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1.  The exocyst and regulatory GTPases in urinary exosomes 
Physiological Reports  2014;2(8):e12116.
Abstract
Cilia, organelles that function as cellular antennae, are central to the pathogenesis of “ciliopathies”, including various forms of polycystic kidney disease (PKD). To date, however, the molecular mechanisms controlling ciliogenesis and ciliary function remain incompletely understood. A recently proposed model of cell–cell communication, called “urocrine signaling”, hypothesizes that a subset of membrane bound vesicles that are secreted into the urinary stream (termed exosome‐like vesicles, or ELVs), carry cilia‐specific proteins as cargo, interact with primary cilia, and affect downstream cellular functions. This study was undertaken to determine the role of the exocyst, a highly conserved eight‐protein trafficking complex, in the secretion and/or retrieval of ELVs. We used Madin–Darby canine kidney (MDCK) cells expressing either Sec10‐myc (a central component of the exocyst complex) or Smoothened‐YFP (a ciliary protein found in ELVs) in experiments utilizing electron gold microscopy and live fluorescent microscopy, respectively. Additionally, human urinary exosomes were isolated via ultracentrifugation and subjected to mass‐spectrometry‐based proteomics analysis to determine the composition of ELVs. We found, as determined by EM, that the exocyst localizes to primary cilia, and is present in vesicles attached to the cilium. Furthermore, the entire exocyst complex, as well as most of its known regulatory GTPases, are present in human urinary ELVs. Finally, in living MDCK cells, ELVs appear to interact with primary cilia using spinning disc confocal microscopy. These data suggest that the exocyst complex, in addition to its role in ciliogenesis, is centrally involved in the secretion and/or retrieval of urinary ELVs.
Our data suggest that the exocyst complex, in addition to its role in ciliogenesis that we previously described, is centrally involved in the secretion and/or retrieval of urinary exosomes. These results could have important implications for PKD, other renal diseases, as well as normal kidney homeostasis.
doi:10.14814/phy2.12116
PMCID: PMC4246586  PMID: 25138791
Cilia; ciliopathies; ELV; exocyst; polycystic kidney disease; urinary exosomes
2.  Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach 
Nephron Extra  2011;1(1):217-223.
Background/Aims
Gene therapy involves delivery of exogenous DNA to provide a therapeutic protein. Ideally, a gene therapy vector should be non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into target cells.
Methods
Adeno-associated virus (AAV) offers these advantages and few, if any, disadvantages, and over 100 isolates exist. We previously showed that AAV-mediated gene therapy can be used to restore vision to patients with Leber's congenital amaurosis, a disease of childhood blindness.
Results
Here we show that novel recombinant AAV2/8 and AAV2/9 transduce kidney tubule cells with high efficiency both in vitroin cell culture and in vivoin mice. In addition, we adapted and modified a retrograde approach to allow for optimal transgene delivery to renal tubular cells that further minimizes the risk of an immunogenic reaction.
Conclusions
We believe that recombinant AAV2, especially AAV2/8, gene delivery to renal tubule cells via a retrograde approach represents a viable method for gene therapy for a multitude of renal disorders ranging from autosomal dominant polycystic kidney disease to acute kidney injury.
doi:10.1159/000333071
PMCID: PMC3290852  PMID: 22470395
Adeno-associated virus; Gene therapy; Kidney injury
3.  Exocyst Sec10 is Involved in Basolateral Protein Translation and Translocation in the Endoplasmic Reticulum 
Nephron. Experimental nephrology  2012;120(4):e134-e140.
Background
Protein translation and translocation at the rough endoplasmic reticulum (RER) are the first steps in the secretory pathway. The translocon through which newly-made proteins are translocated into or across the RER membrane, consists of three main subunits, Sec61α, β, and γ. Sec61β facilitates translocation, and we and others showed that the highly-conserved eight protein exocyst complex interacts with Sec61β. We also showed that the exocyst was involved in basolateral, and not apical, protein synthesis and delivery. Recently, however, exocyst involvement in apical protein delivery was reported. Furthermore, we showed that the exocyst was necessary for formation of primary cilia, organelles found on the apical surface.
Methods
GST pulldown was performed on lysate of renal tubule cells to investigate biochemical interactions. Cell-free assays consisting of cell-free extracts from rabbit reticulocytes, pancreatic ER microsomal membranes, transcripts of cDNA from apical and basolateral proteins, ATP/GTP, amino acids, and 35S-methionine for protein detection, were used to investigate the role of the exocyst in synthesis of polarized proteins. P32-orthophosphate and immunoprecipitation with antibody against Sec61β was used to investigate the Sec61β phosphorylation in exocyst Sec10-overexpressing cells.
