Epithelial Na+ Channel Activation after Bile Duct Ligation with Mineralocorticoid Receptor Blockade.

BACKGROUND: Sodium and fluid retention in liver disease are classically thought to result from reduced effective circulating volume and stimulation of the renin-angiotensin-aldosterone system (RAAS). However, evidence of fluid retention in patients without RAAS activation suggests the involvement of additional mechanisms. In vitro, bile acids activate the epithelial Na+ channel (ENaC) found in the aldosterone-sensitive distal nephron. If this occurs in vivo, ENaC may become activated in liver disease even with antagonism of aldosterone signaling. METHODS: To test this, we performed bile duct ligation to induce liver disease and increase circulating bile acids in mice given spironolactone to antagonize aldosterone signaling. We analyzed effects on blood, urine and body composition. We also determined the effects of taurocholic acid, a primary conjugated bile acid elevated in liver disease, on ion fluxes in microperfused rabbit collecting ducts. RESULTS: Bile duct ligation increased benzamil-sensitive natriuresis compared to sham, indicating ENaC activation. These effects were not explained by effects on ENaC expression, cleavage, or localization. Bile duct ligated mice also gained significantly more fluid than sham-operated animals. Blocking ENaC reversed fluid gains in bile duct ligated mice but had no effect in shams. In dissected collecting ducts from rabbits, which express ENaC, taurocholic acid stimulated net Na+ absorption. CONCLUSIONS: Our results provide experimental evidence for a novel aldosterone-independent mechanism for sodium and fluid retention in liver disease.

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling.

Aldosterone is responsible for maintaining volume and potassium homeostasis. Although high salt consumption should suppress aldosterone production, individuals with hyperaldosteronism lose this regulation, leading to a state of high aldosterone despite dietary sodium consumption. The present study examines the effects of elevated aldosterone, with or without high salt consumption, on the expression of key Na+ transporters and remodelling in the distal nephron. Epithelial sodium channel (ENaC) α-subunit expression was increased with aldosterone regardless of Na+ intake. However, ENaC ß- and γ-subunits unexpectedly increased at both a transcript and protein level with aldosterone when high salt was present. Expression of total and phosphorylated Na+ Cl- cotransporter (NCC) significantly increased with aldosterone, in association with decreased blood [K+ ], but the addition of high salt markedly attenuated the aldosterone-dependent NCC increase, despite equally severe hypokalaemia. We hypothesized this was a result of differences in distal convoluted tubule length when salt was given with aldosterone. Imaging and measurement of the entire pNCC-positive tubule revealed that aldosterone alone caused a shortening of this segment, although the tubule had a larger cross-sectional diameter. This was not true when salt was given with aldosterone because the combination was associated with a lengthening of the tubule in addition to increased diameter, suggesting that differences in the pNCC-positive area are not responsible for differences in NCC expression. Together, our results suggest the actions of aldosterone, and the subsequent changes related to hypokalaemia, are altered in the presence of high dietary Na+ . KEY POINTS: Aldosterone regulates volume and potassium homeostasis through effects on transporters in the kidney; its production can be dysregulated, preventing its suppression by high dietary sodium intake. Here, we examined how chronic high sodium consumption affects aldosterone's regulation of sodium transporters in the distal nephron. Our results suggest that high sodium consumption with aldosterone is associated with increased expression of all three epithelial sodium channel subunits, rather than just the alpha subunit. Aldosterone and its associated decrease in blood [K+ ] lead to an increased expression of Na-Cl cotransporter (NCC); the addition of high sodium consumption with aldosterone partially attenuates this NCC expression, despite similarly low blood [K+ ]. Upstream kinase regulators and tubule remodelling do not explain these results.

SGLT2-independent effects of canagliflozin on NHE3 and mitochondrial complex I activity inhibit proximal tubule fluid transport and albumin uptake.

