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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 Ca

CRISPR/Cas9 approaches were used to generate megalin, cubilin, and Dab2 knockouts in polarized proximal tubule cells. Knockout of each component had different effects on endocytic uptake. Data from cell lines and megalin knockout mice support a novel model whereby each protein contributes to driving apical endocytic flux without compromising the physical integrity of the apical endocytic pathway.

PDF file - 170K, Representative LEC microingjections showing dye transfer. Pre-images obtained using DIC and gfp (a,c,e) showing dsred (pink) for reference cell and lucifer yellow dye transfer in blue. LECs electroporated with empty EGFP vector (a,b); WT- hCx47-EGFP (c,d); H409Y-EGFP (e,f). 40x oil, 1.3 n.a.

Purpose: Secondary lymphedema is a frequent complication of breast cancer associated with surgery, chemotherapy, or radiation following breast cancer treatment. The potential contribution of genetic susceptibility to risk of developing secondary lymphedema following surgical trauma, radiation, and other tissue insults has not been studied.Experimental Design: To determine whether women with breast cancer and secondary lymphedema had mutations in candidate lymphedema genes, we undertook a case–control study of 188 women diagnosed with breast cancer recruited from the University of Pittsburgh Breast Cancer Program (http://www.upmccancercenter.com/breast/index.cfm) between 2000 and 2010.Candidate lymphedema genes, GJC2 (encoding connexin 47 [Cx47]), FOXC2, HGF, MET, and FLT4 (encoding VEGFR3), were sequenced for mutation. Bioinformatics analysis and in vitro functional assays were used to confirm significance of novel mutations.Results: Cx47 mutations were identified in individuals having secondary lymphedema following breast cancer treatment but not in breast cancer controls or normal women without breast cancer. These novel mutations are dysfunctional as assessed through in vitro assays and bioinformatics analysis and provide evidence that altered gap junction function leads to lymphedema.Conclusions: Our findings challenge the view that secondary lymphedema is solely due to mechanical trauma and support the hypothesis that genetic susceptibility is an important risk factor for secondary lymphedema. A priori recognition of genetic risk (i) raises the potential for early detection and intervention for a high-risk group and (ii) allows the possibility of altering surgical approach and/or chemo- and radiation therapy, or direct medical treatment of secondary lymphedema with novel connexin-modifying drugs. Clin Cancer Res; 18(8); 2382–90. ©2012 AACR.

PDF file - 154K, Mutation chromatograms.

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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.

The cells that comprise the proximal tubule (PT) are specialized for high-capacity apical endocytosis necessary to maintain a protein-free urine. Filtered proteins are reclaimed via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. Despite the importance of this pathway, we lack a detailed understanding of megalin trafficking kinetics and how they are regulated. Here, we utilized biochemical and quantitative imaging methods in a highly differentiated model of opossum kidney (OK) cells and in mouse kidney in vivo to develop mathematical models of megalin traffic. A preliminary model based on biochemically quantified kinetic parameters was refined by colocalization of megalin with individual apical endocytic compartment markers. Our model predicts that megalin is rapidly internalized, resulting in primarily intracellular distribution of the receptor at steady state. Moreover, our data show that early endosomes mature rapidly in PT cells and suggest that Rab11 is the primary mediator of apical recycling of megalin from maturing endocytic compartments. Apical recycling represents the rate-limiting component of endocytic traffic, suggesting that this step has the largest impact in determining the endocytic capacity of PT cells. Adaptation of our model to the S1 segment of mouse PT using colocalization data obtained in kidney sections confirms basic aspects of our model and suggests that our OK cell model largely recapitulates in vivo membrane trafficking kinetics. We provide a downloadable application that can be used to adapt our working parameters to further study how endocytic capacity of PT cells may be altered under normal and disease conditions.

Background Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. Methods To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. Results We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. Conclusion This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

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.