Results
Sec10 biochemically interacts with Sec61β using GST pulldown. Using cell-free assays, there is enhanced recruitment to ER membranes following exocyst depletion and basolateral VSVG protein translation, compared to apical HA protein translation. Finally, Sec10 overexpression increases Sec61β phosphorylation.
Conclusion
These data confirm that the exocyst is preferentially involved in basolateral protein translation and translocation, and may well act through the phosphorylation of Sec61β.
doi:10.1159/000342366
PMCID: PMC3740206  PMID: 23037926
exocyst; polarity; translation; endoplasmic reticulum
4.  The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro 
Molecular Biology of the Cell  2009;20(10):2522-2529.
Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, in which they are felt to participate in flow sensing and have been linked to the pathogenesis of cystic renal disorders such as autosomal dominant polycystic kidney disease. We previously localized the exocyst, an eight-protein complex involved in membrane trafficking, to the primary cilium of Madin-Darby canine kidney cells and showed that it was involved in cystogenesis. Here, using short hairpin RNA (shRNA) to knockdown exocyst expression and stable transfection to induce exocyst overexpression, we show that the exocyst protein Sec10 regulates primary ciliogenesis. Using immunofluorescence, scanning, and transmission electron microscopy, primary cilia containing only basal bodies are seen in the Sec10 knockdown cells, and increased ciliogenesis is seen in Sec10-overexpressing cells. These phenotypes do not seem to be because of gross changes in cell polarity, as apical, basolateral, and tight junction proteins remain properly localized. Sec10 knockdown prevents normal cyst morphogenesis when the cells are grown in a collagen matrix, whereas Sec10 overexpression results in increased cystogenesis. Transfection with human Sec10 resistant to the canine shRNA rescues the phenotype, demonstrating specificity. Finally, Par3 was recently shown to regulate primary cilia biogenesis. Par3 and the exocyst colocalized by immunofluorescence and coimmunoprecipitation, consistent with a role for the exocyst in targeting and docking vesicles carrying proteins necessary for primary ciliogenesis.
doi:10.1091/mbc.E08-07-0772
PMCID: PMC2682593  PMID: 19297529
5.  Intracellular Signaling via ERK/MAPK Completes the Pathway for Tubulogenic Fibronectin in MDCK Cells 
A classic in vitro model of branching morphogenesis utilizes the Madin-Darby canine kidney (MDCK) cell line. MDCK Strain II cells form hollow monoclonal cysts in a three-dimensional collagen matrix over the course of ten days and tubulate in response to hepatocyte growth factor (HGF). We and our colleagues previously showed that activation of the extracellular-signal regulated kinase (ERK, aka MAPK) pathway is necessary and sufficient to induce tubulogenesis in MDCK cells. We also showed in a microarray study that one of the genes upregulated by HGF was the known tubulogene fibronectin. Given that HGF activates a multitude of signaling pathways, including ERK/MAPK, to test the intracellular regulatory pathway, we used two distinct inhibitors of ERK activation (U0126 and PD098059). Following induction of MDCK Type II cells with HGF, tubulogenic fibronectin mRNA was upregulated 4-fold by real time PCR, and minimal or no change in fibronectin expression was seen when HGF was added with either U0126 or PD098059. We confirmed these results using an MDCK cell line inducible for Raf, which is upstream of ERK. Following activation of Raf, fibronectin mRNA and protein expression were increased to a similar degree as was seen following HGF induction. Furthermore, MDCK Strain I cells, which originate from collecting ducts and have constitutively active ERK, spontaneously initiate tubulogenesis. We show here that MDCK Strain I cells have high levels of fibronectin mRNA and protein compared to MDCK Strain II cells. When U0126 and PD098059 were added to MDCK Strain I cells, fibronectin mRNA and protein levels were decreased to levels seen in MDCK Strain II cells. These data allow us to complete what we believe is the first description of a tubulogenic pathway from receptor/ligand (HGF/CMET), through an intracellular signaling pathway (ERK/MAPK), to transcription and, finally, secretion of a critical tubuloprotein (fibronectin).
doi:10.1016/j.bbrc.2006.12.106
PMCID: PMC1839983  PMID: 17196167
ERK; MDCK; Tubulogenesis; Fibronectin
6.  High rate of fistula placement in a cohort of dialysis patients in a single payer system 
Arteriovenous fistulas (AVFs) are considered superior to arteriovenous grafts and catheters. Never-theless, AVF prevalence in the United States remains under the established target. The complication rates and financial cost of vascular access continue to rise and disproportionately contribute to the burgeoning health care costs. The relationship between financial incentives for a type of vascular access and rate of access placement is unclear. All chronic hemodialysis patients (n=99) receiving care at Philadelphia Veterans Affairs Medical Center as of August 1, 2008 were participants. Demographic characteristics, vascular access type, and nonrelative value unit compensation were assessed as predictors, and the vascular access prevalence rate, operative times, and frequency of access interventions were analyzed. A 73.7% AVF rate was achieved in this cohort of patients with 51.5% diabetes mellitus. The number of access procedures per patient per year remained constant over time. The Philadelphia Veterans Affairs Medical Center, a single payer system, achieved superior AVF prevalence and exceeded the national AVF target. Financial incentives for arteriovenous graft placement currently exist in the United States, as there is similar Medicare reimbursement for arterio-venous graft and basilic vein transposition, despite longer operative times for basilic vein transpositions. The high AVF prevalence at the Philadelphia Veterans Affairs Medical Center may be due to the VA nonrelative value unit-driven system that allows for interdisciplinary care, priority of AVFs, and frequent use of basilic vein transposition surgery, when appropriate. We have identified an important, hypothesis-generating example of a nonrelative value unit-based approach to vascular access yielding superior results with respect to patient care and cost.
doi:10.1111/j.1542-4758.2010.00479.x
PMCID: PMC3082920  PMID: 20812959
Fistula; hemodialysis; health care economics
7.  The Exocyst Protein Sec10 Interacts with Polycystin-2 and Knockdown Causes PKD-Phenotypes 
PLoS Genetics  2011;7(4):e1001361.
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown—including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation—suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.
Author Summary
ADPKD, the most common potentially lethal monogenetic disorder, is caused by mutations in PKD1 and PKD2. We are beginning to appreciate the important roles these gene products, and others, play in cilia, which are thin rod-like organelles projecting from the cell surface. Defects in cilia function are associated with a variety of human diseases, including all variants of polycystic kidney disease. Despite intense study of cilia and how they influence disease, it is not understood how proteins are targeted and delivered to cilia. Our work provides the first link between the exocyst, a conserved eight-protein complex involved in protein localization, and a disease gene, PKD2. Knockdown of the exocyst protein Sec10 results in a number of cellular- and cilia-related phenotypes that are also seen upon pkd2 loss—both in kidney cells and zebrafish. We then demonstrate specific genetic and biochemical interactions between sec10 and pkd2. We further show that Sec10 interacts with other ciliary proteins, such as IFT20 and IFT88. From this work, we propose that the exocyst is involved in bringing multiple types of ciliary proteins to the cilium. Given that the exocyst is required for cilia structure and function, the exocyst may play a role in cilia-related human diseases.
doi:10.1371/journal.pgen.1001361
PMCID: PMC3072367  PMID: 21490950
8.  The Bioethics and Utility of Selling Kidneys for Renal Transplantation 
Transplantation proceedings  2008;40(5):1264-1270.
In the fifty years since kidney transplantation was first performed, this procedure has evolved from a highly speculative biomedical endeavor to a medically viable and often standard course of therapy (1). Long-term survival is markedly improved among patients who receive a kidney compared with patients who remain on the waiting list for such an organ (2). As outcomes have improved and more clinical indications have emerged, the number of people awaiting transplantation has grown significantly.
In stark contrast to the robust expansion of the waiting list, the number of available deceased donors has remained relatively constant over the last several years (1). The current mechanism for procuring kidneys relies on voluntary donations by the general public, with the primary motivation being altruism. However, in light of the ever-increasing waiting list, it is the authors’ belief that the current system needs to be revised if supply is ever going to meet demand. In response to this critical organ shortage, different programs have been developed in an attempt to increase organ donation. At present, however, no solution to the problem has emerged. This paper begins by outlining the scope of the problem and current legislation governing the procurement of transplantable organs/tissues in the United States. It continues with an overview of different proposals to increase supply. It concludes by exploring some of the controversy surrounding the proposal to increase donation using financial incentives. Though the following discussion certainly has implications for other transplantable organs, this paper will focus on kidney transplantation because the waiting list for kidneys is by far the longest of all the solid organs; and, as it carries the smallest risk to living donors, is the least ethically problematic.
doi:10.1016/j.transproceed.2008.03.095
PMCID: PMC2504358  PMID: 18589084
Bioethics; Transplantation; End Stage Renal Disease
9.  Exocyst Is Involved in Cystogenesis and Tubulogenesis and Acts by Modulating Synthesis and Delivery of Basolateral Plasma Membrane and Secretory Proteins 
Molecular Biology of the Cell  2000;11(12):4259-4275.
Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.
PMCID: PMC15071  PMID: 11102522

Results 1-9 (9)