Insulin use in chronic kidney disease and the risk of hypoglycemic events.

BACKGROUND: We examined in persons with type 2 diabetes (T2D) whether the use of insulin and the risk of serious hypoglycemic events with insulin is higher in persons with more advanced CKD. METHODS: In a national cohort of 855,133 veterans with T2D seen at Veteran Affairs clinics between Jan 1, 2008 and December 31, 2010 with at least two serum creatinine measurements, we defined insulin use from pharmacy records and serious hypoglycemic events by ICD-9/10 codes from emergency room visits or hospitalizations that occurred until December 31, 2016. RESULTS: Mean age was 66 ± 11 years and 97% were men. Mean baseline eGFR was 73 ± 22 ml/min/1.73 m2. In a multivariable Cox regression model of those without insulin use at baseline (N = 653,200), compared to eGFR ≥90 group, eGFR < 30 group had higher hazard (HR 1.80, 95% CI 1.74 to 1.88) of subsequent insulin use. In a multivariable Cox model with propensity score matching for baseline insulin use (N = 305,570), both insulin use (HR 2.34, 95% CI 2.24 to 2.44) and advanced CKD (HR 2.28, 95% CI 2.07 to 2.51 for comparison of eGFR < 30 to eGFR ≥90 ml/min/1.73 m2 groups) were associated with increased risk of subsequent serious hypoglycemic events. CONCLUSIONS AND RELEVANCE: In T2D, more advanced CKD was associated with greater insulin use. Both insulin use and advanced CKD were risk factors for serious hypoglycemic events. The safety of insulin compared to newer glycemic agents in more advanced CKD needs further study.

Structure and allosteric activity of a single-disulfide conopeptide from Conus zonatus at human α3ß4 and α7 nicotinic acetylcholine receptors.

Conopeptides are neurotoxic peptides in the venom of marine cone snails and have broad therapeutic potential for managing pain and other conditions. Here, we identified the single-disulfide peptides Czon1107 and Cca1669 from the venoms of Conus zonatus and Conus caracteristicus, respectively. We observed that Czon1107 strongly inhibits the human α3ß4 (IC50 15.7 ± 3.0 µm) and α7 (IC50 77.1 ± 0.05 µm) nicotinic acetylcholine receptor (nAChR) subtypes, but the activity of Cca1669 remains to be identified. Czon1107 acted at a site distinct from the orthosteric receptor site. Solution NMR experiments revealed that Czon1107 exists in equilibrium between conformational states that are the result of a key Ser4-Pro5cis-trans isomerization. Moreover, we found that the X-Pro amide bonds in the inter-cysteine loop are rigidly constrained to cis conformations. Structure-activity experiments of Czon1107 and its variants at positions P5 and P7 revealed that the conformation around the X-Pro bonds (cis-trans) plays an important role in receptor subtype selectivity. The cis conformation at the Cys6-Pro7 peptide bond was essential for α3ß4 nAChR subtype allosteric selectivity. In summary, we have identified a unique single-disulfide conopeptide with a noncompetitive, potentially allosteric inhibitory mechanism at the nAChRs. The small size and rigidity of the Czon1107 peptide could provide a scaffold for rational drug design strategies for allosteric nAChR modulation. This new paradigm in the "conotoxinomic" structure-function space provides an impetus to screen venom from other Conus species for similar, short bioactive peptides that allosterically modulate ligand-gated receptor function.

Potency- and Selectivity-Enhancing Mutations of Conotoxins for Nicotinic Acetylcholine Receptors Can Be Predicted Using Accurate Free-Energy Calculations.

Nicotinic acetylcholine receptor (nAChR) subtypes are key drug targets, but it is challenging to pharmacologically differentiate between them because of their highly similar sequence identities. Furthermore, α-conotoxins (α-CTXs) are naturally selective and competitive antagonists for nAChRs and hold great potential for treating nAChR disorders. Identifying selectivity-enhancing mutations is the chief aim of most α-CTX mutagenesis studies, although doing so with traditional docking methods is difficult due to the lack of α-CTX/nAChR crystal structures. Here, we use homology modeling to predict the structures of α-CTXs bound to two nearly identical nAChR subtypes, α3ß2 and α3ß4, and use free-energy perturbation (FEP) to re-predict the relative potency and selectivity of α-CTX mutants at these subtypes. First, we use three available crystal structures of the nAChR homologue, acetylcholine-binding protein (AChBP), and re-predict the relative affinities of twenty point mutations made to the α-CTXs LvIA, LsIA, and GIC, with an overall root mean square error (RMSE) of 1.08 ± 0.15 kcal/mol and an R2 of 0.62, equivalent to experimental uncertainty. We then use AChBP as a template for α3ß2 and α3ß4 nAChR homology models bound to the α-CTX LvIA and re-predict the potencies of eleven point mutations at both subtypes, with an overall RMSE of 0.85 ± 0.08 kcal/mol and an R2 of 0.49. This is significantly better than the widely used molecular mechanics-generalized born/surface area (MM-GB/SA) method, which gives an RMSE of 1.96 ± 0.24 kcal/mol and an R2 of 0.06 on the same test set. Next, we demonstrate that FEP accurately classifies α3ß2 nAChR selective LvIA mutants while MM-GB/SA does not. Finally, we use FEP to perform an exhaustive amino acid mutational scan of LvIA and predict fifty-two mutations of LvIA to have greater than 100X selectivity for the α3ß2 nAChR. Our results demonstrate the FEP is well-suited to accurately predict potency- and selectivity-enhancing mutations of α-CTXs for nAChRs and to identify alternative strategies for developing selective α-CTXs.

Collecting duct principal, but not intercalated, cell prorenin receptor regulates renal sodium and water excretion.

The collecting duct is the predominant nephron site of prorenin and prorenin receptor (PRR) expression. We previously demonstrated that the collecting duct PRR regulates epithelial Na+ channel (ENaC) activity and water transport; however, which cell type is involved remains unclear. Herein, we examined the effects of principal cell (PC) or intercalated cell (IC) PRR deletion on renal Na+ and water handling. PC or IC PRR knockout (KO) mice were obtained by crossing floxed PRR mice with mice harboring Cre recombinase under the control of the AQP2 or B1 subunit of the H+ ATPase promoters, respectively. PC KO mice had reduced renal medullary ENaC-α abundance and increased urinary Na+ losses on a low-Na+ diet compared with controls. Conversely, IC KO mice had no apparent differences in Na+ balance or ENaC abundance compared with controls. Acute treatment with prorenin increased ENaC channel number and open probability in acutely isolated cortical collecting ducts from control and IC PRR KO, but not PC PRR KO, mice. Furthermore, compared with controls, PC KO, but not IC KO mice, had increased urine volume, reduced urine osmolality, and reduced abundance of renal medullary AQP2. Taken together, these findings indicate that PC, but not IC, PRR modulates ENaC activity, urinary Na+ excretion, and water transport.

Neuronal Nicotinic Acetylcholine Receptor Modulators from Cone Snails.

Marine cone snails are a large family of gastropods that have evolved highly potent venoms for predation and defense. The cone snail venom has exceptional molecular diversity in neuropharmacologically active compounds, targeting a range of receptors, ion channels, and transporters. These conotoxins have helped to dissect the structure and function of many of these therapeutically significant targets in the central and peripheral nervous systems, as well as unravelling the complex cellular mechanisms modulated by these receptors and ion channels. This review provides an overview of α-conotoxins targeting neuronal nicotinic acetylcholine receptors. The structure and activity of both classical and non-classical α-conotoxins are discussed, along with their contributions towards understanding nicotinic acetylcholine receptor (nAChR) structure and function.

Renal tubular epithelial cell prorenin receptor regulates blood pressure and sodium transport.

The physiological significance of the renal tubular prorenin receptor (PRR) has been difficult to elucidate due to developmental abnormalities associated with global or renal-specific PRR knockout (KO). We recently developed an inducible renal tubule-wide PRR KO using the Pax8/LC1 transgenes and demonstrated that disruption of renal tubular PRR at 1 mo of age caused no renal histological abnormalities. Here, we examined the role of renal tubular PRR in blood pressure (BP) regulation and Na(+) excretion and investigated the signaling mechanisms by which PRR regulates Na(+) balance. No detectable differences in BP were observed between control and PRR KO mice fed normal- or low-Na(+) diets. However, compared with controls, PRR KO mice had elevated plasma renin concentration and lower cumulative Na(+) balance with normal- and low-Na(+) intake. PRR KO mice had an attenuated hypertensive response and reduced Na(+) retention following angiotensin II (ANG II) infusion. Furthermore, PRR KO mice had significantly lower epithelial Na(+) channel (ENaC-α) expression. Treatment with mouse prorenin increased, while PRR antagonism decreased, ENaC activity in isolated split-open collecting ducts (CD). The prorenin effect was prevented by protein kinase A and Akt inhibition, but unaffected by blockade of AT1, ERK1/2, or p38 MAPK pathways. Taken together, these data indicate that renal tubular PRR, likely via direct prorenin/renin stimulation of PKA/Akt-dependent pathways, stimulates CD ENaC activity. Absence of renal tubular PRR promotes Na(+) wasting and reduces the hypertensive response to ANG II.

α-Conotoxin dendrimers have enhanced potency and selectivity for homomeric nicotinic acetylcholine receptors.

Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3ß2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.

MrIC, a novel α-conotoxin agonist in the presence of PNU at endogenous α7 nicotinic acetylcholine receptors.

α-Conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Their high selectivity and affinity for the various subtypes of nAChRs have led to significant advances in our understanding of the structure and function of these key ion channels. Here we report the discovery of a novel 4/7 α-conotoxin, MrIC from the venom duct of Conus marmoreus, which acts as an agonist at the endogenous human α7 nAChR in SH-SY5Y cells pretreated with PNU120596 (PNU). This unique agonist activity of MrIC at α7 nAChRs may guide the development of novel α7 nAChR modulators.

Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors.

αD-conotoxins are 11 kDa homodimers that potently inhibit nicotinic acetylcholine receptors (nAChRs) through a non-competitive (allosteric) mechanism. In this study, we describe the allosteric binding mode of the granulin-like C-terminal (CTD) of VxXXB bound to Lymnea stagnalis acetylcholine binding protein (Ls-AChBP), a soluble homologue of the extracellular ligand-binding domain of nAChRs. This co-crystal complex revealed a novel allosteric binding site for nAChR antagonists outside the C-loop that caps the orthosteric site defined by the nAChR agonist nicotine and the antagonist epibatidine. Mutational and docking studies on Ls-AChBP supported a two-site binding mode for full-length VxXXB, with the first CTD binding site located outside the C-loop as seen in the co-crystal complex, with a second CTD binding site located near the N-terminal end of the adjacent subunit of AChBP. These results provide new structural insight into a novel allosteric mechanism of nAChR inhibition and define the cooperative binding mode of the N-terminal domain linked granulin core domains of αD-conotoxins.

Predicting hospitalization from real-world measures in patients with chronic kidney disease: A proof-of-principle study.

Objective: To investigate if in-clinic measures of physical function and real-world measures of physical behavior and mobility effort are associated with one another and to determine if they predict future hospitalization in participants with chronic kidney disease (CKD). Methods: In this secondary analysis, novel real-world measures of physical behavior and mobility effort, including the best 6-minute step count (B6SC), were derived from passively collected data from a thigh worn actigraphy sensor and compared to traditional in-clinic measures of physical function (e.g. 6-minute walk test (6MWT). Hospitalization status during 2 years of follow-up was determined from electronic health records. Correlation analyses were used to compare measures and Cox Regression analysis was used to compare measures with hospitalization. Results: One hundred and six participants were studied (69 ± 13 years, 43% women). Mean ± SD baseline measures for 6MWT was 386 ± 66 m and B6SC was 524 ± 125 steps. Forty-four hospitalization events over 224 years of total follow-up occurred. Good separation was achieved for tertiles of 6MWT, B6SC and steps/day for hospitalization events. This pattern persisted in models adjusted for demographics (6MWT: HR = 0.63 95% CI 0.43-0.93, B6SC: HR = 0.75, 95% CI 0.56-1.02 and steps/day: HR = 0.75, 95% CI 0.50-1.13) and further adjusted for morbidities (6MWT: HR = 0.54, 95% CI 0.35-0.84, B6SC: HR = 0.70, 95% CI 0.49-1.00 and steps/day: HR = 0.69, 95% CI 0.43-1.09). Conclusion: Digital health technologies can be deployed remotely, passively, and continuously to collect real-world measures of physical behavior and mobility effort that differentiate risk of hospitalization in patients with CKD.

An intervention to decrease sedentary behavior in older adults: A secondary analysis of a randomized controlled trial.

Background: Sedentary behaviors are associated with adverse health outcomes in older adults. The feasibility of behavioral interventions in this population is unclear. Methods: In the Sit Less, Interact, Move More (SLIMM) trial of 106 participants who had obesity, those randomized to the SLIMM intervention (N = 54) were instructed to replace sedentary activities with stepping. An accelerometer was used to measure physical activity. In this secondary analysis, mixed effect models were used to examine the effects of the SLIMM intervention on sedentary and stepping durations and steps/day by age (<70 and ≥ 70 years). Results: Mean ages in the <70 years (N = 47) and ≥70 years (N = 59) groups were 58 ± 11 and 78 ± 5. In the older subgroup, compared to standard-of-care (N = 29), the SLIMM intervention (N = 30) significantly increased stepping duration (13, 95%CI 1-24 min/d, p = 0.038) and steps per day (1330, 95% CI 322-2338, p = 0.01) and non-significantly decreased sedentary duration by (28,95% CI -61-5 min/d, p = 0.09). In the age <70 subgroup, there was no separation between the standard of care (N = 23) and SLIMM (N = 24) groups. Discussion: In older adults who had obesity, SLIMM intervention significantly increased stepping duration and steps per day. Interventions targeting sedentary behaviors by promoting low intensity physical activity may be feasible in this population.

Synthesis of full-length homodimer αD-VxXXB that targets human α7 nicotinic acetylcholine receptors.

αD-Conotoxin VxXXB is a pseudo-homodimer that allosterically inhibits nicotinic acetylcholine receptors (nAChRs) with high potency and selectivity. However, challenges in synthesizing αD-conotoxins have hindered further structure-function studies on this novel class of peptides. To address this gap, we synthesized and characterized its C-terminal domain (CTD) and N-terminal domain (NTD). The CTD inhibited α7 nAChRs (IC50 of 23 nM, measured via FLIPR assays) and bound at the acetylcholine binding protein (Ls-AChBP) through an allosteric binding mode determined from radioligand binding assays. The anti-parallel dimeric NTD synthesised via a regioselective strategy also inhibited α7 nAChRs but with reduced potency (IC50 of 30 µM). The α-ketoacid-hydroxylamine (KAHA) method generated CTD linked to the NTD (VxXXB-NC; α7 IC50 of 27 nM) and full-length synthetic VxXXB variant (α7 IC50 of 11 nM), while the three other native chemical ligation approaches proved unsuccessful. This work underpins further characterisation of the structural components contributing to αD-conotoxin affinity, selectivity and allosteric inhibition of nAChR function that may prove useful in the development of new treatments for nAChR-related disorders.

Influence of Baseline Diastolic Blood Pressure on the Effects of Intensive Systolic Blood Pressure Lowering on the Risk of Stroke.

BACKGROUND: Guidelines recommend lowering systolic blood pressure below 130 mm Hg, irrespective of previous strokes. However, there is a concern that lowering systolic blood pressure in people with low baseline diastolic blood pressure might increase the risk of stroke. METHODS: We conducted a secondary analysis of the Secondary Prevention of Small Subcortical Strokes trial that randomly assigned participants with a history of subcortical strokes to an intensive (<130 mm Hg; N=1519) or standard (130-149 mm Hg; N=1501) systolic targets. We examined the effects of blood pressure intervention on stroke and cardiovascular composite across the range of baseline diastolic blood pressure in spline regression models and tested for interaction of baseline diastolic blood pressure with the intervention on outcomes. RESULTS: Mean baseline systolic and diastolic blood pressures were 143±19 and 78±11 mm Hg, respectively. Within each baseline diastolic blood pressure tertile, the achieved diastolic was lower in the intensive versus standard arm. There were 275 stroke events over 10 889 years of follow-up. Lower baseline diastolic blood pressure was associated with increased risk of stroke in an observational spline regression model. Hazard ratios relating blood pressure intervention with the risk of stroke in the lowest (hazard ratio, 0.78 [95% CI, 0.52-1.16]) and the highest (hazard ratio, 0.80 [95% CI, 0.53-1.21]) baseline diastolic tertiles were similar (P=0.95). Results were similar for the cardiovascular composite. CONCLUSIONS: Intensive systolic control does not appear to increase the risk of stroke in those with low baseline diastolic blood pressure and prior stroke. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT00059306.

Cysteine-Rich α-Conotoxin SII Displays Novel Interactions at the Muscle Nicotinic Acetylcholine Receptor.

α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 310-helix frequently employed by framework I α-conotoxins; α-SII acted at the muscle nAChR with half-maximal inhibitory concentrations (IC50) of 120 nM (adult) and 370 nM (fetal) though weakly at neuronal nAChRs. Truncation of α-SII to a two disulfide bond amidated peptide with framework I disulfide connectivity led to similar activity. Surprisingly, the more constrained α-SII was less stable under mild reducing conditions and displayed a unique docking mode at the nAChR.

Unique Pharmacological Properties of α-Conotoxin OmIA at α7 nAChRs.

OmIA, isolated from Conus omaria venom, is a potent antagonist at α7 nAChRs. We determined the co-crystal structure of OmIA with Lymnae stagnalis acetylcholine binding protein (Ls-AChBP) that identified His5, Val10 and Asn11 as key determinants for the high potency of OmIA at α7 nAChRs. Remarkably, despite a competitive binding mode observed in the co-crystal structure, OmIA and analogues displayed functional insurmountable antagonism at α7 and α3ß4 nAChRs, except OmIA analogues having long side chain at position 10 ([V10Q]OmIA and [V10L]OmIA), which were partial insurmountable antagonist at α7 nAChRs in the presence of type II positive allosteric modulators (PAMs). A "two-state, two-step" model was used to explain these observations, with [V10Q]OmIA and [V10L]OmIA co-existing in a fast reversible/surmountable as well as a tight binding/insurmountable state. OmIA and analogues also showed biphasic-inhibition at α7 nAChRs in the presence of PNU120596, with a preference for the high-affinity binding site following prolonged exposure. The molecular basis of binding and complex pharmacological profile of OmIA at α7 nAChRs presented in here expands on the potential of α-conotoxins to probe the pharmacological properties of nAChRs and may help guide the development novel α7 modulators.

Rigidity of loop 1 contributes to equipotency of globular and ribbon isomers of α-conotoxin AusIA.

α-Conotoxins are small disulfide-rich peptides targeting nicotinic acetylcholine receptors (nAChRs) characterised by a CICII-Xm-CIII-Xn-CIV framework that invariably adopt the native globular conformations which is typically most potent. α-Conotoxins are divided into several structural subgroups based on the number of residues within the two loops braced by the disulfide bonds (m/n), with the 4/7 and 4/3 subgroups dominating. AusIA is a relatively rare α5/5-conotoxin isolated from the venom of Conus australis. Surprisingly, the ribbon isomer displayed equipotency to the wild-type globular AusIA at human α7-containing nAChR. To understand the molecular basis for equipotency, we determined the co-crystal structures of both isomers at Lymnea stagnalis acetylcholine binding protein. The additional residue in the first loop of AusIA was found to be a critical determinant of equipotency, with 11-fold and 86-fold shifts in potency in favour of globular AusIA over ribbon AusIA observed following deletion of Ala4 or Arg5, respectively. This divergence in the potency between globular AusIA and ribbon AusIA was further enhanced upon truncation of the non-conserved Val at the C-termini. Conversely, equipotency could be replicated in LsIA and TxIA [A10L] following insertion of an Ala in the first loop. These findings provide a new understanding of the role the first loop in ribbon and globular α-conotoxins can play in directing α-conotoxin nAChR pharmacology.

Increased frequency of pneumothorax and pneumomediastinum in COVID-19 patients admitted in the ICU: A multicentre study from Mumbai, India.

BACKGROUND: There are limited data regarding the incidence of pneumothorax in COVID-19 patients as well as the impact of the same on patient outcomes. METHODS: A retrospective review of the medical records at three large tertiary care hospitals in Mumbai was performed to identify patients hospitalised with COVID-19 from March 2020 to October 2020. The presence of pneumothorax and/or pneumomediastinum was noted when chest radiographs or CT scans were performed. Demographic and clinical characteristics of patients who developed air leak were recorded. RESULTS: 4,906 patients with COVID-19 were admitted, with 1,324 (27%) having severe COVID-19 disease. The overall incidence of pneumothorax and/or pneumomediastinum in patients with severe disease was 3.2% (42/1,324). Eighteen patients had pneumothorax, 16 had pneumomediastinum and 8 patients had both. Fourteen patients (33.3%) developed this complication breathing spontaneously, 28 patients (66.6%) developed it during mechanical ventilation. Overall mortality in this cohort was 74%, compared with 17% in the COVID-19 patients without pneumothorax (p<0.001). CONCLUSIONS: Our study demonstrates that air leaks occur with a higher frequency in patients with COVID-19 than in other ICU patients. When present, such air leaks contributed to poor outcomes with almost 74% mortality rates in these patients.

Structure-Function of Neuronal Nicotinic Acetylcholine Receptor Inhibitors Derived From Natural Toxins.

Neuronal nicotinic acetylcholine receptors (nAChRs) are prototypical cation-selective, ligand-gated ion channels that mediate fast neurotransmission in the central and peripheral nervous systems. nAChRs are involved in a range of physiological and pathological functions and hence are important therapeutic targets. Their subunit homology and diverse pentameric assembly contribute to their challenging pharmacology and limit their drug development potential. Toxins produced by an extensive range of algae, plants and animals target nAChRs, with many proving pivotal in elucidating receptor pharmacology and biochemistry, as well as providing templates for structure-based drug design. The crystal structures of these toxins with diverse chemical profiles in complex with acetylcholine binding protein (AChBP), a soluble homolog of the extracellular ligand-binding domain of the nAChRs and more recently the extracellular domain of human α9 nAChRs, have been reported. These studies have shed light on the diverse molecular mechanisms of ligand-binding at neuronal nAChR subtypes and uncovered critical insights useful for rational drug design. This review provides a comprehensive overview and perspectives obtained from structure and function studies of diverse plant and animal toxins and their associated inhibitory mechanisms at neuronal nAChRs.