Kidney proximal tubule cells cultured under shear stress become remarkably well differentiated and endocytic capacity is rapidly tuned in response to acute changes in shear stress. The results have implications for understanding how proximal tubule function is regulated acutely by daily variations in glomerular filtration rate., Cells lining the proximal tubule (PT) have unique membrane specializations that are required to maintain the high-capacity ion transport and endocytic functions of this nephron segment. PT cells in vivo acutely regulate ion transport in response to changes in glomerular filtration rate (GFR) to maintain glomerulotubular balance. PT cells in culture up-regulate endocytic capacity in response to acute changes in fluid shear stress (FSS); however, it is not known whether GFR modulates PT endocytosis to enable maximally efficient uptake of filtered proteins in vivo. Here, we show that cells cultured under continuous FSS develop an expanded apical endocytic pathway and increased endocytic capacity and lysosomal biogenesis. Furthermore, endocytic capacity in fully differentiated cells is rapidly modulated by changes in FSS. PT cells exposed to continuous FSS also acquired an extensive brush border and basolateral membrane invaginations resembling those observed in vivo. Culture under suboptimal levels of FSS led to intermediate phenotypes, suggesting a threshold effect. Cells exposed to FSS expressed higher levels of key proteins necessary for PT function, including ion transporters, receptors, and membrane-trafficking machinery, and increased adenine nucleotide levels. Inhibition of the mechanistic target of rapamycin (mTOR) using rapamycin prevented the increase in cellular energy levels, lysosomal biogenesis, and endocytic uptake, suggesting that these represent a coordinated differentiation program. In contrast, rapamycin did not prevent the FSS-induced increase in Na+/K+-ATPase levels. Our data suggest that rapid tuning of the endocytic response by changes in FSS may contribute to glomerulotubular balance in vivo. Moreover, FSS provides an essential stimulus in the differentiation of PT cells via separate pathways that up-regulate endocytosis and ion transport capacity. Variations in FSS may also contribute to the maturation of PT cells during kidney development and during repair after kidney injury.

Rationale: Soluble guanylate cyclase (sGC) heme iron, in its oxidized state (Fe 3+ ), 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.

Several model systems have been used to study signaling cascades in kidney epithelial cells, including kidney histology after systemic treatments, ex vivo isolated tubule perfusion, epithelial cell lines in culture, kidney micropuncture, and ex vivo kidney slices. We and others have found the ex vivo kidney slice method useful to study the signaling cascades involved in the regulation of kidney transport proteins. In this chapter we describe our adaptations to this classic method for the study of the regulation of kinases and endocytosis in rodent kidney epithelial cells. Briefly, slices are obtained by sectioning of freshly harvested rat or mouse kidneys using a Stadie-Riggs tissue slicer. Alternatively, a vibratome can be used to obtain slices at a more consistent and finer thickness. The harvested kidney and kidney slices are kept viable in either cell culture media or in buffers that mimic physiological conditions equilibrated with 5% CO

Several model systems have been used to study signaling cascades in kidney epithelial cells, including kidney histology after systemic treatments, ex vivo isolated tubule perfusion, epithelial cell lines in culture, kidney micropuncture, and ex vivo kidney slices. We and others have found the ex vivo kidney slice method useful to study the signaling cascades involved in the regulation of kidney transport proteins. In this chapter we describe our adaptations to this classic method for the study of the regulation of kinases and endocytosis in rodent kidney epithelial cells. Briefly, slices are obtained by sectioning of freshly harvested rat or mouse kidneys using a Stadie-Riggs tissue slicer. Alternatively, a vibratome can be used to obtain slices at a more consistent and finer thickness. The harvested kidney and kidney slices are kept viable in either cell culture media or in buffers that mimic physiological conditions equilibrated with 5% CO2 at body temperature (37 °C). These buffers keep the slices viable during hours for incubations in the presence/absence of different pharmacological agents. After the incubation period the slices can be used for biochemistry experiments by preparing tissue lysates or for histological evaluation after fixation. Moreover, the fixed slices can be used to evaluate changes in subcellular trafficking of epithelial proteins or endosomes via immunolabeling followed by confocal microscopy. The resulting micrographs can then be used for systematic quantification of protein- or compartment-specific changes in subcellular localization under each condition.

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 O(2) 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 O(2)-dependent and mechanosensitive crosstalk to regulate PT cell function.

