Localization of natriuretic peptide receptors A, B, and C in healthy and diseased mouse kidneys.

The natriuretic peptides (NPs) ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide) mediate their widespread effects by activating the natriuretic peptide receptor-A (NPR-A), while C-type natriuretic peptide (CNP) acts via natriuretic peptide receptor-B (NPR-B). NPs are removed from the circulation by internalization via the natriuretic peptide clearance receptor natriuretic peptide receptor-C (NPR-C). In addition to their well-known functions, for instance on blood pressure, all three NPs confer significant cardioprotection and renoprotection. Since neither the NP-mediated renal functions nor the renal target cells of renoprotection are completely understood, we performed systematic localization studies of NP receptors using in situ hybridization (RNAscope) in mouse kidneys. NPR-A mRNA is highly expressed in glomeruli (mainly podocytes), renal arterioles, endothelial cells of peritubular capillaries, and PDGFR-receptor ß positive (PDGFR-ß) interstitial cells. No NPR-A mRNA was detected by RNAscope in the tubular system. In contrast, NPR-B expression is highest in proximal tubules. NPR-C is located in glomeruli (mainly podocytes), in endothelial cells and PDGFR-ß positive cells. To test for a possible regulation of NPRs in kidney diseases, their distribution was studied in adenine nephropathy. Signal intensity of NPR-A and NPR-B mRNA was reduced while their spatial distribution was unaltered compared with healthy kidneys. In contrast, NPR-C mRNA signal was markedly enhanced in cell clusters of myofibroblasts in fibrotic areas of adenine kidneys. In conclusion, the primary renal targets of ANP and BNP are glomerular, vascular, and interstitial cells but not the tubular compartment, while the CNP receptor NPR-B is highly expressed in proximal tubules. Further studies are needed to clarify the function and interplay of this specific receptor expression pattern.

The potential of two different Avena sativa L. cultivars to alleviate Cu toxicity.

Agronomic strategies as intercropping might be applied to reduce plant-available copper (Cu) in Cu-contaminated soils. Thus, our aim was to characterize two different oat cultivars, Avena sativa L. cv. Fronteira and cv. Perona for their ability to tolerate and/or phytostabilize Cu. Copper toxicity reduced plant biomass of both cultivars. The exudate analysis revealed the presence of phenolic compounds and phytosiderophores, yet with a different pattern between the cultivars: cv. Fronteira showed a Cu-concentration and time-dependent release of phenolic compounds, while cv. Perona down-regulated this release during the second week of treatment. Copper concentration increased linearly in all the tissues analysed with increasing Cu concentration showing yet a different compartmentalization: cv. Fronteira and cv. Perona preferentially accumulated Cu in the apoplasm and symplast, respectively. This higher accumulation of Cu in the apoplasm strongly reduces the available binding sites, leading to a competitive absorption with other macro-and micronutrients (e.g. Ca, Mn, Zn). Furthermore, in both cultivars Cu toxicity led to a significant reduction of shoot phosphorus content. The ionomic profile and compartmentalization of Cu together with the root activities demonstrate the different tolerance mechanism towards Cu toxicity of the two oat cultivars. In particular, cv. Fronteira seems to adopt an exclusion strategy based on accumulating Cu in the apoplasm and on the exudation of phenolic compounds. Thus, this cultivar could reduce the mobility and the consequent soil bioavailability of Cu playing an important role as phytostabilizer plant in intercropping systems in Cu-contaminated vineyards or orchards.

Membrane/Water Partition Coefficients of Bile Salts Determined Using Laurdan as a Fluorescent Probe.

The interaction of liposomal membranes composed of soybean phosphatidylcholine with the bile salts (BSs) cholate (Ch), glycocholate (GC), chenodeoxycholate (CDC), and glycochenodeoxycholate (GCDC) was studied. The BSs differed with regard to their lipophilicity, pKa values, and the size of their hydrophilic moiety. Their membrane interactions were investigated using Laurdan as a membrane-anchored fluorescent dye. The apparent membrane/water partition coefficient, D, at pH 7.4 was calculated from binding plots and compared with direct binding measurements using ultracentrifugation as a reference. The Laurdan-derived LogD values at pH 7.4 were found to be 2.10 and 2.25 for the trihydroxy BSs, i.e., Ch and GC, and 2.85 and 2.75 for the dihydroxy BSs, i.e., CDC and GCDC, respectively. For the membrane-associated glycine-conjugated GC and GCDC (pKa values of ∼3.9), no differences in the Laurdan spectra of the respective BS were found at pH 6.8, 7.4, and 8.2. Unconjugated Ch and CDC (pKa values of ∼5.0) showed pronounced differences at the three pH values. Furthermore, the kinetics of membrane adsorption and transbilayer movement differed between conjugated and unconjugated BSs as determined with Laurdan-labeled liposomes.