Beyond glycemic control, SGLT2 inhibitors (SGLT2is) have protective effects on cardiorenal function. Renoprotection has been suggested to involve inhibition of NHE3 leading to reduced ATP-dependent tubular workload and mitochondrial oxygen consumption. NHE3 activity is also important for regulation of endosomal pH, but the effects of SGLT2i on endocytosis are unknown. We used a highly differentiated cell culture model of proximal tubule (PT) cells to determine the direct effects of SGLT2i on Na+-dependent fluid transport and endocytic uptake in this nephron segment. Strikingly, canagliflozin but not empagliflozin reduced fluid transport across cell monolayers and dramatically inhibited endocytic uptake of albumin. These effects were independent of glucose and occurred at clinically relevant concentrations of drug. Canagliflozin acutely inhibited surface NHE3 activity, consistent with a direct effect, but did not affect endosomal pH or NHE3 phosphorylation. In addition, canagliflozin rapidly and selectively inhibited mitochondrial complex I activity. Inhibition of mitochondrial complex I by metformin recapitulated the effects of canagliflozin on endocytosis and fluid transport, whereas modulation of downstream effectors AMPK and mTOR did not. Mice given a single dose of canagliflozin excreted twice as much urine over 24 h compared with empagliflozin-treated mice despite similar water intake. We conclude that canagliflozin selectively suppresses Na+-dependent fluid transport and albumin uptake in PT cells via direct inhibition of NHE3 and of mitochondrial function upstream of the AMPK/mTOR axis. These additional targets of canagliflozin contribute significantly to reduced PT Na+-dependent fluid transport in vivo.NEW & NOTEWORTHY Reduced NHE3-mediated Na+ transport has been suggested to underlie the cardiorenal protection provided by SGLT2 inhibitors. We found that canagliflozin, but not empagliflozin, reduced NHE3-dependent fluid transport and endocytic uptake in cultured proximal tubule cells. These effects were independent of SGLT2 activity and resulted from inhibition of mitochondrial complex I and NHE3. Studies in mice are consistent with greater effects of canagliflozin versus empagliflozin on fluid transport. Our data suggest that these selective effects of canagliflozin contribute to reduced Na+-dependent transport in proximal tubule cells.

Impaired Endosome Maturation Mediates Tubular Proteinuria in Dent Disease Cell Culture and Mouse Models.

SIGNIFICANCE STATEMENT: Loss of function of the 2Cl - /H + antiporter ClC-5 in Dent disease causes an unknown impairment in endocytic traffic, leading to tubular proteinuria. The authors integrated data from biochemical and quantitative imaging studies in proximal tubule cells into a mathematical model to determine that loss of ClC-5 impairs endosome acidification and delays early endosome maturation in proximal tubule cells, resulting in reduced megalin recycling, surface expression, and half-life. Studies in a Dent mouse model also revealed subsegment-specific differences in the effects of ClC-5 knockout on proximal tubule subsegments. The approach provides a template to dissect the effects of mutations or perturbations that alter tubular recovery of filtered proteins from the level of individual cells to the entire proximal tubule axis. BACKGROUND: Loss of function of the 2Cl - /H + antiporter ClC-5 in Dent disease impairs the uptake of filtered proteins by the kidney proximal tubule, resulting in tubular proteinuria. Reduced posttranslational stability of megalin and cubilin, the receptors that bind to and recover filtered proteins, is believed to underlie the tubular defect. How loss of ClC-5 leads to reduced receptor expression remains unknown. METHODS: We used biochemical and quantitative imaging data to adapt a mathematical model of megalin traffic in ClC-5 knockout and control cells. Studies in ClC-5 knockout mice were performed to describe the effect of ClC-5 knockout on megalin traffic in the S1 segment and along the proximal tubule axis. RESULTS: The model predicts that ClC-5 knockout cells have reduced rates of exit from early endosomes, resulting in decreased megalin recycling, surface expression, and half-life. Early endosomes had lower [Cl - ] and higher pH. We observed more profound effects in ClC-5 knockout cells expressing the pathogenic ClC-5 E211G mutant. Alterations in the cellular distribution of megalin in ClC-5 knockout mice were consistent with delayed endosome maturation and reduced recycling. Greater reductions in megalin expression were observed in the proximal tubule S2 cells compared with S1, with consequences to the profile of protein retrieval along the proximal tubule axis. CONCLUSIONS: Delayed early endosome maturation due to impaired acidification and reduced [Cl - ] accumulation is the primary mediator of reduced proximal tubule receptor expression and tubular proteinuria in Dent disease. Rapid endosome maturation in proximal tubule cells is critical for the efficient recovery of filtered proteins.