Benzyl isothiocyanate (BITC) is a highly promising phytochemical abundant in cruciferous vegetables with preclinical evidence of in vivo efficacy against breast cancer in xenograft and transgenic mouse models. Mammary cancer chemoprevention by BITC is associated with apoptotic cell death but the underlying mechanism is not fully understood. Herein, we demonstrate for the first time that altered mitochondrial dynamics is an early and critical event in BITC-induced apoptosis in breast cancer cells. Exposure of MCF-7 and MDA-MB-231 cells to plasma achievable doses of BITC resulted in rapid collapse of mitochondrial filamentous network. BITC treatment also inhibited polyethyleneglycol-induced mitochondrial fusion. In contrast, a normal human mammary epithelial cell line (MCF-10A) that was derived from fibrocystic breast disease, was resistant to BITC-mediated alterations in mitochondrial dynamics as well as apoptosis. Transient or sustained decrease in levels of proteins engaged in regulation of mitochondrial fission and fusion was clearly evident after BITC treatment in both cancer cell lines. A trend for a decrease in the levels of mitochondrial fission- and fusion-related proteins was also observed in vivo in tumors of BITC-treated mice compared with control. Immortalized mouse embryonic fibroblasts from Drp1 knockout mice were resistant to BITC-induced apoptosis when compared with those from wild-type mice. Upon treatment with BITC, Bak dissociated from mitofusin 2 in both MCF-7 and MDA-MB-231 cells suggesting a crucial role for interaction of Bak and mitofusins in BITC-mediated inhibition of fusion and morphological dynamics. In conclusion, the present study provides novel insights into the molecular complexity of BITC-induced cell death.

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.

Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased the delivery of mitochondria to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1 light chain 3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modelling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an 'eat-me' signal for the elimination of damaged mitochondria from neuronal cells.

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.

Purpose: Secondary lymphedema is a frequent complication of breast cancer associated with surgery, chemotherapy, or radiation following breast cancer treatment. The potential contribution of genetic susceptibility to risk of developing secondary lymphedema following surgical trauma, radiation, and other tissue insults has not been studied. Experimental Design: To determine whether women with breast cancer and secondary lymphedema had mutations in candidate lymphedema genes, we undertook a case–control study of 188 women diagnosed with breast cancer recruited from the University of Pittsburgh Breast Cancer Program (http://www.upmccancercenter.com/breast/index.cfm) between 2000 and 2010. Candidate lymphedema genes, GJC2 (encoding connexin 47 [Cx47]), FOXC2, HGF, MET, and FLT4 (encoding VEGFR3), were sequenced for mutation. Bioinformatics analysis and in vitro functional assays were used to confirm significance of novel mutations. Results: Cx47 mutations were identified in individuals having secondary lymphedema following breast cancer treatment but not in breast cancer controls or normal women without breast cancer. These novel mutations are dysfunctional as assessed through in vitro assays and bioinformatics analysis and provide evidence that altered gap junction function leads to lymphedema. Conclusions: Our findings challenge the view that secondary lymphedema is solely due to mechanical trauma and support the hypothesis that genetic susceptibility is an important risk factor for secondary lymphedema. A priori recognition of genetic risk (i) raises the potential for early detection and intervention for a high-risk group and (ii) allows the possibility of altering surgical approach and/or chemo- and radiation therapy, or direct medical treatment of secondary lymphedema with novel connexin-modifying drugs. Clin Cancer Res; 18(8); 2382–90. ©2012 AACR.

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.

Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H(2)O(2) was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.

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.