Two-pore domain potassium channels in the adrenal cortex.

The physiological control of steroid hormone secretion from the adrenal cortex depends on the function of potassium channels. The "two-pore domain K(+) channels" (K2P) TWIK-related acid sensitive K(+) channel 1 (TASK1), TASK3, and TWIK-related K(+) channel 1 (TREK1) are strongly expressed in adrenocortical cells. They confer a background K(+) conductance to these cells which is important for the K(+) sensitivity as well as for angiotensin II and adrenocorticotropic hormone-dependent stimulation of aldosterone and cortisol synthesis. Mice with single deletions of the Task1 or Task3 gene as well as Task1/Task3 double knockout mice display partially autonomous aldosterone synthesis. It appears that TASK1 and TASK3 serve different functions: TASK1 affects cell differentiation and prevents expression of aldosterone synthase in the zona fasciculata, while TASK3 controls aldosterone secretion in glomerulosa cells. TREK1 is involved in the regulation of cortisol secretion in fasciculata cells. These data suggest that a disturbed function of K2P channels could contribute to adrenocortical pathologies in humans.

Connexin 40 is dispensable for vascular renin cell recruitment but is indispensable for vascular baroreceptor control of renin secretion.

Defects of the gap junction protein connexin 40 (Cx40) in renin-secreting cells (RSCs) of the kidney lead to a shift of the localization of RSCs from the media layer of afferent arterioles to the periglomerular interstitium. The dislocation of RSCs goes in parallel with elevated plasma renin levels, impaired pressure control of renin secretion, and hypertension. The reasons for the extravascular shift of RSCs and the blunted pressure regulation of renin secretion caused by the absence of Cx40 are still unclear. We have therefore addressed the question if Cx40 is essential for the metaplastic transformation of preglomerular vascular smooth muscle cells (SMCs) into RSCs and if Cx40 is essential for the pressure control of renin secretion from RSCs located in the media layer of afferent arterioles. For our study, we used mice lacking the angiotensin II type 1A (AT1A) receptors, which display a prominent and reversible salt-sensitive metaplastic transformation of SMCs into RSCs. This mouse line was crossed with Cx40-deficient mice to obtain AT1A and Cx40 double deleted mice. The kidneys of AT1A (-/-)Cx40(-/-) mice kept on normal salt (0.3 %) displayed RSCs both in the inner media layer of preglomerular vessels and in the periglomerular interstitium. In contrast to hypotensive AT1A (-/-) (mean bp syst 112 mmHg) and hypertensive Cx40(-/-) (mean bp syst 160 mmHg) mice AT1A (-/-)Cx40(-/-) mice were normotensive(mean bp syst 130 mmHg). Pressure regulation of renin secretion from isolated kidneys was normal in AT1A (-/-) mice, but was absent in AT1A (-/-)Cx40(-/-) mice alike in Cx40(-/-) mice. Low-salt diet (0.02 %) increased RSC numbers in the media layer, whilst high-salt diet (4 %) caused disappearance of RSCs in the media layer but not in the periglomerular interstitium. Blood pressure was clearly salt sensitive both in AT1A (-/-) and in AT1A (-/-)Cx40(-/-) mice but was shifted to higher pressure values in the latter genotype. Our data indicate that Cx40 is not a requirement for intramural vascular localization of RSCs nor for reversible metaplastic transformation of SMCs into RSCs. Therefore, the ectopic localization of RSCs in Cx40(-/-) kidneys is more likely due to a disturbed intercellular communication rather than being the result of chronic overactivation of the renin-angiotensin-aldosterone system or hypertension. Moreover, our findings suggest that Cx40 is a requirement for the pressure control of renin secretion irrespective of the localization of RSCs.

Function of cGMP-dependent protein kinase II in volume load-induced diuresis.

