Glycemic response to meals with a high glycemic index differs between morning and evening: a randomized cross-over controlled trial among students with early or late chronotype.

PURPOSE: Glycemic response to the same meal depends on daytime and alignment of consumption with the inner clock, which has not been examined by individual chronotype yet. This study examined whether the 2-h postprandial and 24-h glycemic response to a meal with high glycemic index (GI) differ when consumed early or late in the day among students with early or late chronotype. METHODS: From a screening of 327 students aged 18-25 years, those with early (n = 22) or late (n = 23) chronotype participated in a 7-day randomized controlled cross-over intervention study. After a 3-day observational phase, standardized meals were provided on run-in/washout (days 4 and 6) and intervention (days 5 and 7), on which participants received a high GI meal (GI = 72) in the morning (7 a.m.) or in the evening (8 p.m.). All other meals had a medium GI. Continuous glucose monitoring was used to measure 2-h postprandial and 24-h glycemic responses and their variability. RESULTS: Among students with early chronotype 2-h postprandial glucose responses to the high GI meal were higher in the evening than in the morning (iAUC: 234 (± 92) vs. 195 (± 91) (mmol/L) × min, p = 0.042). Likewise, mean and lowest 2-h postprandial glucose values were higher when the high GI meal was consumed in the evening (p < 0.001; p = 0.017). 24-h glycemic responses were similar irrespective of meal time. Participants with late chronotype consuming a high GI meal in the morning or evening showed similar 2-h postprandial (iAUC: 211 (± 110) vs. 207 (± 95) (mmol/L) × min, p = 0.9) and 24-h glycemic responses at both daytimes. CONCLUSIONS: Diurnal differences in response to a high GI meal are confined to those young adults with early chronotype, whilst those with a late chronotype seem vulnerable to both very early and late high GI meals. Registered at clinicaltrials.gov (NCT04298645; 22/01/2020).

The phosphorylation site T613 in the ß-subunit of rat epithelial Na+ channel (ENaC) modulates channel inhibition by Nedd4-2.

The epithelial Na+ channel (ENaC) is a heteromeric channel composed of three subunits (α, ß, γ). At the C-terminus of each subunit, a PY-motif allows binding of the ubiquitin ligase Nedd4-2 which plays a key role in promoting ENaC retrieval from the plasma membrane. Phosphorylation of Nedd4-2 by the serum and glucocorticoid-inducible kinase 1 (Sgk1) reduces Nedd4-2 binding to the PY-motifs. In ß and γENaC, threonine residues (ßT613, γT623) belong to an extracellular signal-regulated kinase (ERK) motif and directly precede the PY-motifs. Thus, phosphorylation of these residues may modulate the interaction of their adjacent PY-motifs with Nedd4-2. In this study, a phosphospecific antibody was used to demonstrate phosphorylation of ßT613 in Xenopus laevis oocytes heterologously expressing rat αßγENaC. Treating the oocytes with progesterone to stimulate ERK increased phosphorylation of ßT613. Inactivation of the putative phosphorylation sites by mutating both threonine residues to alanine (ßT613A/γT623A) increased ENaC-mediated amiloride-sensitive whole-cell currents (ΔIami) and expression of ßENaC at the cell surface. Co-expression of Nedd4-2 largely reduced ΔIami in oocytes expressing αßγENaC or channels with mutated PY-motifs in α and γENaC or in α and ßENaC. Importantly, the inhibitory effect of co-expressed Nedd4-2 was largely reduced in channels with mutated PY-motifs in α and γENaC when combined with the ßT613A mutation but conserved in channels with mutated PY-motifs in α and ßENaC combined with the γT623A mutation. These results suggest that phosphorylation and dephosphorylation of ßT613 play a prominent role in regulating Nedd4-2-mediated ENaC retrieval from the plasma membrane.

Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites.

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK(178)), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.

A mutation in the ß-subunit of ENaC identified in a patient with cystic fibrosis-like symptoms has a gain-of-function effect.

In some patients with atypical cystic fibrosis (CF), only one allele of the CF transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology of the disease in these patients. To functionally characterize a mutation in the ß-subunit of ENaC (ßV348M) recently identified in a patient with severe CF-like symptoms (Mutesa et al. 2009), we expressed wild-type (wt) αßγENaC or mutant αßV348MγENaC in Xenopus laevis oocytes. The ßV348M mutation stimulated amiloride-sensitive whole-cell current (ΔI(ami)) by ∼40% but had no effect on surface expression or single-channel conductance of ENaC. Instead the mutation increased channel open probability (P(o)). Proteolytic activation of mutant ENaC by chymotrypsin was reduced compared with that of wt ENaC (∼3.0-fold vs. ∼4.2-fold), which is consistent with the increased baseline P(o) of mutant ENaC. Similarly, the ENaC activator S3969 stimulated mutant ENaC currents to a lesser degree (by ∼2.6-fold) than wt ENaC currents (by ∼3.5-fold). The gain-of-function effect of the ßV348M mutation was confirmed by whole-cell current measurements in HEK293 cells transiently transfected with wt or mutant ENaC. Computational channel modeling in combination with functional expression of different ßV348 mutants in oocytes suggests that the ßV348M mutation increases channel P(o) by destabilizing the closed channel state. Our findings indicate that the gain-of-function effect of the ßV348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.

The association of chronotype and social jet lag with body composition in German students: The role of physical activity behaviour and the impact of the pandemic lockdown.

Young adults with a later chronotype are vulnerable for a discrepancy in sleep rhythm between work- and free days, called social jet lag (SJL). This study analysed (i) chronotype/SJL association with visceral fat/skeletal muscle mass, (ii) the attribution to physical activity behaviour, and (iii) chronotype-specific changes in physical activity behaviour in young adults during the Covid-19 pandemic lockdown. Chronotype and SJL were derived from the Munich-Chrono-Type-Questionnaire in 320 German students (age 18-25 years) from September 2019 to January 2020, 156 of these participated in an online follow-up survey in June 2020. Body composition was assessed by bioimpedance analysis at baseline. Multivariable linear regression analyses were used to relate chronotype/SJL to body composition; the contribution of self-reported physical activity was tested by mediation analysis. At baseline, a later chronotype and a larger SJL were associated with a higher visceral fat mass (P<0.05), this relation was notably mediated by the attention to physical activity (P<0.05). Chronotype (P = 0.02) but not SJL (P = 0.87) was inversely associated with skeletal muscle mass. During the pandemic lockdown, chronotype hardly changed, but SJL was reduced. Timing and physical activity behaviour remained in most participants and changes were unrelated to chronotype (all P>0.07). A later chronotype/higher SJL may increase the risk of a higher visceral fat mass even in this relatively healthy sample, which may be partly due to their physical activity behaviour. Despite a reduction in SJL during the pandemic lockdown, later chronotypes did not change their physical activity behaviour more than earlier chronotypes.

Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron.

Aldosterone is thought to be the main hormone to stimulate the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the entire collecting duct (CD). There is immunohistochemical evidence for an axial gradient of ENaC expression along the ASDN with highest expression in the DCT2 and CNT. However, most of our knowledge about renal ENaC function stems from studies in the cortical collecting duct (CCD). Here we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice maintained on different sodium diets to vary plasma aldosterone levels. Single-channel recordings demonstrated amiloride-sensitive Na(+) channels in DCT2/CNT with biophysical properties typical for ENaC previously described in CNT/CCD. In animals maintained on a standard salt diet, the average ENaC-mediated whole cell current (ΔI(ami)) was higher in DCT2/CNT than in CNT/CCD. A low salt diet increased ΔI(ami) in CNT/CCD but had little effect on ΔI(ami) in DCT2/CNT. To investigate whether aldosterone is necessary for ENaC activity in the DCT2/CNT, we used aldosterone synthase knockout (AS(-/-)) mice that lack aldosterone. In CNT/CCD of AS(-/-) mice, ΔI(ami) was lower than that in wild-type (WT) animals and was not stimulated by a low salt diet. In contrast, in DCT2/CNT of AS(-/-) mice, ΔI(ami) was similar to that in DCT2/CNT of WT animals both on a standard and on a low salt diet. We conclude that ENaC function in the DCT2/CNT is largely independent of aldosterone which is in contrast to its known aldosterone sensitivity in CNT/CCD.

Vectorial secretion of CTGF as a cell-type specific response to LPA and TGF-ß in human tubular epithelial cells.