Background: Lymphedema is the abnormal accumulation of protein-rich fluid in the interstitial space. Primary lymphedema is a rare genetic condition with both autosomal dominant and autosomal recessive modes of inheritance. Three genes, FLT4 (VEGFR3), FOXC2, and SOX18 cause varying forms of primary lymphedema. In industrialized countries, secondary lymphedema is usually associated with cancer therapy and/or trauma. Recent observations suggested that hepatocyte growth factor/high affinity hepatocyte growth factor receptor (HGF/MET) were new candidate lymphedema genes. Methods and Results: The coding exons and flanking regions of HGF and MET were directly sequenced in 145 lymphedema probands, 59 unrelated women with secondary lymphedema following treatment for breast cancer, 21 individual patients with lymphedema and intestinal lymphangiectasia, and at least 159 unrelated ethnic matched control individuals. Mutations leading to truncation or missense changes in evolutionarily conserved residues of HGF and MET were identified. These mutations were not polymorphic in control individuals. Conclusions: The identification of HGF/MET mutations in primary lymphedema, lymphedema/lymphangiectasia, and breast cancer-associated secondary lymphedema suggests that the HGF/MET pathway is causal or alters susceptibility for a broad range of lymphedema phenotypes. The HGF/MET pathway provides a new target for the prevention and/or treatment of lymphedema.

Cell migration is an essential process in organ development, differentiation, and wound healing, and it has been hypothesized that gap junctions play a pivotal role in these cell processes. However, the changes in gap junctions and the capacity for cell communication as cells migrate are unclear. To monitor gap junction plaques during cell migration, adrenocortical cells were transfected with cDNA encoding for the connexin 43-green fluorescent protein. Time-lapse imaging was used to analyze cell movements and concurrent gap junction plaque dynamics. Immunocytochemistry was used to analyze gap junction morphology and distribution. Migration was initiated by wounding the cell monolayer and diffusional coupling was demonstrated by monitoring Lucifer yellow dye transfer and fluorescence recovery after photobleaching (FRAP) in cells at the wound edge and in cells located some distance from the wound edge. Gap junction plaques were retained at sites of contact while cells migrated in a "sheet-like" formation, even when cells dramatically changed their spatial relationship to one another. Consistent with this finding, cells at the leading edge retained their capacity to communicate with contacting cells. When cells detached from one another, gap junction plaques were internalized just prior to cell process detachment. Although gap junction plaque internalization clearly was a method of gap junction removal during cell separation, cells retained gap junction plaques and continued to communicate dye while migrating.

We previously demonstrated that the ATP/PKA-dependent activation of the human intermediate conductance, Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal motif. The NH2-terminus of hIK1 contains a multi-basic 13RRRKR17 motif, known to be important in the trafficking and function of ion channels. While individual mutations within this domain have no effect on channel function, the triple mutation (15RKR17/AAA), as well as additional double mutations, result in a near complete loss of functional channels, as assessed by whole-cell patch-clamp. However, cell-surface immunoprecipitation studies confirmed expression of these mutated channels at the plasma membrane. To elucidate the functional consequences of the (15)RKR(17)/AAA mutation we performed inside-out patch clamp recordings where we observed no difference in Ca2+ affinity between the wild-type and mutated channels. However, in contrast to wild-type hIK1, channels expressing the 15RKR17/AAA mutation exhibited rundown, which could not be reversed by the addition of ATP. Wild-type hIK1 channel activity was reduced by alkaline phosphatase both in the presence and absence of ATP, indicative of a phosphorylation event, whereas the 15RKR17/AAA mutation eliminated this effect of alkaline phosphatase. Further, single channel analysis demonstrated that the 15RKR17/AAA mutation resulted in a four-fold lower channel open probability (P(o)), in the presence of saturating Ca2+ and ATP, compared to wild-type hIK1. In conclusion, these results represent the first demonstration for a role of the NH2-terminus in the second messenger-dependent regulation of hIK1 and, in combination with our previous findings, suggest that this regulation is dependent upon a close NH2/C-terminal association.

Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP(2)) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P(2) but not PI(4)P(1) or PIP(3), releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP(2) in the plasma membrane and eliminated EGF-induced calpain activation. This PIP(2)-binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP(2) in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP(2) concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-gamma hydrolyzes PIP(2) in the protruding lamellipodia.