Atrial natriuretic peptide (ANP)/cGMPs cause diuresis and natriuresis. Their downstream effectors beyond cGMP remain unclear. To elucidate a probable function of cGMP-dependent protein kinase II (cGKII), we investigated renal parameters in different conditions (basal, salt diets, starving, water load) using a genetically modified mouse model (cGKII-KO), but did not detect any striking differences between WT and cGKII-KO. Thus, cGKII is proposed to play only a marginal role in the adjustment of renal concentration ability to varying salt loads without water restriction or starving conditions. When WT mice were subjected to a volume load (performed by application of a 10-mM glucose solution (3% of BW) via feeding needle), they exhibited a potent diuresis. In contrast, urine volume was decreased significantly in cGKII-KO. We showed that AQP2 plasma membrane (PM) abundance was reduced for about 50% in WT upon volume load, therefore, this might be a main cause for the enhanced diuresis. In contrast, cGKII-KO mice almost completely failed to decrease AQP2-PM distribution. This significant difference between both genotypes is not induced by an altered p-Ser256-AQP2 phosphorylation, as phosphorylation at this site decreases similarly in WT and KO. Furthermore, sodium excretion was lowered in cGKII-KO mice during volume load. In summary, cGKII is only involved to a minor extent in the regulation of basal renal concentration ability. By contrast, cGKII-KO mice are not able to handle an acute volume load. Our results suggest that membrane insertion of AQP2 is inhibited by cGMP/cGKII.

Empagliflozin increases kidney weight due to increased cell size in the proximal tubule S3 segment and the collecting duct.

The inhibition of renal SGLT2 glucose reabsorption has proven its therapeutic efficacy in chronic kidney disease. SGLT2 inhibitors (SGLTi) have been intensively studied in rodent models to identify the mechanisms of SGLT2i-mediated nephroprotection. So far, the overwhelming effects from clinical trials, could only partially be reproduced in rodent models of renal injury. However, a commonly disregarded observation from these studies, is the increase in kidney weight after SGLT2i administration. Increased kidney mass often relies on tubular growth in response to reabsorption overload during glomerular hyperfiltration. Since SGLT2i suppress hyperfiltration but concomitantly increase renal weight, it seems likely that SGLT2i have a growth promoting effect on the kidney itself, independent of GFR control. This study aimed to investigate the effect of SGLT2i on kidney growth in wildtype animals, to identify enlarged nephron segments and classify the size increase as hypertrophic/hyperplastic growth or cell swelling. SGLT2i empagliflozin increased kidney weight in wildtype mice by 13% compared to controls, while bodyweight and other organs were not affected. The enlarged nephron segments were identified as SGLT2-negative distal segments of proximal tubules and as collecting ducts by histological quantification of tubular cell area. In both segments protein/DNA ratio, a marker for hypertrophic growth, was increased by 6% and 12% respectively, while tubular nuclei number (hyperplasia) was unchanged by empagliflozin. SGLT2-inhibition in early proximal tubules induces a shift of NaCl resorption along the nephron causing compensatory NaCl and H2O reabsorption and presumably cell growth in downstream segments. Consistently, in collecting ducts of empagliflozin-treated mice, mRNA expression of the Na+-channel ENaC and the H2O-channels Aqp-2/Aqp-3 were increased. In addition, the hypoxia marker Hif1α was found increased in intercalated cells of the collecting duct together with evidence for increased proton secretion, as indicated by upregulation of carbonic anhydrases and acidified urine pH in empagliflozin-treated animals. In summary, these data show that SGLT2i induce cell enlargement by hypertrophic growth and possibly cell swelling in healthy kidneys, probably as a result of compensatory glucose, NaCl and H2O hyperreabsorption of SGLT2-negative segments. Particularly affected are the SGLT2-negative proximal tubules (S3) and the collecting duct, areas of low O2 availability.

Empagliflozin Reduces Renal Hyperfiltration in Response to Uninephrectomy, but Is Not Nephroprotective in UNx/DOCA/Salt Mouse Models.