BACKGROUND: Increased expression of the pro-fibrotic protein connective tissue growth factor (CTGF) has been detected in injured kidneys and elevated urinary levels of CTGF are discussed as prognostic marker of chronic kidney disease. There is evidence that epithelial cells lining the renal tubular system contribute to uptake and secretion of CTGF. However, the role of different types of tubular epithelial cells in these processes so far has not been addressed in primary cultures of human cells. RESULTS: Tubular epithelial cells of proximal and distal origin were isolated from human kidneys and cultured as polarized cells in insert wells. The pro-fibrotic stimuli lysophosphatidic acid (LPA) and transforming growth factor ß (TGF-ß) were used to induce CTGF secretion.LPA activated CTGF secretion in proximal tubular cells when applied from either the apical or the basolateral side as shown by immunocytochemistry. CTGF was secreted exclusively to the apical side. Signaling pathways activated by LPA included MAP kinase and Rho kinase signaling. TGF-ß applied from either side also stimulated CTGF secretion primarily to the apical side with little basolateral release.Interestingly, TGF-ß activation induced different signaling pathways depending on the side of TGF-ß application. Smad signaling was almost exclusively activated from the basolateral side most prominently in cells of distal origin. Only part of these cells also synthesized CTGF indicating that Smad activation alone was not sufficient for CTGF induction. MAP kinases were involved in apical TGF-ß-mediated activation of CTGF synthesis in proximal cells and a subset of epithelial cells of distal origin. This subpopulation of distal tubular cells was also able to internalize recombinant apical CTGF, in addition to proximal cells which were the main cells to take up exogenous CTGF. CONCLUSIONS: Analysis of polarized human primary renal epithelial cells in a transwell system shows that vectorial secretion of the pro-fibrotic protein CTGF depends on the cell type, the stimulus and the signaling pathway activated. In all conditions, CTGF was secreted mainly to the apical side upon TGF-ß and LPA treatment and therefore, likely contributes to increased urinary CTGF levels in vivo. Moreover, CTGF secreted basolaterally may be active as paracrine pro-fibrotic mediator.

The delta-subunit of the epithelial sodium channel (ENaC) enhances channel activity and alters proteolytic ENaC activation.

The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (alphabetagamma). In humans an additional delta-subunit (delta-hENaC) exists but little is known about its function. Using the Xenopus laevis oocyte expression system, we compared the functional properties of alphabetagamma- and deltabetagamma-hENaC and investigated whether deltabetagamma-hENaC can be proteolytically activated. The amiloride-sensitive ENaC whole-cell current (DeltaI(ami)) was about 11-fold larger in oocytes expressing deltabetagamma-hENaC than in oocytes expressing alphabetagamma-hENaC. The 2-fold larger single-channel Na(+) conductance of deltabetagamma-hENaC cannot explain this difference. Using a chemiluminescence assay, we demonstrated that an increased channel surface expression is also not the cause. Thus, overall channel activity of deltabetagamma-hENaC must be higher than that of alphabetagamma-hENaC. Experiments exploiting the properties of the known betaS520C mutant ENaC confirmed this conclusion. Moreover, chymotrypsin had a reduced stimulatory effect on deltabetagamma-hENaC whole-cell currents compared with its effect on alphabetagamma-hENaC whole-cell currents (2-fold versus 5-fold). This suggests that the cell surface pool of so-called near-silent channels that can be proteolytically activated is smaller for deltabetagamma-hENaC than for alphabetagamma-hENaC. Proteolytic activation of deltabetagamma-hENaC was associated with the appearance of a delta-hENaC cleavage product at the cell surface. Finally, we demonstrated that a short inhibitory 13-mer peptide corresponding to a region of the extracellular loop of human alpha-ENaC inhibited DeltaI(ami) in oocytes expressing alphabetagamma-hENaC but not in those expressing deltabetagamma-hENaC. We conclude that the delta-subunit of ENaC alters proteolytic channel activation and enhances base-line channel activity.

Up-regulation of connective tissue growth factor in endothelial cells by the microtubule-destabilizing agent combretastatin A-4.