Background— Conduction system defects and slowed ventricular conduction are common in patients with systolic dysfunction and contribute to arrhythmias and sudden death. In animal models of heart failure, cardiac α 1 -adrenergic signaling is constitutively activated. Here, we report the effects of constitutive activation of α 1 -adrenergic signaling on connexin phosphorylation and cardiac conduction. Methods and Results— Transgenic mice were generated with cardiac-specific overexpression of the transcription factor RTEF-1 (transcription enhancer factor-1-related factor), which mediates α 1 -adrenergic signaling in cardiac myocytes. Surface and intracardiac ECGs revealed prolongation of the PR, QRS, and AH intervals and the appearance of progressive atrial arrhythmias in RTEF-1 mice. Optical mapping using voltage-sensitive dye revealed slower conduction velocities across the atrial and ventricular myocardium. Intercellular dye transfer between RTEF-1 transgenic cardiac myocytes confirmed impaired conduction at the cellular level. Conduction defects were correlated with dephosphorylation of connexin40 and connexin43 and upregulation of protein phosphatase 1β (PP1β). Overexpression of PP1β in HeLa cells dephosphorylated cardiac connexin. Confocal microscopy revealed increased levels of dephosphorylated connexin43 at the cardiac gap junctions in RTEF-1 mice, suggesting that defective conduction is a result of impaired gap-junction conductance rather than assembly. Conclusion— Constitutive activation of α 1 -adrenergic signaling through the RTEF-1 transcription factor results in chronic elevation of PP1β expression and connexin dephosphorylation. This mechanism may underlie some defects in cardiac conduction.

HCM continues to be a challenging disease for veterinarians. Acute cases with ATE or CHF are difficult to manage, and we still lack the tools to advise owners well with regard to their pet's prognosis. Nevertheless, it appears that the historical view of HCM as a serious disease with a poor prognosis is now being adjusted to accommodate the apparently large numbers of asymptomatic cats with much longer survival times. Although there is evidence of a genetic cause of the disease in at least several families of cats, no disease-associated mutation has been found to be causative of feline HCM. Prophylactic treatment of asymptomatic or mildly affected cats continues to be empiric, but a randomized, double-blind, placebo-controlled, multicenter study on chronic therapy of symptomatic HCM cats should provide new guidance for practitioners managing these cases.

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.

Hyperthyroidism was diagnosed in 80 cats with thyroid scintigraphy using technetium pertechnetate. These cats were subsequently treated with radioiodine using a modified fixed dose method based on the volume of hyperfunctioning thyroid tissue calculated from the pertechnetate scans. The medical records and thyroid scintigrams were evaluated retrospectively. Follow-up was obtained on the cats to evaluate treatment success. Several parameters were evaluated in an attempt to identify a difference between treatment success and failure. Cats that failed to become euthyroid after one dose of radioiodine had a significantly higher pretreatment serum thyroxine level, had a significantly larger volume of hyperfunctioning thyroid tissue on scintigrams, and cats receiving oral versus intravenous radioiodine were over represented. Based on our results we conclude: 1) the administration of a dose of radioiodine based solely on the volume of hyperfunctioning thyroid tissue as estimated from the pertechnetate scan may be inadequate for those patients with extremely elevated serum thyroxine levels or large thyroid glands, and 2) oral administration of radioiodine is not recommended for the treatment of feline hyperthyroidism.

A 4-month-old male Labrador Retriever was presented for recurrent bouts of pulmonary edema associated with tachycardia. Initial physical examination and echocardiography were unremarkable, and the electrocardiogram revealed only an intraventricular conduction disturbance. Subsequent recordings showed paroxysmal supraventricular tachycardia (SVT) (340 beats/min), which consistently produced pulmonary edema. The supraventricular tachycardia was unresponsive to adenosine, esmolol, and propranolol; was variable and transiently responsive to various vagal maneuvers and precordial thumps; and was always responsive to IV diltiazem. Multiple life-threatening episodes of SVT occurred, however, despite the chronic administration of oral diltiazem, propranolol, and procainamide. Diastolic cardiac dysfunction was documented by Doppler echocardiography and was thought to contribute to the development of pulmonary edema. A subsequent electrophysiologic study confirmed the presence of an atrioventricular posteroseptal accessory pathway that participated in orthodromic reciprocating tachycardia. This pathway was determined to conduct only in the retrograde direction ("concealed accessory pathway"). Intraoperative IV procainamide titration terminated the arrhythmia, which could not be reinduced when procainamide blood concentration approximated 20 micrograms/dL. Increasing the oral procainamide dose to achieve such plasma concentrations was successful in eliminating orthodromic reciprocating tachycardia, preventing heart failure, and returning Doppler indices of diastolic function to normal.

