursting to spiking (28), and dendritic spiking (29, 30) makes the development of accurate models of prolonged inactivation important for computational simulations of neuronal function.     

Robustness of topological order and formation of quantum well states in topological insulators exposed to ambient environment

The physical property investigation (like transport measurements) and ultimate application of the topological insulators usually involve surfaces that are exposed to ambient environment (1 atm and room temperature). One critical issue is how the topological surface state will behave under such ambient conditions. We report high resolution angle-resolved photoemission measurements to directly probe the surface state of the prototypical topological insulators, Bi₂Se₃ and Bi₂Te₃, upon exposing to various environments. We find that the topological order is robust even when the surface is exposed to air at room temperature. However, the surface state is strongly modified after such an exposure. Particularly, we have observed the formation of two-dimensional quantum well states near the exposed surface of the topological insulators. These findings provide key information in understanding the surface properties of the topological insulators under ambient environment and in engineering the topological surface state for applications.The topological insulators represent a novel state of matter where the bulk is insulating but the surface is metallic, which is expected to be robust due to topological protection (1–5). The topological surface state exhibits unique electronic structure and spin texture that provide a venue not only to explore novel quantum phenomena in fundamental physics (6–10) but also to show potential applications in spintronics and quantum computing (2,5,11). The angle-resolved photoemission spectroscopy (ARPES) is a powerful experimental tool to directly identify and characterize topological insulators (12). A number of three-dimensional topological insulators have been theoretically predicted and experimentally identified by ARPES (13–21); some of their peculiar properties have been revealed by scanning tunneling microscopy (STM) (22–26). The application of the topological surface states depends on the surface engineering that can be manipulated by incorporation of nonmagnetic (27–31) or magnetic (27, 28, 31–33) impurities or gas adsorptions (27, 33–35). While the ARPES and STM measurements usually involve the fresh surface obtained by cleaving samples in situ under ultrahigh vacuum, for the transport and optical techniques, which are widely used to investigate the intrinsic quantum behaviors of the topological surface state (36–40), and particularly the ultimate applications of the topological insulators, the surface is usually exposed to ambient conditions (1 atm air and room temperature) or some gas protection environment. It is therefore crucial to investigate whether the topological order can survive under the ambient conditions and, furthermore, whether and how the surface state may be modified after such exposures.     

Prevalence of Atrial Fibrillation and Its Predictors in Nondialysis Patients with Chronic Kidney Disease

Background and objectives: Chronic kidney disease (CKD) increases systemic inflammation, which is implicated in development and maintenance of atrial fibrillation (AF); therefore, we hypothesized that the prevalence of AF would be increased among nondialysis patients with CKD. This study also reports independent predictors of the presence of AF in this population.Design, setting, participants, & measurements: A retrospective, cross-sectional analysis of 1010 consecutive nondialysis patients with CKD from two community-based hospitals was conducted. Estimated GFRs (eGFRs) were calculated using the Modification of Diet in Renal Disease (MDRD) equation. Multivariate logistic regression was used to determine independent predictors.Results: Of 1010 nondialysis patients with CKD, 214 (21.2%) had AF. Patients with AF were older than patients without AF (76 ± 11 versus 63 ± 15 yr). The prevalence of AF among white patients (42.7%) was higher than among black patients (12.7%) or other races (5.7%). In multivariate analyses, age, white race, increasing left atrial diameter, lower systolic BP, and congestive heart failure were identified as independent predictors of the presence of AF. Although serum high-sensitivity C-reactive protein levels were elevated in our population (5.2 ± 7.4 mg/L), levels did not correlate with the presence of AF or with eGFR. Finally, eGFR did not correlate with the presence of AF in our population.Conclusions: The prevalence of AF was increased in our population, and independent predictors were age, white race, increasing left atrial diameter, lower systolic BP, and congestive heart failure.Atrial fibrillation (AF) is the most common arrhythmia in clinical practice (1). Cardiac comorbidities that are associated with AF include hypertension, coronary artery disease (CAD), valvular heart disease (VHD), congestive heart failure (CHF), cardiomyopathy, pericarditis, congenital heart disease (CHD), and cardiac surgery (2–9). Noncardiac comorbidities that are associated with AF include acute pulmonary embolism, chronic obstructive pulmonary disease (COPD), obstructive sleep apnea, hyperthyroidism, and obesity (10–14).Evidence suggests that inflammation is involved in the pathogenesis of AF (15–20). For example, AF after cardiac surgery is associated with proinflammatory cytokine and complement activation (16,19). Moreover, patients with refractory lone AF have inflammatory infiltrates, myocyte necrosis, and fibrosis on biopsy (18). Several studies also reported elevated serum high-sensitivity C-reactive protein (hsCRP) levels in patients with AF (15–17,20).Evidence suggests that inflammation is associated with renal dysfunction (21–24). Proposed mechanisms include decreased proinflammatory cytokine clearance, endotoxemia, oxidative stress, and reduced antioxidant levels (23,24). Moreover, hsCRP levels are higher among elderly patients with renal insufficiency (24). In hemodialysis (HD) patients with ESRD, hsCRP, IL-6, and fibrinogen levels are elevated (21,22).HD patients with ESRD have an increased prevalence of AF; however, prevalence among nondialysis patients with CKD has not been investigated (25–30). Because CKD promotes inflammation, which promotes AF, we hypothesized the prevalence of AF would be increased among nondialysis patients with CKD. This study reports the prevalence and independent predictors of the presence of AF in a nondialysis population with CKD.     