Incubation of microvascular endothelial cells with combretastatin A-4 phosphate (CA-4P), a microtubule-destabilizing compound that preferentially targets tumor vessels, altered cell morphology and induced scattering of Golgi stacks. Concomitantly, CA-4P up-regulated connective tissue growth factor (CTGF/CCN2), a pleiotropic factor with antiangiogenic properties. In contrast to the effects of other microtubule-targeting agents such as colchicine or nocodazole, up-regulation of CTGF was only detectable in sparse cells, which were not embedded in a cell monolayer. Furthermore, CA-4P induced CTGF expression in endothelial cells, forming tube-like structures on basement membrane gels. Up-regulation of CTGF by CA-4P was dependent on Rho kinase signaling and was increased when p42/44 mitogen-activated protein kinase was inhibited. Additionally, FoxO transcription factors were identified as potent regulators of CTGF expression in endothelial cells. Activation of FoxO transcription factors by inhibition of phosphatidylinositol 3-kinase/AKT signaling resulted in a synergistic increase in CA-4P-mediated CTGF induction. CA-4P-mediated expression of CTGF was thus potentiated by the inhibition of kinase pathways, which are targets of novel antineoplastic drugs. Up-regulation of CTGF by low concentrations of CA-4P may thus occur in newly formed tumor vessels and contribute to the microvessel destabilization and antiangiogenic effects of CA-4P observed in vivo.

Functional characterization of a partial loss-of-function mutation of the epithelial sodium channel (ENaC) associated with atypical cystic fibrosis.

Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the alpha-subunit of ENaC (alphaF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type alpha beta gamma-ENaC or mutant alpha(F61L) beta gamma-ENaC in Xenopus laevis oocytes. The alphaF61L mutation reduced the ENaC mediated whole-cell currents by approximately 90%. In contrast, the mutation decreased channel surface expression only by approximately 40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (P(o)). This was confirmed by experiments using the betaS520C mutant ENaC which can be converted to a channel with a P(o) of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average P(o) of ENaC by approximately 75%. Na(+) self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel P(o). The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the alphaF61L mutation. We conclude that the alphaF61L mutation may contribute to respiratory symptoms in atypical CF patients.

Plasmin in nephrotic urine activates the epithelial sodium channel.

Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC gamma subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.

Cholesterol depletion of the plasma membrane prevents activation of the epithelial sodium channel (ENaC) by SGK1.

The lipid environment of the epithelial sodium channel (ENaC) and its possible association with so-called lipid rafts may be relevant to its function. The aim of our study was to confirm the association of ENaC with lipid rafts and to analyze the effect of cholesterol depletion of the plasma membrane by methyl-beta-cyclodextrin (MbetaCD) on channel function and regulation. Using sucrose density gradient centrifugation we demonstrated that a significant portion of ENaC protein distributes to low density fractions thought to be typical lipid raft fractions. Importantly, cholesterol depletion of cell lysate by MbetaCD shifted ENaC to non-raft fractions of higher density. Live cell imaging demonstrated that treatment with MbetaCD largely reduced filipin staining over time, confirming cholesterol depletion of the plasma membrane. For electrophysiological studies intact oocytes were exposed to 20 mM MbetaCD for three hours. MbetaCD treatment had no consistent effect on baseline whole-cell ENaC currents. In addition to the typical single channel conductance of about 5 pS, subconductance states of ENaC were occasionally observed in patches from MbetaCD treated but not from control oocytes. Importantly, in outside-out patch clamp recordings the stimulatory effect of recombinant SGK1 in the pipette solution was essentially abolished in oocytes pretreated with MbetaCD. These results indicate that ENaC activation by cytosolic SGK1 is compromised by removing cholesterol from the plasma membrane. Thus, ENaC activation by SGK1 may require the presence of an intact lipid environment and/or of lipid rafts as signalling platform.

Cleavage in the {gamma}-subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near-silent channels.

The mechanisms by which proteases activate the epithelial sodium channel (ENaC) are not yet fully understood. We investigated the effect of extracellular proteases on rat ENaC heterologously expressed in Xenopus laevis oocytes. Application of trypsin increased ENaC whole-oocyte currents by about 8-fold without a concomitant increase in channel surface expression. The stimulatory effect of trypsin was preserved in oocytes expressing alphagamma-ENaC, but was abolished in oocytes expressing alphabeta-ENaC. Thus, the gamma-subunit appears to be essential for channel activation by extracellular proteases. Site-directed mutagenesis of a putative prostasin cleavage site in the extracellular loop of the gamma-subunit revealed that mutating the 181Lys residue to alanine (gammaK181A) increases ENaC baseline whole-oocyte currents, decreases channel surface expression, and largely reduces the stimulatory effect of extracellular proteases (trypsin, chymotrypsin and human neutrophil elastase). In single-channel recordings from outside-out patches we demonstrated that the gammaK181A mutation essentially abolishes the activation of near-silent channels by trypsin, while a stimulatory effect of trypsin on channel gating is preserved. This apparent dual effect of trypsin on channel gating and on the recruitment of near-silent channels was confirmed by experiments using the beta518C mutant ENaC which can be converted to a channel with an open probability of nearly one by exposure to a sulfhydryl reagent. Interestingly, the gammaK181A mutation results in the spontaneous appearance of a 67 kDa fragment of the gamma-subunit in the plasma membrane which can be prevented by a furin inhibitor and also occurs after channel activation by extracellular trypsin. This suggests that the mutation promotes channel cleavage and activation by endogenous proteases. This would lower the pool of near-silent channels and explain the constitutive activation and reduced responsiveness of the mutant channel to extracellular proteases. We conclude that the mutated site (K181A) affects a region in the gamma-subunit of ENaC that is functionally important for the activation of near-silent channels by extracellular proteases.