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.

BH3-interacting domain death agonist (Bid), a BH3-only B cell lymphoma 2 family molecule, is generally known for its importance in activating the mitochondrial apoptosis pathway after death receptor engagement, particularly in hepatocytes. However, Bid also promotes hepatocyte proliferation during liver regeneration and carcinogenesis. This study was designed to examine the hypothesis that Bid regulates endoplasmic reticulum calcium concentration ([Ca2+]ER) homeostasis to affect hepatocyte proliferation. We found that serum-stimulated hepatocyte proliferation was dependent on calcium, and the depletion of calcium with thapsigargin or ethylene glycol tetraacetic acid (EGTA) inhibited the proliferation. Subcellular fractionation showed that a portion of Bid was inserted into the endoplasmic reticulum (ER)–enriched membranes, and single-cell calcium imaging indicated that Bid was important for maintaining the [Ca2+]ER level. Bid-deficient hepatocytes manifested delayed and reduced serum-stimulated proliferation, which was corrected by ionomycin or reconstitution of Bid, particularly an ER-targeted Bid. Finally, B cell lymphoma 2–associated X protein (Bax) could also be found in the ER-enriched membranes, and Bax deficiency caused the same proliferation defect. However, Bid/Bax double deletion in hepatocytes did not further augment the defect, which suggested that Bid and Bax worked by the same regulatory mechanism in [Ca2+]ER control. Conclusion: Bid regulates hepatocyte proliferation by positively affecting [Ca2+]ER homeostasis, and this could be important for liver regeneration and carcinogenesis. (HEPATOLOGY 2010)

The vacuolar H(+)-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V(1) sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO(3)(-)-containing culture medium compared with cells expressing vector alone or a PKA phosphorylation-deficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H(+) secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO(3)(-)-free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175.

Bid, a BH3-only Bcl-2 family molecule, is generally known for its importance in activating the mitochondrial apoptosis pathway following death receptor engagement, particularly in hepatocytes. Howe...

Background— Carbon monoxide (CO) has emerged as a vascular homeostatic molecule that prevents balloon angioplasty–induced stenosis via antiproliferative effects on vascular smooth muscle cells. The effects of CO on reendothelialization have not been evaluated. Methods and Results— Exposure to CO has diametrically opposite effects on endothelial cell (EC) and vascular smooth muscle cell proliferation in rodent models of carotid injury. In contrast to its effect of blocking vascular smooth muscle cell growth, CO administered as a gas or as a CO-releasing molecule enhances proliferation and motility of ECs in vitro by >50% versus air controls, and in vivo, it accelerates reendothelialization of the denuded artery by day 4 after injury versus day 6 in air-treated animals. CO enhanced EC proliferation via rapid activation of RhoA (Ras homolog gene family, member A), followed by downstream phosphorylation of Akt, endothelial nitric oxide (NO) synthase phosphorylation, and a 60% increase in NO generation by ECs. CO drives cell cycle progression through phosphorylation of retinoblastoma, which is dependent in part on endothelial NO synthase–generated NO. Similarly, endothelial repair in vivo requires NO-dependent mobilization of bone marrow–derived EC progenitors, and CO yielded a 4-fold increase in the number of mobilized green fluorescent protein–Tie2–positive endothelial progenitor cells versus controls, with a corresponding accelerated deposition of differentiated green fluorescent protein–Tie2–positive ECs at the site of injury. CO was ineffective in augmenting EC repair and the ensuing development of intimal hyperplasia in eNOS −/− mice. Conclusions— Collectively, the present data demonstrate that CO accelerates EC proliferation and vessel repair in a manner dependent on NO generation and enhanced recruitment of bone marrow–derived endothelial progenitor cells.

The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel and epithelial Na(+) channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (I(sc)), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2-5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent I(sc) in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red-dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03-1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease.