Development of a DNA transfer technique for human lung cells with synthetic peptide Tat-RGD and its application for regulatory and functional analysis of RELMB

The presented study is organized in two sections. In the first part a DNA-transfection procedure for human lung cells employing a peptide conjugate has been established and the corresponding transfection process characterized. In the second part this new transfection strategy has been applied for studying functional aspects of the human RELMBbeta gene. Cell-lines and primary cells exhibit a varying degree of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide TatRGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into human pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded in significant transgene expression. When combining TRD with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by ~30-fold over TRD mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL mediated transfection efficiency was observed by a factor of ~ 2 and ~ 3 over that of DL. Laser scanning confocal microscopy for visualizing TR dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the TatRGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with standard cationic lipid based transfection reagent. The enhancement of DNA uptake by TatRGD is suggested to be mediated by caveoli dependent endocytosis. RELMBbeta(resistin-like molecule) represents the most related human homologue of mouse RELMBbeta also known as hypoxic-induced-mitogenic-factor (HIMF) for which no human orthologue gene exist. In this part of the study, we isolated RELMBbeta cDNA from human lung tissue and performed regulatory and functional expression studies with the TatRGD procedure. RELMBbeta mRNA was upregulated in hypoxia in human lung A549 cell line as well as primary cultured adventitial fibroblasts (FB) and smooth muscle cells (SMC) of pulmonary artery. Upon transfection of a RELMBbeta encoding expression plasmid into these cells, we observed significant induction of proliferation particularly in SMC and A549 cells. The results suggest that human RELMBbeta may contribute to hypoxic induced pulmonary vascular remodeling processes or hypoxia related fibrotic lung disease.Die vorgelegte Studie ist in zwei Abschnitte gegliedert. Im ersten Teil wird eine DNATransfektions Prozedur mit Hilfe eines Peptid-Konjugats etabliert und der zugehörige Transfektions Prozess charakterisiert. Im zweiten Teil wird diese Transfektions Strategie angewendet, um funktionelle Aspekte des humanen RELMBbeta Gens zu untersuchen. Zelllinien und primäre Zellen zeigen eine unterschiedliche Resistenz gegenüber DNATransfektions Methoden. In dieser Studie wurde das synthetische Peptid TatRGD (TR), das aus dem von HIV-1 abgeleitetem Translokations Peptid Tat und dem Integrin bindenden RGD Motiv zusammengesetzt ist, genutzt, um den DNA Transfer in humane pulmonale Zellen zu begünstigen. Bindungsexperimente zwischen DNA und TR, sowie Zytotoxizitäts Messungen von TR behandelten Zellen wurden durchgeführt, um die zu verwendenden DNA und TR Konzentrationen zu optimieren. Zugabe eines Komplexes von TR und DNA (TRD) zu A549 Zellen resultierte in signifikanter transgener Expression. Die Kombination von TRD und Lipofectamin (TRDL) verstärkte die Expression 5-fach gegebenüber Lipofectamin (DL) und 30-fach gegenüber TRD vermittelter Transfektion. Ebenso in primär kultivierten glatten Muskelzellen (SMC) und adventitialen Fibroblasten (FB), die aus Pulmonalarterien gewonnen wurden, wurde eine Steigerung der Transfektionseffizienz um den Faktor 2 bzw. 3 gegenüber DL beobachtet. Laser-Scan basierte konfokale Mikroskopie zeigte, dass die TR abhängige DNA Aufnahme abhängig von Caveolae in der Plasmamembran ist. Inhibition der Caveolae Bildung durch Methyl-b-Cyclo-Dextrin verminderte drastisch die Transfektions Effizienz durch TR. Zusammengefasst, vermittelt TatRGD einen effizienten DNA Transfer in humane pulmonale Zellen, insbesondere wenn es mit Transfektions Reagenzien, die auf kationischen Lipiden basieren, kombiniert wird. Die Steigerung der DNA Aufnahme wird vermutlich durch Caveolae-abhängige Endozytose vermittelt. RELMBbeta(resistin-like molecule) repräsentiert das am nächsten homologe Gen von RELMBbeta der Maus, das auch unter dem Namen HIMF (hypoxic-induced-mitogenicfactor) bekannt ist, für das aber kein orthologes humanes Gen existiert. In diesem Teil der Studie isolierten wir RELMBbeta cDNA von humanem Lungen Gewebe und führten funktionelle Expressions Studien mittels der TatRGD Methode sowie Untersuchungen zu seiner Regulation durch. Wir beobachteten eine Hochregulation von RELMBbeta mRNA in Hypoxie in der epithelialen humanen Lungenzellinie A549 sowie in primär kultivierten FB und SMC der Pulmonalarterie. Nach Transfektion der Zellen mit einem Expressions plasmid kodierend für RELMBbeta beobachteten wir eine signifikante Induktion der Proliferation insbesondere in SMC und A549 Zellen. Die Ergebnisse weisen darauf hin, dass humanes RELMBbeta am pulmonal-arteriellen Remodeling Prozess in Hypoxie oder an Hypoxie-assozierten fibrotischen Lungenerkrankungen beteiligt sein könnte