Modelling normal and nephrotic axial uptake of albumin and other filtered proteins along the proximal tubule.

Recent studies indicate that filtered albumin is retrieved in the proximal tubule (PT) via three pathways: receptor-mediated endocytosis via cubilin (high affinity) and megalin (low affinity), and fluid-phase uptake. Expression of megalin is required to maintain all three pathways, making it challenging to determine their respective contributions. Moreover, uptake of filtered molecules varies between the sub-segments (S1, S2 and S3) that make up the PT. Here we used new and published data to develop a mathematical model that predicts the rates of albumin uptake in mouse PT sub-segments in normal and nephrotic states, and partially accounts for competition by ß2 -microglobulin (ß2m) and immunoglobulin G (IgG). Our simulations indicate that receptor-mediated, rather than fluid-phase, uptake accounts for the vast majority of ligand recovery. Our model predicts that ∼75% of normally filtered albumin is reabsorbed via cubilin; however, megalin-mediated uptake predominates under nephrotic conditions. Our results also suggest that ∼80% of albumin is normally recovered in S1, whereas nephrotic conditions or knockout of cubilin shifts the bulk of albumin uptake to S2. The model predicts ß2m and IgG axial recovery profiles qualitatively similar to those of albumin under normal conditions. In contrast with albumin, however, the bulk of IgG and ß2m uptake still occurs in S1 under nephrotic conditions. Overall, our model provides a kinetic rationale for why tubular proteinuria can occur even though a large excess in potential PT uptake capacity exists, and suggests testable predictions to expand our understanding of the recovery profile of filtered proteins along the PT. KEY POINTS: We used new and published data to develop a mathematical model that predicts the profile of albumin uptake in the mouse proximal tubule in normal and nephrotic states, and partially accounts for competitive inhibition of uptake by normally filtered and pathological ligands. Three pathways, consisting of high-affinity uptake by cubilin receptors, low-affinity uptake by megalin receptors and fluid phase uptake, contribute to the overall retrieval of filtered proteins. The axial profile and efficiency of protein uptake depend on the initial filtrate composition and the individual protein affinities for megalin and cubilin. Under normal conditions, the majority of albumin is retrieved in sub-segment S1 but shifts to sub-segment S2 under nephrotic conditions. Other proteins exhibit different uptake profiles. Our model explains how tubular proteinuria can occur despite a large excess in potential proximal tubule uptake capacity.

Functional characterization of ion channels expressed in kidney organoids derived from human induced pluripotent stem cells.

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca2+-activated K+ (BKCa) channel, the renal outer medullary K+ (ROMK, Kir1.1) channel, and the epithelial Na+ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BKCa channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BKCa and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K+ and Na+ channels.NEW & NOTEWORTHY Our results show that human kidney organoids express key K+ and Na+ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca2+-activated K+ channel, renal outer medullary K+ channel, and epithelial Na+ channel.

Shear stress and oxygen availability drive differential changes in opossum kidney proximal tubule cell metabolism and endocytosis.