Interferon for Hepatitis C Virus in Hemodialysis—an Individual Patient Meta-analysis of Factors Associated with Sustained Virological Response

Background and objectives: Hepatitis C virus (HCV) infection is prevalent in hemodialysis patients and causes excess mortality. Interferon (IFN) treatment of chronic HCV infection in hemodialysis patients results in high sustained virological response (SVR) rates 6 mo after treatment. The authors aimed to identify factors associated with SVR in hemodialysis patients through analysis of individual patient data obtained from systematic review of published literature.Design, setting, participants & measurements: Medline was searched from 1966 through February 2009, and prospective studies describing IFN treatment of hemodialysis patients with chronic HCV infection with published individual patient data were included. To identify factors associated with SVR, logistic regression was applied with adjustment for study.Results: Twenty studies of IFN treatment provided data on 428 patients. Overall SVR was 45% and in univariate analyses was higher with: 1) three million units or higher three times weekly of IFN; 2) treatment for at least 6 mo; 3) treatment completion; 4) lower baseline HCV RNA; 5) female gender; and 6) early virological negativity. Although limited by missing data, these relationships persisted in multivariate regression.Conclusions: SVR is more likely with larger IFN dose, longer treatment duration, treatment completion, female gender, lower HCV RNA and early virological negativity. For appropriate treatment candidates, regimens should consist of three million units of IFN three times weekly for at least 6 mo, with patients encouraged to complete the full course.Hepatitis C virus (HCV) infects an estimated 170 million people worldwide (1). The prevalence of HCV in hemodialysis (HD) patients ranges from 3 to 23% in developed countries (2) and exceeds 50% in some developing countries (3). HCV-infected HD patients have higher mortality rates than noninfected HD patients, with reported relative risks from 1.25 to 1.57 (4,5). Untreated, spontaneous viral clearance occurs in only 0.5% of chronic HCV-infected patients per year (6). The standard measure of treatment success, sustained virological response (SVR), is defined as achieving HCV RNA negativity six months after treatment completion. In non-HD patients, interferon (IFN) monotherapy achieves SVR in 9 to 22% of patients (7–9) but combination pegylated IFN and ribavirin achieves SVR in 50 to 60% (10,11). However, IFN and ribavirin are associated with significant toxicity including influenza-like symptoms, anemia and depression with IFN (7–9), and hemolytic anemia with ribavirin (7,8).Most studies of HCV-infected HD patients have investigated IFN monotherapy. Only recently, studies have explored pegylated IFN or ribavirin (12–18). IFN treatment after kidney transplantation is associated with increased rates of allograft rejection (19,20), so treatment before transplantation is advised (21,22). Our recent meta-analysis of summary data in HCV-infected HD patients demonstrated an overall SVR rate of 41% with IFN (22), higher than rates in IFN-treated non-HD patients (7,8), but rates of treatment discontinuation due to adverse events were also higher (22).Identifying factors associated with a higher likelihood of SVR among HD patients has important implications for selecting treatment candidates and the optimal treatment regimen. In non-HD patients, higher SVR rates are associated with younger age, female gender, lower patient weight, HCV genotypes other than 1, lower baseline HCV RNA, and absence of cirrhosis on liver biopsy (7–11). Early virological response (EVR), defined as a 2-log₁₀ or larger decrease in HCV RNA by the 12^th week of treatment, is a powerful predictor of SVR (23,24).We investigated whether these previously identified factors associated with SVR in non-HD patients could be validated in the HD population. The majority of studies of IFN in HD patients reported individual patient data, allowing us to extend our prior subgroup analysis and meta-regression of summary data (22) to identify factors associated with SVR.     