In Liddle Syndrome, Epithelial Sodium Channel Is Hyperactive Mainly in the Early Part of the Aldosterone-Sensitive Distal Nephron.

The epithelial sodium channel (ENaC) is rate limiting for Na(+) absorption in the aldosterone-sensitive distal nephron comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT), and the entire collecting duct. Liddle syndrome (pseudohyperaldosteronism), a severe form of salt-sensitive hypertension, is caused by gain-of-function mutations of ENaC, but the precise tubular site of increased ENaC function is unknown. In the cortical collecting duct (CCD), ENaC is known to be regulated by aldosterone. In contrast, we recently reported aldosterone-independent ENaC regulation in the early part of the aldosterone-sensitive distal nephron. Here, we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice homozygous for Liddle syndrome mutation or from wild-type control mice. Whole-cell patch-clamp recordings were used to measure amiloride-sensitive ENaC currents in nephron fragments from mice maintained on different sodium diets to vary plasma aldosterone levels. Our data indicate that in mice with Liddle syndrome, the primary site of increased Na(+) reabsorption is the DCT2/CNT. In addition, increased aldosterone responsiveness of ENaC in CNT/CCD may contribute to salt-sensitive hypertension in Liddle syndrome. Single channel properties of ENaC were similar in Liddle syndrome mutation and wild-type mice, but ENaC expression at the apical membrane was increased in Liddle syndrome mutation when compared with wild-type mice, in particular, in animals maintained on a high salt diet. Our findings highlight the importance of ENaC function and regulation in the early part of the aldosterone-sensitive distal nephron for the maintenance of sodium balance and blood pressure control.

Distinct mesenchymal alterations in N-cadherin and E-cadherin positive primary renal epithelial cells.

BACKGROUND: Renal tubular epithelial cells of proximal and distal origin differ markedly in their physiological functions. Therefore, we hypothesized that they also differ in their capacity to undergo epithelial to mesenchymal alterations. RESULTS: We used cultures of freshly isolated primary human tubular cells. To distinguish cells of different tubular origin we took advantage of the fact that human proximal epithelial cells uniquely express N-cadherin instead of E-cadherin as major cell-cell adhesion molecule. To provoke mesenchymal alteration we treated these cocultures with TGF-ß for up to 6 days. Within this time period, the morphology of distal tubular cells was barely altered. In contrast to tubular cell lines, E-cadherin was not down-regulated by TGF-ß, even though TGF-ß signal transduction was initiated as demonstrated by nuclear localization of Smad2/3. Analysis of transcription factors and miRNAs possibly involved in E-cadherin regulation revealed high levels of miRNAs of the miR200-family, which may contribute to the stability of E-cadherin expression in human distal tubular epithelial cells. By contrast, proximal tubular epithelial cells altered their phenotype when treated with TGF-ß. They became elongated and formed three-dimensional structures. Rho-kinases were identified as modulators of TGF-ß-induced morphological alterations. Non-specific inhibition of Rho-kinases resulted in stabilization of the epithelial phenotype, while partial effects were observed upon downregulation of Rho-kinase isoforms ROCK1 and ROCK2. The distinct reactivity of proximal and distal cells was retained when the cells were cultured as polarized cells. CONCLUSIONS: Interference with Rho-kinase signaling provides a target to counteract TGF-ß-mediated mesenchymal alterations of epithelial cells, particularly in proximal tubular epithelial cells. Furthermore, primary distal tubular cells differed from cell lines by their high phenotypic stability which included constant expression of E-cadherin. Our cell culture system of primary epithelial cells is thus suitable to understand and modulate cellular remodeling processes of distinct tubular cells relevant for human renal disease.