Kidney proximal tubule (PT) cells have high-metabolic demands to drive the extraordinary ion and solute transport, water reabsorption, and endocytic uptake that occur in this nephron segment. Increases in renal blood flow alter glomerular filtration rate and lead to rapid mechanosensitive adaptations in PT transport, impacting metabolic demand. Although the PT reabsorbs essentially all of the filtered glucose, PT cells rely primarily on oxidative metabolism rather than glycolysis to meet their energy demands. We lack an understanding of how PT functions are impacted by changes in O2 availability via cortical capillaries and mechanosensitive signaling in response to alterations in luminal flow. Previously, we found that opossum kidney (OK) cells recapitulate key features of PT cells in vivo, including enhanced endocytic uptake and ion transport, when exposed to mechanical stimulation by culture on an orbital shaker. We hypothesized that increased oxygenation resulting from orbital shaking also contributes to this more physiologic phenotype. RNA seq of OK cells maintained under static conditions or exposed to orbital shaking for up to 96 hours showed significant time- and culture-dependent changes in gene expression. Transcriptional and metabolomics data were consistent with a decrease in glycolytic flux and with an increased utilization of aerobic metabolic pathways in cells exposed to orbital shaking. Moreover, we found spatial differences in the pattern of mitogenesis vs development of ion transport and endocytic capacities in our culture system that highlight the complexity of O2 -dependent and mechanosensitive crosstalk to regulate PT cell function.

Paraoxonase 3 functions as a chaperone to decrease functional expression of the epithelial sodium channel.

The paraoxonase (PON) family comprises three highly conserved members: PON1, PON2, and PON3. They are orthologs of Caenorhabditis elegans MEC-6, an endoplasmic reticulum-resident chaperone that has a critical role in proper assembly and surface expression of the touch-sensing degenerin channel in nematodes. We have shown recently that MEC-6 and PON2 negatively regulate functional expression of the epithelial Na+ channel (ENaC), suggesting that the chaperone function is conserved within this family. We hypothesized that other PON family members also modulate ion channel expression. Pon3 is specifically expressed in the aldosterone-sensitive distal tubules in the mouse kidney. We found here that knocking down endogenous Pon3 in mouse cortical collecting duct cells enhanced Na+ transport, which was associated with increased γENaC abundance. We further examined Pon3 regulation of ENaC in two heterologous expression systems, Fisher rat thyroid cells and Xenopus oocytes. Pon3 coimmunoprecipitated with each of the three ENaC subunits in Fisher rat thyroid cells. As a result of this interaction, the whole-cell and surface abundance of ENaC α and γ subunits was reduced by Pon3. When expressed in oocytes, Pon3 inhibited ENaC-mediated amiloride-sensitive Na+ currents, in part by reducing the surface expression of ENaC. In contrast, Pon3 did not alter the response of ENaC to chymotrypsin-mediated proteolytic activation or [2-(trimethylammonium)ethyl]methanethiosulfonate-induced activation of αßS518Cγ, suggesting that Pon3 does not affect channel open probability. Together, our results suggest that PON3 regulates ENaC expression by inhibiting its biogenesis and/or trafficking.

Activation of AMP-activated protein kinase during sepsis/inflammation improves survival by preserving cellular metabolic fitness.

The purpose was to determine the role of AMPK activation in the renal metabolic response to sepsis, the development of sepsis-induced acute kidney injury (AKI) and on survival. In a prospective experimental study, 167 10- to 12-week-old C57BL/6 mice underwent cecal ligation and puncture (CLP) and human proximal tubule epithelial cells (TEC; HK2) were exposed to inflammatory mix (IM), a combination of lipopolysaccharide (LPS) and high mobility group box 1 (HMGB1). Renal/TEC metabolic fitness was assessed by monitoring the expression of drivers of oxidative phosphorylation (OXPHOS), the rates of utilization of OXPHOS/glycolysis in response to metabolic stress, and mitochondrial function by measuring O2 consumption rates (OCR) and the membrane potential (Δψm ). Sepsis/IM resulted in AKI, increased mortality, and in renal AMPK activation 6-24 hours after CLP/IM. Pharmacologic activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or metformin during sepsis improved the survival, while AMPK inhibition with Compound C increased mortality, impaired mitochondrial respiration, decreased OCR, and disrupted TEC metabolic fitness. AMPK-driven protection was associated with increased Sirt 3 expression and restoration of metabolic fitness. Renal AMPK activation in response to sepsis/IM is an adaptive mechanism that protects TEC, organs, and the host by preserving mitochondrial function and metabolic fitness likely through Sirt3 signaling.

Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model.