The Origin of Multiple Molecular Forms in Urine of HNL/NGAL

Background and objectives: Several molecular forms of human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), a novel biomarker for acute kidney injury (AKI), have been found in urine. The origin of these different forms and the effect of antibody configuration on assay performances were investigated in this report.Design, setting, participants, & measurements: The molecular forms of HNL/NGAL from human neutrophils and present in urine obtained from cardiac surgery patients and patients with urinary tract infection (UTI), as well as secreted from HK-2 cells, were studied by Western blotting. The levels of HNL/NGAL in urine were measured by ELISAs. Kidney injury was simulated by incubation of HK-2 cells under stressful conditions.Results: The major molecular form of HNL/NGAL secreted by neutrophils is dimeric, whereas the major form secreted by HK-2 cells is monomeric. This was reflected by a predominance of the monomeric form in urine from patients with AKI and the dimeric form in patients with UTIs. The epitope specificities of the antibody used in the ELISAs had a profound effect on assay performance and paralleled differences of the antibodies to identify the different forms of urine HNL/NGAL.Conclusions: The monomeric form is the predominant form secreted by tubular epithelial cells, and the dimeric form is the predominant form secreted by neutrophils. The development of molecular form-specific assays for HNL/NGAL may be a means to identify the origin of HNL/NGAL in urine and construct more specific tools for the diagnosis of AKI.Human neutrophil lipocalin(HNL) (1), also named neutrophil gelatinase-associated lipocalin (NGAL) (2), is a ubiquitous glycoprotein originally isolated from human neutrophils and localized in their specific granules. HNL/NGAL exists as a 25-kD monomer, or as a 45-kD disulfide-linked homodimer, and it is covalently conjugated with gelatinase (matrix metalloproteinase 9) via an intermolecular disulfide bridge as a 135-kD heterodimeric form (2).Although HNL/NGAL was originally identified in and purified from human neutrophils, it is also expressed in kidney, liver, and epithelial cells under certain conditions (3,4). Pathologic or stressful conditions such as inflammation, infection, cancer, intoxication, ischemia, kidney injury, and cardiac surgery can induce the upregulation of the synthesis of HNL/NGAL (5–13). In addition, several studies have shown that upregulation of HNL/NGAL in human cell lines (A459 (14), MCF-7 (15), and HepG2 (11)) is induced by oxidative stress, cytokines, or other stimuli.HNL/NGAL has recently been highlighted as a novel and early biomarker of acute kidney injury (AKI) (12,13,16–19). Thus, the levels of HNL/NGAL were significantly increased in serum/plasma and urine after cardiac surgery and paralleled reduction in renal function (12,16,19). Several immunoassays have been developed for the measurement of HNL/NGAL. The assays are based on different formats and include RIA (20), Western blotting (21), ELISA (22,23), Triage device (24), and the Architect platform (16). Several research groups used one of these assays to determine the levels of HNL/NGAL in urine and drew the conclusion that HNL/NGAL is a biomarker of AKI (12,13,16–18). Our previous results indicated that the antibody configuration had an effect on the clinical performance of the assay (19,25). We also reported, for the first time, the existence of several molecular forms of HNL/NGAL in urine obtained from patients after cardiac surgery and that the presence of dimeric and monomeric forms and their ratios changed after operation (19). The source of the different molecular forms of HNL/NGAL and what they might reflect has not yet been elucidated. The aim of this report was therefore to study the possible cellular source of these different molecular forms and to investigate the possible effect of these different forms on the assay performances of HNL/NGAL assays using several different monoclonal and polyclonal antibodies with different epitope specificities.     

Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-Å crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.Glypicans are heparan sulfate proteoglycans (HSPGs) that consist of an approximately 450 amino acid N-terminal protein domain followed by an approximately 100 amino acid stalk region that is attached to the outer cell membrane via a glycosyl-phosphatidylinositol anchor (1). The N-terminal domain of most glypicans is proteolytically processed by a furin-like convertase to produce two chains that remain connected by disulfide bonds (2). This processing appears required for some but not all glypican activity (2, 3). The stalk regions of glypicans are predicted to be largely random coil and typically contain 1–5 heparan sulfate attachment sites (1, 4). Six glypicans are present in humans and mice (glypican-1, -2, -3, -4, -5, and -6); two are present in Drosophila [Dally and Dally-like (Dlp)] (1). Based on sequence similarity, glypicans assort into two subfamilies with glypican-1, -2, -4, -6, and Dlp in one family and glypican-3, -5, and Dally in another (1).Glypicans are active in development in both vertebrates and invertebrates. Loss of Dally in fruit flies results in defects in brain, eye, wings, antennae, and genitalia (5). Loss of glypican-3 in humans is responsible for Simpson–Golabi–Behmel overgrowth syndrome, in which widespread visceral and skeletal abnormalities are present along with a predisposition to tumor formation (6). Loss of glypican-6 has recently been shown to cause omodysplasia, a genetic disorder characterized by variable heart defects, cognitive delay, skeletal and facial abnormalities, and shortness of stature (7). Much of the function of glypicans is attributable to modulation of signaling by several heparin-binding growth factors active during development including members of the fibroblast growth factor, Hedgehog (Hh), Wnt, and transforming growth factor-β families (8–15). Each of these factors functions as a morphogen to elicit distinct concentration-dependent responses within target cells, and glypicans have been shown to be required both for normal response to these factors as well as to establish their proper distribution (9, 10, 12, 16–21). The heparan sulfate attachments of glypicans are clearly important for mediating interactions with these growth factors and downstream signaling components (22, 23), but recent work has demonstrated a role for the N-terminal protein domain, which lacks heparan sulfate modifications, in mediating responsiveness to at least Wnt and Hh signals (23–26).Curiously, glypicans appear able to play both positive and negative roles in mediating Hh signaling. The protein region of Dally-like contributes positively to Drosophila Hh responsiveness, and the developmental defects in omodysplasia, particularly the bone growth defects, are suggestive of a positive role for glypican-6 function in response to Indian hedgehog (7). Notably, glypican-4 and glypican-6 are most similar to Dlp (vs. Dally) and complement Dlp function in a Drosophila cultured cell-based Hh signaling assay (25). In contrast, the protein region of glypican-3, which is more similar to Dally than Dally-like, is a negative regulator of Hh responsiveness in the mouse (24, 25, 27, 28). Based on sequence homology and functional phenotypes, it has thus been speculated that the two major subfamilies of glypicans have evolved opposing activities in Hh signal responsiveness (25).To investigate the molecular basis for glypican function, we have undertaken structural and functional characterization of the N-terminal protein domain of Dlp and report here its 2.4-Å crystal structure. We show that the N-terminal protein domains of glypicans adopt an elongated α-helical structure with no evident homology to any known structure. We have used structure-guided mutagenesis to identify a localized region on the Dlp surface important for the ability of Dlp to mediate Hh signal response. These results are most consistent with Dlp functioning as a binding protein in Hh signaling, but we are unable to detect high-affinity interactions between Dlp and either Hh or an Hh:Ihog complex. These results establish a molecular basis for mapping and comparing functional regions of different glypicans.     

Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1

Store-operated Ca²⁺ entry (SOCE) is activated by redistribution of STIM1 into puncta in discrete ER-plasma membrane junctional regions where it interacts with and activates store-operated channels (SOCs). The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined. Here we report that caveolin-1 (Cav1) is a critical plasma membrane scaffold that retains TRPC1 within the regions where STIM1 puncta are localized following store depletion. This enables the interaction of TRPC1 with STIM1 that is required for the activation of TRPC1-SOCE. Silencing Cav1 in human submandibular gland (HSG) cells decreased plasma membrane retention of TRPC1, TRPC1-STIM1 clustering, and consequently reduced TRPC1-SOCE, without altering STIM1 puncta. Importantly, activation of TRPC1-SOCE was associated with an increase in TRPC1-STIM1 and a decrease in TRPC1-Cav1 clustering. Consistent with this, overexpression of Cav1 decreased TRPC1-STIM1 clustering and SOCE, both of which were recovered when STIM1 was expressed at higher levels relative to Cav1. Silencing STIM1 or expression of ΔERM-STIM1 or STIM1(⁶⁸⁴EE⁶⁸⁵) mutant prevented dissociation of TRPC1-Cav1 and activation of TRPC1-SOCE. However expression of TRPC1-(⁶³⁹KK⁶⁴⁰) with STIM1(⁶⁸⁴EE⁶⁸⁵) restored function and the dissociation of TRPC1 from Cav1 in response to store depletion. Further, conditions that promoted TRPC1-STIM1 clustering and TRPC1-SOCE elicited corresponding changes in SOCE-dependent NFkB activation and cell proliferation. Together these data demonstrate that Cav1 is a critical plasma membrane scaffold for inactive TRPC1. We suggest that activation of TRPC1-SOC by STIM1 mediates release of the channel from Cav1.Store-operated calcium entry (SOCE) is activated by depletion of endoplasmic reticulum (ER) Ca²⁺ stores and regulates a variety of critical cellular functions (1). Ca²⁺ depletion in the ER lumen is detected by the Ca²⁺-binding protein STIM1, which oligomerizes into puncta and relocates to discrete ER-plasma membrane (ER-PM) junctional regions (2, 3) where it associates with and activates store-operated channels including Orai1 and TRPC1, which are components of CRAC and SOC channels, respectively (4–13). Therefore, the location of these channels in the plasma membrane is likely to be critical for their interaction with peripheral STIM1 and activation. However, mechanisms involved in the precise targeting and retention of the channels at the domains where STIM1 puncta are located are not well-understood.Distinct regions of STIM1 determine aggregation and targeting of the protein to ER-PM junctional domains as well as its clustering with and gating of Orai1 and TRPC1 at these sites. The SAM and coiled-coiled domains are involved in STIM1 aggregation while the polybasic C-terminal region of STIM1 is suggested to target STIM1 to ER-PM junctional regions, which is the likely site for SOCE in native cells (3, 9–11, 14). Thus, it can be predicted that SOCs are either localized in this region or in close proximity to it so that they can be readily recruited following store depletion. Clustering of STIM1 in ER-PM junctional regions results in relatively high local concentrations of STIM1 at these sites, which appears to be sufficient to drive the diffusion of Orai1 into this region where it binds to STIM1, resulting in CRAC channel activation (11, 12).STIM1 also associates with and regulates store-operated TRPC channels, including TRPC1-SOC (6–8, 13, 15–19). Several studies show that association between TRPC1 and STIM1 increases following store depletion (6, 16, 19, 20), although a recent study was unable to demonstrate this (21). While the ezrin/radixin/moesin (ERM) domain of STIM1 appears to bind to TRPC1, the C-terminal ⁶⁸⁴KK⁶⁸⁵ residues are involved in gating the channel via electrostatic interaction with TRPC1(⁶³⁹DD⁶⁴⁰) (8, 13). We have reported earlier that lipid raft domains (LRD) provide a platform for regulation of TRPC1-SOCE (22). Further, we demonstrated that peripheral STIM1 puncta are anchored in LRD which facilitates TRPC1-STIM1 association required for activation of TRPC1-SOCE (6). This has now been suggested in several other studies (10, 18, 23). The role of LRD in the regulation of TRPC1-SOC suggested by these recent findings are consistent with previous reports which showed that plasma membrane localization of TRPC1 depends on its association with the cholesterol-binding protein caveolin-1 (Cav1), which promotes retention of TRPC1 within the LRD (6, 18, 22–25). Together, these findings suggest a critical role for Cav1 and LRD in the association of TRPC1 with STIM1 within ER-PM junctional regions that is required for SOCE (10, 26–28). However, the precise molecular interactions involving Cav1, STIM1, and TRPC1 that are triggered by store depletion and are critical for TRPC1-SOCE have not yet been defined.Here we have examined the contribution of Cav1 and STIM1 in the regulation of TRPC1-SOCE that occurs within the ER-PM junctional region in response ER-Ca²⁺ store depletion. We report that Cav1 is a critical plasma membrane scaffold that retains TRPC1-SOC within the ER-PM region where STIM1 puncta are localized following store depletion. Retention of TRPC1 in this region facilitates the interaction of STIM1 with the channel that is required for activation of SOCE. Activation of TRPC1 by STIM1 also releases the channel from Cav1.     