Four subunits (αßγδ) of the epithelial sodium channel (ENaC) are expressed in the human eye in various locations.

PURPOSE: The epithelial sodium channel (ENaC) is typically expressed in sodium-absorbing epithelia. Several reports suggest that ENaC is also expressed in ocular tissues and may play a role in aqueous humor secretion and glaucoma. However, the precise localization of ENaC in the human eye is still unclear. Here, the authors studied ENaC expression in 12 normal human donor eyes and in six eyes of patients with glaucoma. METHODS: Quantitative real-time PCR was used to investigate the expression of α-, ß-, γ-, and δ-ENaC transcripts in ocular tissues. In addition, the authors performed immunohistochemical studies using recently generated antibodies against human ß- and γ-ENaC. RESULTS: At the mRNA level, all four ENaC subunits were found to be expressed in a wide range of ocular tissues from normal and glaucomatous human eyes, with the cornea, ciliary body, iris, and retina showing the highest expression levels. At the protein level, ß- and γ-ENaC subunits showed distinct distribution patterns and could be immunolocalized primarily to the cell membranes of epithelial cells of the cornea and to the conjunctiva, iris, ciliary body, lens, and retinal pigment epithelium but also to vascular endothelial cells, smooth muscle cells, stromal cells, and retinal neurons. The authors found no altered mRNA level of any subunit in glaucomatous eyes. CONCLUSIONS: All four ENaC subunits (αßγδ) are expressed in the normal human eye, with distinct localization of subunits possibly reflecting different functional states of the channel. The (patho-)physiological roles of ENaC in the various localizations in the eye remain to be determined.

Actin-dependent regulation of connective tissue growth factor.

Expression of connective tissue growth factor (CTGF) in endothelial cells is modulated by shear stress affecting the organization of the cytoskeleton. The molecular connection between alterations of actin and CTGF expression was investigated in human umbilical vein endothelial cells (HUVEC) and a microvascular endothelial cell line. Overexpression of nonpolymerizable monomeric actin R62D interfered with stress fiber formation in HUVEC and concomitantly reduced immunoreactive CTGF. In microvascular endothelial cells, flow-dependent upregulation of CTGF was prevented by this actin mutant. In contrast, overexpression of actin S14C strengthened filamentous actin and increased CTGF expression. These data indicated an inverse relationship between CTGF expression and monomeric actin. Coexpression of the mutant actins and different CTGF promoter constructs revealed an actin-sensitive site between 3 and 4.5 kb of the CTGF promoter. A CArG-like box at -3791 bp was responsible for actin-dependent CTGF induction as shown by mutagenesis. Overexpression of actin S14C activated the nonmutated promoter significantly more strongly than the mutated promoter. Actin polymerization is regulated by the small GTPase RhoA and activation of serum response factor (SRF). Overexpression of constitutively active RhoA or SRF significantly increased CTGF protein synthesis. The 4.5-kb promoter construct, but not the construct with a mutation in the CArG box, was activated by SRF or RhoA, providing evidence for a functional role of this site in CTGF induction. These findings provide novel evidence that monomeric actin is the connecting link between alterations in the cytoskeleton and CTGF gene expression and demonstrate the importance of SRF in regulating CTGF transcription.

VEGF induces proliferation, migration, and TGF-beta1 expression in mouse glomerular endothelial cells via mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

The role of glomerular endothelial cells in kidney fibrosis remains incompletely understood. While endothelia are indispensable for repair of acute damage, they can produce extracellular matrix proteins and profibrogenic cytokines that promote fibrogenesis. We used a murine cell line with all features of glomerular endothelial cells (glEND.2), which dissected the effects of vascular endothelial growth factor (VEGF) on cell migration, proliferation, and profibrogenic cytokine production. VEGF dose-dependently induced glEND.2 cell migration and proliferation, accompanied by up-regulation of VEGFR-2 phosphorylation and mRNA expression. VEGF induced a profibrogenic gene expression profile, including up-regulation of TGF-beta1 mRNA, enhanced TGF-beta1 secretion, and bioactivity. VEGF-induced endothelial cell migration and TGF-beta1 induction were mediated by the phosphatidyl-inositol-3 kinase pathway, while proliferation was dependent on the Erk1/2 MAP kinase pathway. This suggests that differential modulation of glomerular angiogenesis by selective inhibition of the two identified VEGF-induced signaling pathways could be a therapeutic approach to treat kidney fibrosis.