BACKGROUND: Lowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn't clear. METHODS: We examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish. RESULTS: OCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment. CONCLUSIONS: Our data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.

Distinct functions of megalin and cubilin receptors in recovery of normal and nephrotic levels of filtered albumin.

Proximal tubule (PT) cells express a single saturable albumin-binding site whose affinity matches the estimated tubular concentration of albumin; however, albumin uptake capacity is greatly increased under nephrotic conditions. Deciphering the individual contributions of megalin and cubilin to the uptake of normal and nephrotic levels of albumin is impossible in vivo, as knockout of megalin in mice globally disrupts PT endocytic uptake. We quantified concentration-dependent albumin uptake in an optimized opossum kidney cell culture model and fit the kinetic profiles to identify albumin-binding affinities and uptake capacities. Mathematical deconvolution fit best to a three-component model that included saturable high- and low-affinity uptake sites for albumin and underlying nonsaturable uptake consistent with passive uptake of albumin in the fluid phase. Knockdown of cubilin or its chaperone amnionless selectively reduced the binding capacity of the high-affinity site, whereas knockdown of megalin impacted the low-affinity site. Knockdown of disabled-2 decreased the capacities of both binding sites. Additionally, knockdown of megalin or disabled-2 profoundly inhibited the uptake of a fluid phase marker, with cubilin knockdown having a more modest effect. We propose a novel model for albumin retrieval along the PT in which cubilin and megalin receptors have different functions in recovering filtered albumin in proximal tubule cells. Cubilin binding to albumin is tuned to capture normally filtered levels of the protein. In contrast, megalin binding to albumin is of lower affinity, and its expression is also essential for enabling the recovery of high concentrations of albumin in the fluid phase.

Cytochrome b5 Reductase 3 Modulates Soluble Guanylate Cyclase Redox State and cGMP Signaling.

RATIONALE: Soluble guanylate cyclase (sGC) heme iron, in its oxidized state (Fe3+), is desensitized to NO and limits cGMP production needed for downstream activation of protein kinase G-dependent signaling and blood vessel dilation. OBJECTIVE: Although reactive oxygen species are known to oxidize the sGC heme iron, the basic mechanism(s) governing sGC heme iron recycling to its NO-sensitive, reduced state remain poorly understood. METHODS AND RESULTS: Oxidant challenge studies show that vascular smooth muscle cells have an intrinsic ability to reduce oxidized sGC heme iron and form protein-protein complexes between cytochrome b5 reductase 3, also known as methemoglobin reductase, and oxidized sGC. Genetic knockdown and pharmacological inhibition in vascular smooth muscle cells reveal that cytochrome b5 reductase 3 expression and activity is critical for NO-stimulated cGMP production and vasodilation. Mechanistically, we show that cytochrome b5 reductase 3 directly reduces oxidized sGC required for NO sensitization as assessed by biochemical, cellular, and ex vivo assays. CONCLUSIONS: Together, these findings identify new insights into NO-sGC-cGMP signaling and reveal cytochrome b5 reductase 3 as the first identified physiological sGC heme iron reductase in vascular smooth muscle cells, serving as a critical regulator of cGMP production and protein kinase G-dependent signaling.

Lemmings into the sea or back across the bridge? The fate of albumin in nephrotic syndrome.

An intact glomerular filtration barrier is essential for maintaining plasma albumin levels. However, the capacity of the proximal tubule to reabsorb filtered albumin and the subsequent fate of this protein are hotly debated. Weyer et al. find that knockout of megalin and cubilin receptors in a nephrotic mouse model causes no further reduction in plasma albumin levels, suggesting that albumin retrieval by the proximal tubule does not contribute significantly to albumin homeostasis.

Rab11a-positive compartments in proximal tubule cells sort fluid-phase and membrane cargo.

The proximal tubule (PT) reabsorbs the majority of sodium, bicarbonate, and chloride ions, phosphate, glucose, water, and plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell (PTC) function, the organization of the endocytic pathway in these cells remains poorly understood. We have used immunofluorescence and live-cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PTCs isolated from mouse kidney cortex. Cells from the S1 segment could be distinguished from those from more distal PT segments by their robust uptake of albumin and comparatively low expression of γ-glutamyltranspeptidase. Rab11a in these cells is localized to variously sized spherical compartments that resemble the apical vacuoles observed by electron microscopy analysis of PTCs in vivo. These Rab11a-positive structures are highly dynamic and receive membrane and fluid-phase cargo. In contrast, fluid-phase cargoes are largely excluded from Rab11a-positive compartments in immortalized kidney cell lines. The unusual morphology and sorting capacity of Rab11a compartments in primary PTCs may reflect a unique specialization of these cells to accommodate the functional demands of handling a high endocytic load.

Fibrosis reduces severity of acute-on-chronic pancreatitis in humans.

BACKGROUND & AIMS: Acute pancreatitis (AP) and chronic pancreatitis (CP) share etiologies, but AP can be more severe and is associated with a higher rate of mortality. We investigated features of CP that protect against severe disease. The amount of intrapancreatic fat (IPF) is increased in obese patients and fibrosis is increased in patients with CP, so we studied whether fibrosis or fat regulate severity of AP attacks in patients with CP. METHODS: We reviewed records from the University of Pittsburgh Medical Center/Presbyterian Hospital Autopsy Database (1998-2008) for patients with a diagnosis of AP (n = 23), CP (n = 35), or both (AP-on-CP; n = 15). Pancreatic histology samples from these patients and 50 randomly selected controls (no pancreatic disease) were analyzed, and IPF data were correlated with computed tomography data. An adipocyte and acinar cell Transwell coculture system, with or without collagen type I, was used to study the effects of fibrosis on acinar-adipocyte interactions. We studied the effects of nonesterified fatty acids (NEFAs) and adipokines on acinar cells in culture. RESULTS: Levels of IPF were significantly higher in nonobese patients with CP than in nonobese controls. In patients with CP or AP-on-CP, areas of IPF were surrounded by significantly more fibrosis than in controls or patients with AP. Fat necrosis-associated peri-fat acinar necrosis (PFAN, indicated by NEFA spillage) contributed to most of the necrosis observed in samples from patients with AP; however, findings of peri-fat acinar necrosis and total necrosis were significantly lower in samples from patients with CP or AP-on-CP. Fibrosis appeared to wall off the fat necrosis and limit peri-fat acinar necrosis, reducing acinar necrosis. In vitro, collagen I limited the lipolytic flux between acinar cells and adipocytes and prevented increases in adipokines in the acinar compartment. This was associated with reduced acinar cell necrosis. However, NEFAs, but not adipokines, caused acinar cell necrosis. CONCLUSIONS: Based on analysis of pancreatic samples from patients with CP, AP, or AP-on-CP and in vitro studies, fibrosis reduces the severity of acute exacerbations of CP by reducing lipolytic flux between adipocytes and acinar cells.

Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

Cigarette smoking affects oxidative protein folding in endoplasmic reticulum by modifying protein disulfide isomerase.

The endoplasmic reticulum (ER) stress response (ERSR) and associated protein aggregation, is under investigation for its role in human diseases, including chronic obstructive pulmonary disease (COPD) where cigarette smoking (CS) is a risk factor for disease development. Our hypothesis states that CS-associated oxidative stress interferes with oxidative protein folding in the ER and elicits ERSR. We investigated ERSR induction following acute CS exposure and delineated mechanisms of CS-induced ERSR. Lung lysates from mice exposed or not to one cigarette were tested for activation of the ERSR. Up to 4-fold increase in phosphorylation of eIF2α and nuclear form of ATF6 was detected in CS-exposed animals. CS affected the formation of disulfide bonds through excessive posttranslational oxidation of protein disulfide isomerase (PDI). Increased amounts of complexes between PDI and its client proteins persisted in CS-exposed samples. BiP was not a constituent of these complexes, demonstrating the specificity of the early effects of CS exposure on ER. Disturbances in protein folding were accompanied by changes in the organization of ER network and ER exit sites. Our results provide evidence that ERSR is induced early in response to CS exposure and identifies the first known ER-resident target of CS PDI, demonstrating that CS affects oxidative protein folding.