Variations in methanobactin structure influences copper utilization by methane-oxidizing bacteria

Methane-oxidizing bacteria are nature’s primary biological mechanism for suppressing atmospheric levels of the second-most important greenhouse gas via methane monooxygenases (MMOs). The copper-containing particulate enzyme is the most widespread and efficient MMO. Under low-copper conditions methane-oxidizing bacteria secrete the small copper-binding peptide methanobactin (mbtin) to acquire copper, but how variations in the structures of mbtins influence copper metabolism and species selection are unknown. Methanobactins have been isolated from Methylocystis strains M and hirsuta CSC1, organisms that can switch to using an iron-containing soluble MMO when copper is limiting, and the nonswitchover Methylocystis rosea. These mbtins are shorter, and have different amino acid compositions, than the characterized mbtin from Methylosinus trichosporium OB3b. A coordinating pyrazinedione ring in the Methylocystis mbtins has little influence on the Cu(I) site structure. The Methylocystis mbtins have a sulfate group that helps stabilize the Cu(I) forms, resulting in affinities of approximately 10²¹ M^-1. The Cu(II) affinities vary over three orders of magnitude with reduction potentials covering approximately 250 mV, which may dictate the mechanism of intracellular copper release. Copper uptake and the switchover from using the iron-containing soluble MMO to the copper-containing particulate enzyme is faster when mediated by the native mbtin, suggesting that the amino acid sequence is important for the interaction of mbtins with receptors. The differences in structures and properties of mbtins, and their influence on copper utilization by methane-oxidizing bacteria, have important implications for the ecology and global function of these environmentally vital organisms.Copper is an essential protein cofactor involved in many important cellular processes (1, 2), and copper-trafficking systems have been extensively studied (1, 3–8). Although copper uptake by eukaryotes is well defined (1, 4, 9), acquisition of this metal by prokaryotes remains poorly understood. Methane-oxidizing bacteria secrete the small copper-binding molecule methanobactin (mbtin) when copper is limiting (10–18), presumably for sequestration of this metal. These organisms have conditionally high requirements for copper (19), primarily for the active site (20) of the particulate methane monooxygenase (pMMO). Almost all known methane-oxidizing bacteria use pMMO for the consumption of methane (19), an important greenhouse gas. A subclass of “switchover” organisms exists that can also produce a less efficient iron-containing soluble MMO (sMMO) under copper-deficient conditions, with pMMO expression up-regulated in response to an increase in the copper-to-cell ratio (15, 21).Methanobactin production has been examined in a number of methane-oxidizing bacteria (22–24), but mbtins from only two organisms have been characterized (13, 18). The mbtin (two forms) from Methylosinus trichosporium OB3b (a switchover organism) is the most extensively studied (13, 15–17, 25–29), and binds a single copper ion coordinated in a distorted tetrahedral arrangement by the nitrogens from two oxazolone rings (29) and the sulfurs from two enethiolate groups. The molecule has a compact arrangement stabilized by a disulfide bridge. The very high affinities for copper that have been determined for the M. trichosporium OB3b molecules are consistent with mbtins playing a role in the acquisition of copper (17). Direct evidence of uptake and cytoplasmic localization has recently been obtained for Cu(I)-mbtin from M. trichosporium OB3b (30). These studies confirm that mbtin is the primary component of an active copper-acquisition system in methane-oxidizing bacteria. Comparisons have been made (13, 15, 16) between mbtins and iron-sequestering siderophores (31, 32), particularly the structurally related pyoverdines. Whereas detailed information is available for siderophore-mediated iron uptake and utilization, almost nothing is known about how mbtins acquire and deliver copper.In this work mbtins have been isolated and characterized from three Methylocystis strains, including switchover and nonswitchover organisms. All of these mbtins have high Cu(I) affinities that are similar to those of the M. trichosporium OB3b mbtins. The N-terminal group present in the Methylocystis mbtins alters the Cu(II) affinity, which will influence acquisition of the metal, and results in different reduction potentials (E_m values) that could dictate the copper release mechanism. Variations in the structures of mbtins affect their ability to provide copper to methane-oxidizing bacteria and to initiate the transition from using sMMO to pMMO in switchover organisms. We have identified features of mbtins that influence how methane-oxidizing bacteria uptake and utilize copper that may influence their capacity to suppress methane in the natural environment.     

Coronary Calcification in Patients with Chronic Kidney Disease and Coronary Artery Disease

Background and objectives: A close linkage between chronic kidney disease (CKD) and cardiovascular disease (CVD) has been demonstrated. Coronary artery calcification (CAC) is considered to be the causal link connecting them. The aim of the study is to determine the relationship between level of kidney function and the prevalence of CAC.Design, setting, participants, & measurements: Autopsy subjects known to have coronary artery disease and a wide range of kidney function were studied. Patients without CKD were classified into five groups depending on estimated GFR (eGFR) and proteinuria: eGFR ≥60 ml/min/1.73 m² without proteinuria; CKD1/2: eGFR ≥60 ml/min/1.73 m² with proteinuria; CKD3: 60 ml/min/1.73 m² >eGFR ≥30 ml/min/1.73 m²; CKD4/5: eGFR <30 ml/min/1.73 m²; and CKD5D: on hemodialysis. Intimal and medial calcification of the coronary arteries was evaluated. Risk factors for CVD and uremia were identified as relevant to CAC using logistic regression analysis.Results: Intimal calcification of plaques was present in all groups, but was most frequent and severe in the CKD5D group and less so in the CKD4/5 and CKD3 groups. Risk factors included luminal stenosis, age, smoking, diabetes, calcium-phosphorus product, inflammation, and kidney function. Medial calcification was seen in a small number of CKD4/5 and CKD5D groups. Risk factors were use of calcium-containing phosphate binders, hemodialysis treatment, and duration.Conclusions: It was concluded that CAC was present in the intimal plaque of both nonrenal and renal patients. Renal function and traditional risks were linked to initimal calcification. Medial calcification occurred only in CKD patients.Cardiovascular disease (CVD) is the main cause of morbidity and mortality in patients with end-stage renal disease (ESRD) (1,2) or chronic kidney disease (CKD) (3–7). The mechanisms underlying this increased cardiovascular risk are not clearly understood. In the general population, traditional risk factors for CVD have been well characterized (8), and these are also present in CKD (3–6,9). The mechanisms involved in the connection between CKD and CVD are probably numerous (3–6). Vascular calcification, such as coronary artery calcification (CAC) (10,11), is considered to be the causal link between them.Vascular calcification is common in physiologic and pathologic conditions such as aging, diabetes, dyslipidemia, genetic diseases, and diseases with disturbances of calcium metabolism (12–14). In CKD patients, vascular calcification is even more common, developing early and contributing to the markedly increased cardiovascular risk. Pathomorphologically, atherosclerosis (plaque-forming degenerative changes of the aorta and of large elastic arteries) and arteriosclerosis (concentric medial thickening and hyalinosis of muscular arteries) can be distinguished. Increased knowledge about the mechanisms of calcification together with improved imaging techniques have provided evidence that vascular calcification should be divided into two distinct entities according to the specific site of calcification within the vascular wall: plaque calcification, involving patchy calcification of the intima in the vicinity of lipid or cholesterol deposits, and calcification of the media in the absence of such lipid or cholesterol deposits, known as Mönckeberg-type atherosclerosis (12–14). These two types of calcification may vary in terms of the type of vessel affected, the location along the arterial tree (proximal versus distal), clinical presentation, and treatment and prognosis (12–14). In the general population and in patients with CKD, electron-beam computed tomography (EBCT) has proven CAC as a potent predictor of cardiac events (15–18). Both the prevalence and intensity of CAC are increased in patients with CKD (19–27). Several studies have been undertaken to investigate whether calcification occurs in the intima or media of the coronaries and whether the morphologic details of calcified plaques differ between renal and nonrenal patients (12–14,24). Causal elements for either type of CAC have not been definitively determined (12–14).Autopsy studies are limited in terms of patient selection, but have a major advantage in terms of being able to distinguish intimal from medial calcification. Therefore, our primary goal is to determine whether, among autopsy subjects known to have CAD, there exists a direct relationship between level of kidney function and the prevalence of intimal or medial calcification.