Large-scale clinical outcome studies demonstrated the efficacy of SGLT2 inhibitors in patients with type II diabetes. Besides their therapeutic efficacy in diabetes, significant renoprotection was observed in non-diabetic patients with chronic kidney disease (CKD), suggesting the existence of glucose-independent beneficial effects of SGLT2 inhibitors. However, the relevant mechanisms by which SGLT2 inhibition delays the progression of renal injury are still largely unknown and speculative. Previous studies showed that SGLT2 inhibitors reduce diabetic hyperfiltration, which is likely a key element in renoprotection. In line with this hypothesis, this study aimed to investigate the nephroprotective effects of the SGLT2 inhibitor empagliflozin (EMPA) in different mouse models with non-diabetic hyperfiltration and progressing CKD to identify the underlying diabetes-independent cellular mechanisms. Non-diabetic hyperfiltration was induced by unilateral nephrectomy (UNx). Since UNx alone does not result in renal damage, renal disease models with varying degrees of glomerular damage and albuminuria were generated by combining UNx with high NaCl diets ± deoxycorticosterone acetate (DOCA) in different mouse strains with and without genetic predisposition for glomerular injury. Renal parameters (GFR, albuminuria, urine volume) were monitored for 4-6 weeks. Application of EMPA via the drinking water resulted in sufficient EMPA plasma concentration and caused glucosuria, diuresis and in some models renal hypertrophy. EMPA had no effect on GFR in untreated wildtype animals, but significantly reduced hyperfiltration after UNx by 36%. In contrast, EMPA did not reduce UNx induced hyperfiltration in any of our kidney disease models, regardless of their degree of glomerular damage caused by DOCA/salt treatment. Consistent with the lack of reduction in glomerular hyperfiltration, EMPA-treated animals developed albuminuria and renal fibrosis to a similar extent as H2O control animals. Taken together, the data clearly indicate that blockade of SGLT2 has the potential to reduce non-diabetic hyperfiltration in otherwise untreated mice. However, no effects on hyperfiltration or progression of renal injury were observed in hypervolemic kidney disease models, suggesting that high salt intake and extracellular volume might attenuate the protective effects of SGLT2 blockers.

An R-Package for the Deconvolution and Integration of 1D NMR Data: MetaboDecon1D.

NMR spectroscopy is a widely used method for the detection and quantification of metabolites in complex biological fluids. However, the large number of metabolites present in a biological sample such as urine or plasma leads to considerable signal overlap in one-dimensional NMR spectra, which in turn hampers both signal identification and quantification. As a consequence, we have developed an easy to use R-package that allows the fully automated deconvolution of overlapping signals in the underlying Lorentzian line-shapes. We show that precise integral values are computed, which are required to obtain both relative and absolute quantitative information. The algorithm is independent of any knowledge of the corresponding metabolites, which also allows the quantitative description of features of yet unknown identity.

Adenovirus-Mimetic Nanoparticles: Sequential Ligand-Receptor Interplay as a Universal Tool for Enhanced In Vitro/In Vivo Cell Identification.

Viral infection patterns often rely on precisely coordinated sequences of distinct ligand-receptor interactions, leading in many cases to an outstanding target cell specificity. A successful mimicry of viral targeting strategies to create more site-specific nanoparticles (NPs) would therefore require particle-cell interactions to also be adequately controllable. In the present study, hetero-multivalent block-copolymer NPs present their attached ligands in a sterically controlled manner to create a sequential NP-cell interaction similar to the cell infiltration strategy of human adenovirus type 2. Targeting renal mesangial cells, particles therefore initially bind angiotensin II receptor type 1 (AT1r) on the cell surface via a structurally flexible AT1r antagonist. After a mandatory spatial approach, particle endocytosis is realized via binding of immobile αVß3 integrins with a previously concealed secondary ligand, thereby creating a stepwise particle-cell interplay of primary NP attachment and subsequent uptake. Manufactured adenovirus-mimetic NPs show great avidity for both target motifs in vitro, leading to a substantial binding as well as subsequent cell uptake into target mesangial cells. Additionally, steric shielding of secondary ligand visibility leads to a highly controllable, sequential ligand-receptor interaction, whereby hetero-functional NPs activate mesangial cell surface integrins only after a successful prior binding to the AT1r. This stepwise cell identification significantly enhances mesangial cell specificity in co-culture assays with different off-target cells. Additionally, described NPs display excellent in vivo robustness by efficiently accumulating in the mesangium upon injection, thereby opening new paths for possible drug delivery applications.

Nanoparticles Mimicking Viral Cell Recognition Strategies Are Superior Transporters into Mesangial Cells.