GJC2 missense mutations cause human lymphedema.

Lymphedema is the clinical manifestation of defects in lymphatic structure or function. Mutations identified in genes regulating lymphatic development result in inherited lymphedema. No mutations have yet been identified in genes mediating lymphatic function that result in inherited lymphedema. Survey microarray studies comparing lymphatic and blood endothelial cells identified expression of several connexins in lymphatic endothelial cells. Additionally, gap junctions are implicated in maintaining lymphatic flow. By sequencing GJA1, GJA4, and GJC2 in a group of families with dominantly inherited lymphedema, we identified six probands with unique missense mutations in GJC2 (encoding connexin [Cx] 47). Two larger families cosegregate lymphedema and GJC2 mutation (LOD score = 6.5). We hypothesize that missense mutations in GJC2 alter gap junction function and disrupt lymphatic flow. Until now, GJC2 mutations were only thought to cause dysmyelination, with primary expression of Cx47 limited to the central nervous system. The identification of GJC2 mutations as a cause of primary lymphedema raises the possibility of novel gap-junction-modifying agents as potential therapy for some forms of lymphedema.

Megalin, cubilin, and Dab2 drive endocytic flux in kidney proximal tubule cells.

The kidney proximal tubule (PT) elaborates a uniquely high-capacity apical endocytic pathway to retrieve albumin and other proteins that escape the glomerular filtration barrier. Megalin and cubilin/amnionless (CUBAM) receptors engage Dab2 in these cells to mediate clathrin-dependent uptake of filtered ligands. Knockout of megalin or Dab2 profoundly inhibits apical endocytosis and is believed to atrophy the endocytic pathway. We generated CRISPR/Cas9 knockout (KO) clones lacking cubilin, megalin, or Dab2 expression in highly differentiated PT cells and determined the impact on albumin internalization and endocytic pathway function. KO of each component had different effects on the concentration dependence of albumin uptake as well its distribution within PT cells. Reduced uptake of a fluid phase marker was also observed, with megalin KO cells having the most dramatic decline. Surprisingly, protein levels and distribution of key endocytic proteins were preserved in KO PT cell lines and in megalin KO mice, despite the reduced endocytic activity. Our data highlight specific functions of megalin, cubilin, and Dab2 in apical endocytosis and demonstrate that these proteins drive endocytic flux without compromising the physical integrity of the apical endocytic pathway. Our studies suggest a novel model to explain how these components coordinate endocytic uptake in PT cells.

Mineralocorticoid receptor-independent activation of ENaC in bile duct ligated mice.

Sodium and fluid retention in liver disease is classically thought to result from reduced effective circulating volume and stimulation of the renin-angiotensin-aldosterone system (RAAS). Aldosterone dives Na+ retention by activating the mineralocorticoid receptor and promoting the maturation and apical surface expression of the epithelial Na+ channel (ENaC), found in the aldosterone-sensitive distal nephron. However, evidence of fluid retention without RAAS activation suggests the involvement of additional mechanisms. Liver disease can greatly increase plasma and urinary bile acid concentrations and have been shown to activate ENaC in vitro. We hypothesize that elevated bile acids in liver disease activate ENaC and drive fluid retention independent of RAAS. We therefore increased circulating bile acids in mice through bile duct ligation (BDL) and measured effects on urine and body composition, while using spironolactone to antagonize the mineralocorticoid receptor. We found BDL lowered blood [K+] and hematocrit, and increased benzamil-sensitive natriuresis compared to sham, consistent with ENaC activation. BDL mice also gained significantly more body water. Blocking ENaC reversed fluid gains in BDL mice but had no effect in shams. In isolated collecting ducts from rabbits, taurocholic acid stimulated net Na+ absorption but had no effect on K+ secretion or flow-dependent ion fluxes. Our results provide experimental evidence for a novel aldosterone-independent mechanism for sodium and fluid retention in liver disease which may provide additional therapeutic options for liver disease patients.