Herpes simplex virus as a model vector system for gene therapy in renal disease

Herpes simplex virus as a model vector system for gene therapy in renal disease. The past decade has been marked by significant advances in the application of gene transfer into living cells of animals and humans. These approaches have been tested in a few animal models of inherited and acquired renal diseases, including carbonic anhydrase II deficiency 1 and experimental glomerulonephritis2,3. Gene transfer into proximal tubular cells has been successfully accomplished by intrarenal arterial infusion of a liposomal complex4 or an adenoviral vector5. Tubular cells from the papilla and medulla have been selectively transduced by retrograde infusion into the pelvi-calyceal system of an adenoviral vector containing a reporter for β-galactosidase5. Although the results of these initial studies are promising, further studies to optimize viral vectors, maximize gene delivery, minimize side-effects, and develop cell-specific and long-term regulated gene expression are critical to the success of gene therapy targeted to specific compartments of the kidney. Our recent efforts have focused on defining the cellular pathways responsible for viral entry and infection into renal epithelial cells using herpes simplex virus (HSV) as a model vector. We anticipate that a solid understanding of the basic scientific principles underlying viral entry and gene expression into specific populations of renal cells will facilitate the design of successful therapeutic viral-based gene transfer strategies

Herpes simplex virus triggers activation of calcium-signaling pathways

The cellular pathways required for herpes simplex virus (HSV) invasion have not been defined. To test the hypothesis that HSV entry triggers activation of Ca2+-signaling pathways, the effects on intracellular calcium concentration ([Ca2+]i) after exposure of cells to HSV were examined. Exposure to virus results in a rapid and transient increase in [Ca2+]i. Pretreatment of cells with pharmacological agents that block release of inositol 1,4,5-triphosphate (IP3)–sensitive endoplasmic reticulum stores abrogates the response. Moreover, treatment of cells with these pharmacological agents inhibits HSV infection and prevents focal adhesion kinase (FAK) phosphorylation, which occurs within 5 min after viral infection. Viruses deleted in glycoprotein L or glycoprotein D, which bind but do not penetrate, fail to induce a [Ca2+]i response or trigger FAK phosphorylation. Together, these results support a model for HSV infection that requires activation of IP3-responsive Ca2+-signaling pathways and that is associated with FAK phosphorylation. Defining the pathway of viral invasion may lead to new targets for anti-viral therapy

Effects of biomechanical forces on signaling in the cortical collecting duct (CCD)

An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm2 of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2 release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD

Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpkTg737) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca2+ primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca2+ derived from both extracellular and intracellular stores. This flow-induced Ca2+ signal was less robust in cilium-deficient monolayers. Flow-induced Ca2+ signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca2+. Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na+) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases

Herpes simplex virus as a model vector system for gene therapy in renal disease

Herpes simplex virus as a model vector system for gene therapy in renal disease. The past decade has been marked by significant advances in the application of gene transfer into living cells of animals and humans. These approaches have been tested in a few animal models of inherited and acquired renal diseases, including carbonic anhydrase II deficiency 1 and experimental glomerulonephritis2,3. Gene transfer into proximal tubular cells has been successfully accomplished by intrarenal arterial infusion of a liposomal complex4 or an adenoviral vector5. Tubular cells from the papilla and medulla have been selectively transduced by retrograde infusion into the pelvi-calyceal system of an adenoviral vector containing a reporter for β-galactosidase5. Although the results of these initial studies are promising, further studies to optimize viral vectors, maximize gene delivery, minimize side-effects, and develop cell-specific and long-term regulated gene expression are critical to the success of gene therapy targeted to specific compartments of the kidney. Our recent efforts have focused on defining the cellular pathways responsible for viral entry and infection into renal epithelial cells using herpes simplex virus (HSV) as a model vector. We anticipate that a solid understanding of the basic scientific principles underlying viral entry and gene expression into specific populations of renal cells will facilitate the design of successful therapeutic viral-based gene transfer strategies