Human RELMβ is a mitogenic factor in lung cells and induced in hypoxia

AbstractRELMβ (resistin-like molecule) represents the most related human homologue of mouse RELMα, also known as hypoxic-induced mitogenic factor (HIMF). In this study, we isolated RELMβ cDNA from human lung tissue and performed regulatory and functional expression studies. RELMβ mRNA was upregulated in hypoxia in human lung A549 cell line as well as primary cultured adventitial fibroblasts and smooth muscle cells (SMC) of pulmonary arteries. Upon transfection of a RELMβ encoding expression plasmid into these cells, we observed significant induction of proliferation particularly in SMC and A549 cells, which could be blocked by phosphatidyl-inositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin. The results suggest that human RELMβ may contribute to hypoxic-induced pulmonary vascular remodeling processes or hypoxia related fibrotic lung disease

Defects in KCNJ16 Cause a Novel Tubulopathy with Hypokalemia, Salt Wasting, Disturbed Acid-Base Homeostasis, and Sensorineural Deafness

Background: The transepithelial transport of electrolytes, solutes, and water in the kidney is a well-orchestrated process involving numerous membrane transport systems. Basolateral potassium channels in tubular cells not only mediate potassium recycling for proper Na+,K+-ATPase function but are also involved in potassium and pH sensing. Genetic defects in KCNJ10 cause EAST/SeSAME syndrome, characterized by renal salt wasting with hypokalemic alkalosis associated with epilepsy, ataxia, and sensorineural deafness. Methods: A candidate gene approach and whole-exome sequencing determined the underlying genetic defect in eight patients with a novel disease phenotype comprising a hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Electrophysiologic studies and surface expression experiments investigated the functional consequences of newly identified gene variants. Results: We identified mutations in the KCNJ16 gene encoding KCNJ16, which along with KCNJ15 and KCNJ10, constitutes the major basolateral potassium channel of the proximal and distal tubules, respectively. Coexpression of mutant KCNJ16 together with KCNJ15 or KCNJ10 in Xenopus oocytes significantly reduced currents. Conclusions: Biallelic variants in KCNJ16 were identified in patients with a novel disease phenotype comprising a variable proximal and distal tubulopathy associated with deafness. Variants affect the function of heteromeric potassium channels, disturbing proximal tubular bicarbonate handling as well as distal tubular salt reabsorption. Keywords: KCNJ10; KCNJ15; KCNJ16; acid-base homeostasis; deafness; distal tubule; hypokalemia; potassium channels; proximal tubule; tubulopathy

Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl– blocker, whereas CLDN16 functioned as a Na+ channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl– permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC

Tamm-Horsfall Glycoprotein Interacts with Renal Outer Medullary Potassium Channel ROMK2 and Regulates Its Function*

Tamm-Horsfall glycoprotein (THGP) or Uromodulin is a membrane protein exclusively expressed along the thick ascending limb (TAL) and early distal convoluted tubule (DCT) of the nephron. Mutations in the THGP encoding gene result in Familial Juvenile Hyperuricemic Nephropathy (FJHN), Medullary Cystic Kidney Disease type 2 (MCKD-2), and Glomerulocystic Kidney Disease (GCKD). The physicochemical and biological properties of THGP have been studied extensively, but its physiological function in the TAL remains obscure. We performed yeast two-hybrid screening employing a human kidney cDNA library and identified THGP as a potential interaction partner of the renal outer medullary potassium channel (ROMK2), a key player in the process of salt reabsorption along the TAL. Functional analysis by electrophysiological techniques in Xenopus oocytes showed a strong increase in ROMK current amplitudes when co-expressed with THGP. The effect of THGP was specific for ROMK2 and did not influence current amplitudes upon co-expression with Kir2.x, inward rectifier potassium channels related to ROMK. Single channel conductance and open probability of ROMK2 were not altered by co-expression of THGP, which instead increased surface expression of ROMK2 as determined by patch clamp analysis and luminometric surface quantification, respectively. Despite preserved interaction with ROMK2, disease-causing THGP mutants failed to increase its current amplitude and surface expression. THGP−/− mice exhibited increased ROMK accumulation in intracellular vesicular compartments when compared with WT animals. Therefore, THGP modulation of ROMK function confers a new role of THGP on renal ion transport and may contribute to salt wasting observed in FJHN/MCKD-2/GCKD patients

Defects in KCNJ16 cause a novel tubulopathy with hypokalemia, salt wasting, disturbed acid-base homeostasis, and sensorineural deafness

Background The transepithelial transport of electrolytes, solutes, and water in the kidney is a wellorchestrated process involving numerous membrane transport systems. Basolateral potassium channels in tubular cells not onlymediate potassiumrecycling for proper Na+, K+-ATPase function but are also involved in potassium and pH sensing. Genetic defects in KCNJ10 cause EAST/SeSAME syndrome, characterized by renal salt wasting with hypokalemic alkalosis associated with epilepsy, ataxia, and sensorineural deafness. Methods A candidate gene approach and whole-exome sequencing determined the underlying genetic defect in eight patients with a novel disease phenotype comprising a hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineuraldeafness. Electrophysiologic studies and surface expression experiments investigated the functional consequences of newly identified gene variants. ResultsWeidentifiedmutations in the KCNJ16 gene encoding KCNJ16, which along with KCNJ15 and KCNJ10, constitutes the major basolateral potassium channel of the proximal and distal tubules, respectively. Coexpression of mutant KCNJ16 together with KCNJ15 or KCNJ10 in Xenopus oocytes significantly reduced currents. Conclusions Biallelic variants inKCNJ16were identified in patients with a novel disease phenotype comprising a variable proximal and distal tubulopathy associated with deafness. Variants affect the function of heteromeric potassium channels, disturbing proximal tubular bicarbonate handling aswell as distal tubular salt reabsorption