Poor drug availability in the tissue of interest is a frequent cause of therapy failure. While nanotechnology has developed a plethora of nanocarriers for drug transport, their ability to unequivocally identify cells of interest remains moderate. Viruses are the ideal nanosized carriers as they are able to address their embedded nucleic acids with high specificity to their host cells. Here, it is reported that particles endowed with a virus-like ability to identify cells by three consecutive checks have a superior ability to recognize mesangial cells (MCs) in vivo compared to conventional nanoparticles. Mimicking the initial viral attachment followed by a stepwise target cell recognition process leads to a 5- to 15-fold higher accumulation in the kidney mesangium and extensive cell uptake compared to particles lacking one or both of the viral traits. These results highlight the relevance that the viral cell identification process has on specificity and its application on the targeting strategies of nanomaterials. More so, these findings pave the way for transporting drugs into the mesangium, a tissue that is pivotal in the development of diabetic nephropathy and for which currently no efficient pharmacotherapy exists.

Cellular Pathophysiology of an Adrenal Adenoma-Associated Mutant of the Plasma Membrane Ca(2+)-ATPase ATP2B3.

Adrenal aldosterone-producing adenomas (APAs) are a main cause for primary aldosteronism leading to arterial hypertension. Physiologically, aldosterone production in the adrenal gland is stimulated by angiotensin II and high extracellular potassium. These stimuli lead to a depolarization of the plasma membrane and, as a consequence, an increase of intracellular Ca(2+). Mutations of the plasma membrane Ca(2+)-ATPase ATP2B3 have been found in APAs with a prevalence of 0.6%-3.1%. Here, we investigated the effects of the APA-associated ATP2B3(Leu425_Val426del) mutation in adrenocortical NCI-H295R and human embryonic kidney (HEK-293) cells. Ca(2+) measurements revealed a higher basal Ca(2+) level in cells expressing the mutant ATP2B3. This rise in intracellular Ca(2+) was even more pronounced under conditions with high extracellular Ca(2+) pointing to an increased Ca(2+) influx associated with the mutated protein. Furthermore, cells with the mutant ATP2B3 appeared to have a reduced capacity to export Ca(2+) suggesting a loss of the physiological pump function. Surprisingly, expression of the mutant ATP2B3 caused a Na(+)-dependent inward current that strongly depolarized the plasma membrane and compromised the cytosolic cation composition. In parallel to these findings, mRNA expression of the cytochrome P450, family 11, subfamily B, polypeptide 2 (aldosterone synthase) was substantially increased and aldosterone production was enhanced in cells overexpressing mutant ATP2B3. In summary, the APA-associated ATP2B3(Leu425_Val426del) mutant promotes aldosterone production by at least 2 different mechanisms: 1) a reduced Ca(2+) export due to the loss of the physiological pump function; and 2) an increased Ca(2+) influx due to opening of depolarization-activated Ca(2+) channels as well as a possible Ca(2+) leak through the mutated pump.

Severe hyperaldosteronism in neonatal Task3 potassium channel knockout mice is associated with activation of the intraadrenal renin-angiotensin system.

Task3 K(+) channels are highly expressed in the adrenal cortex and contribute to the angiotensin II and K(+) sensitivity of aldosterone-producing glomerulosa cells. Adult Task3(-/-) mice display a partially autonomous aldosterone secretion, subclinical hyperaldosteronism, and salt-sensitive hypertension. Here, we investigated the age dependence of the adrenal phenotype of Task3(-/-) mice. Compared with adults, newborn Task3(-/-) mice displayed a severe adrenal phenotype with strongly increased plasma levels of aldosterone, corticosterone, and progesterone. This adrenocortical dysfunction was accompanied by a modified gene expression profile. The most strongly up-regulated gene was the protease renin. Real-time PCR corroborated the strong increase in adrenal renin expression, and immunofluorescence revealed renin-expressing cells in the zona fasciculata. Together with additional factors, activation of the local adrenal renin system is probably causative for the severely disturbed steroid hormone secretion of neonatal Task3(-/-) mice. The changes in gene expression patterns of neonatal Task3(-/-) mice could also be relevant for other forms of hyperaldosteronism.

Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension.

Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na(+)/K(+) ATPase α subunit) and ATP2B3 (encoding a Ca(2+) ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation-positive cases showed male dominance, increased plasma aldosterone concentrations and lower potassium concentrations compared with mutation-negative cases. In summary, dominant somatic alterations in two members of the ATPase gene family result in autonomous aldosterone secretion.

Task3 potassium channel gene invalidation causes low renin and salt-sensitive arterial hypertension.

Task1 and Task3 potassium channels (Task: tandem of P domains in a weak inward rectifying K(+) channel-related acid-sensitive K(+) channel) are believed to control the membrane voltage of aldosterone-producing adrenal glomerulosa cells. This study aimed at understanding the role of Task3 for the control of aldosterone secretion. The adrenal phenotype of Task3(-/-) mice was investigated using electrophysiology, adrenal slices, and blood pressure measurements. Primary adrenocortical cells of Task3(-/-) mice were strongly depolarized compared with wild-type (-52 vs. -79 mV), and in fresh adrenal slices Ca(2+) signaling of Task3(-/-) glomerulosa cells was abnormal. In living Task3(-/-) mice, the regulation of aldosterone secretion showed specific deficits: Under low Na(+) and high K(+) diets, protocols known to increase aldosterone, and under standard diet, Task3 inactivation was compensated and aldosterone was normal. However, high Na(+) and low K(+) diets, two protocols known to lower aldosterone, failed to lower aldosterone in Task3(-/-) mice. The physiological regulation of aldosterone was disturbed: aldosterone-renin ratio, an indicator of autonomous aldosterone secretion, was 3-fold elevated at standard and high Na(+) diets. Isolated adrenal glands of Task3(-/-) produced 2-fold more aldosterone. As a consequence, Task3(-/-) mice showed salt-sensitive arterial hypertension (plus 10 mm Hg). In conclusion, Task3 plays an important role in the adaptation of aldosterone secretion to dietary salt intake.

KCNJ5 mutations in European families with nonglucocorticoid remediable familial hyperaldosteronism.

Primary aldosteronism is the most frequent cause of endocrine hypertension. Three forms of familial hyperaldosteronism (FH) have been described, named FH-I to -III. Recently, a mutation of KCNJ5 has been shown to be associated with FH-III, whereas the cause of FH-II is still unknown. In this study we searched for mutations in KCNJ5 in 46 patients from 21 families with FH, in which FH-I was excluded. We identified a new germline G151E mutation in 2 primary aldosteronism-affected subjects from an Italian family and 3 somatic mutations in aldosterone-producing adenomas, T158A described previously as a germline mutation associated with FH-III, and G151R and L168R both described as somatic mutations in aldosterone-producing adenoma. The phenotype of the family with the G151E mutation was remarkably milder compared with the previously described American family, in terms of both clinical and biochemical parameters. Furthermore, patients with somatic KCNJ5 mutations displayed a phenotype indistinguishable from that of sporadic primary aldosteronism. The functional characterization of the effects of the G151E mutation in vitro showed a profound alteration of the channel function, with loss of K(+) selectivity, Na(+) influx, and membrane depolarization. These alterations have been postulated to be responsible for voltage gate Ca(2+) channel activation, increase in cytosolic calcium, and stimulation of aldosterone production and adrenal cell proliferation. In conclusion, we describe herein a new mutation in the KCNJ5 potassium channel associated with FH-III, responsible for marked alterations of channel function but associated with a mild clinical and hormonal phenotype.

Insect cells for antibody production: evaluation of an efficient alternative.

In recent years there has been an increase in both availability and demand for therapeutic monoclonal antibodies. Currently, most of these antibodies are produced by stably transfected mammalian cells. In this study we evaluated the use of different baculoviral insect cell systems as an alternative for commonly used production schemes. We expressed the human anti-gp41 antibody 3D6 in Spodoptera frugiperda Sf9, Trichoplusia ni BTI-TN5B1-4 "High Five", and Spodoptera frugiperda SfSWT-1 "Mimic™" insect cells and compared product yield, specificity and glycosylation patterns with a 3D6 antibody expressed in Chinese hamster ovary cells. Using "High Five" cells we achieved amounts of secreted antibody comparable to those resulting from transient expression in mammalian cells. We determined the N-linked oligosaccharide structures present on asparagine-297 in IgG1 heavy chains and tested the functionality in terms of antigen binding and the ability to elicit effector functions. Antibodies expressed in all insect cell lines displayed highly specific antigen binding. In general, the insect-produced antibodies carried, as the CHO-produced form, fucosylated N-glycans, including, in the case of "High Five" cells, high levels of core α1,3-fucose. This indicates that in all systems glycoengineering may be required in order to produce optimal glycoforms of this antibody.