Kurt Amsler

PRKX, a phylogenetically and functionally distinct cAMP-dependent protein kinase, activates renal epithelial cell migration and morphogenesis

The human protein kinase X gene (PRKX) is a member of an ancient family of cAMP-dependent serine/threonine kinases here shown to be phylogenetically distinct from the classical PKA, PKB/Akt, PKC, SGK, and PKG gene families. Renal expression of the PRKX gene is developmentally regulated and restricted to the ureteric bud epithelium of the fetal metanephric kidney. Aberrant adult kidney expression of PRKX was found in autosomal dominant polycystic kidney disease. PRKX kinase expression markedly activated migration of cultured renal epithelial cells in the presence of cAMP; this effect was blocked by cell treatment with the PKA inhibitor H89 and was not observed in PKA-transfected cells. In addition, expression of PRKX kinase activated branching morphogenesis of Madin–Darby canine kidney cells in collagen gels even in the absence of cAMP and/or hepatocyte growth factor, an effect not seen with either PKA expression or expression of a mutant, kinase-inactivated PRKX. These results suggest that the PRKX kinase may regulate epithelial morphogenesis during mammalian kidney development. Because another member of the PRKX gene family (the Dictyostelium discoideum gene KAPC-DICDI) also plays a role in cellular migration, these studies suggest that regulation of morphogenesis may be a distinctive property of these genes that has been conserved in evolution that is not shared with PKA family genes.

Abnormalities in focal adhesion complex formation, regulation, and function in human autosomal recessive polycystic kidney disease epithelial cells

Integrin-associated focal adhesion complex formation and turnover plays an essential role in directing interactions between epithelial cells and the extracellular matrix during organogenesis, leading to appropriate cell spreading, cell-matrix adhesion, and migration. Autosomal recessive polycystic kidney disease (ARPKD) is associated with loss of function of PKHD1-encoded protein fibrocystin-1 and is characterized by cystic dilation of renal collecting tubules (CT) in utero and loss of renal function in patients if they survive the perinatal period. Normal polycystin-1 (PC-1)/focal adhesion complex function is required for control of CT diameter during renal development, and abnormalities in these complexes have been demonstrated in human PC-1 mutant cystic cells. To determine whether loss of fibrocystin-1 was associated with focal adhesion abnormalities, ARPKD cells or normal age-matched human fetal (HF)CT cells in which fibrocystin-1 had been decreased by 85% by small interfering RNA inhibition were compared with normal HFCT. Accelerated attachment and spreading on collagen matrix and decreased motility of fibrocystin-1-deficient cells were associated with longer paxillin-containing focal adhesions, more complex actin-cytoskeletal rearrangements, and increased levels of total beta(1)-integrin, c-Src, and paxillin. Immunoblot analysis of adhesive cells using site-specific phospho-antibodies demonstrated ARPKD-associated loss of activation of focal adhesion kinase (FAK) by phosphorylation at its autophosphorylation site (Y397); accelerated FAK inhibition by phosphorylation at Y407, S843, and S910; as well as increased activation of c-Src at pY418. Paxillin coimmunoprecipitation analyses suggested that fibrocystin-1 was a component of the normal focal adhesion complex and that actin and fibrocystin-1 were lost from ARPKD complexes.

Midkine promotes selective expansion of the nephrogenic mesenchyme during kidney organogenesis.

During kidney development, the growth and development of the stromal and nephrogenic mesenchyme cell populations and the ureteric bud epithelium is tightly coupled through intricate reciprocal signaling mechanisms between these three tissue compartments. Midkine, a target gene activated by retinoid signaling in the metanephros, encodes a secreted polypeptide with mitogenic and anti-apoptotic activities in a wide variety of cell types. Using immmunohistochemical methods we demonstrated that Midkine is found in the uninduced mesenchyme at the earliest stages of metanephric kidney development and only subsequently concentrated in the ureteric bud epithelium and basement membrane. The biological effects of purified recombinant Midkine were analyzed in metanephric organ culture experiments carried out in serum-free defined media. These studies revealed that Midkine selectively promoted the overgrowth of the Pax-2 and N-CAM positive nephrogenic mesenchymal cells, failed to stimulate expansion of the stromal compartment and suppressed branching morphogenesis of the ureteric bud. Midkine suppressed apoptosis and stimulated cellular proliferation of the nephrogenic mesenchymal cells, and was capable of maintaining the viability of isolated mesenchymes cultured in the absence of the ureteric bud. These results suggest that Midkine may regulate the balance of epithelial and stromal progenitor cell populations of the metanephric mesenchyme during renal organogenesis.

Cyclic adenosine monophosphate modulates cell morphology and behavior of a cultured renal epithelial

The role of cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) in modulating functions of differentiated renal cells is well established. Its importance in controlling their growth and differentiation is less clear. We have used somatic cell genetic techniques to probe the role of PKA in controlling morphology and behavior of a renal epithelial cell line, LLC-PK1, which acquires many properties characteristic of the renal proximal tubular cell. Mutants of this line altered in PKA activity have been isolated and their behavior compared to that of the parent line. The results indicate that PKA is involved, either directly or indirectly, in maintenance of cell morphology, cell-cell and cell-substratum interactions, density-dependent growth regulation, and expression of one function characteristic of the renal proximal tubular cell, Na-hexose symport. The relevance of these results to the role of PKA in controlling growth and differentiation of renal epithelial cells in vivo is discussed.

Occludin Content Modulates Hydrogen Peroxide‐Induced Increase in Renal Epithelial Paracellular Permeability

The ability of hydrogen peroxide (H2O2) to increase paracellular permeability of renal epithelial cell monolayers was examined and the role of occludin in this regulation was investigated. H2O2 treatment increased the paracellular movement of calcein, a marker for the leak pathway permeability, across monolayers of two renal epithelial cell lines, MDCK and LLC‐PK1, in a concentration‐dependent manner. At the same concentrations, H2O2 did not alter transepithelial resistance (TER) nor increase cell death. The magnitude of the H2O2‐induced increase in leak pathway permeability was inversely related to cellular occludin protein content. H2O2 treatment did not produce any major change in total cellular content or Triton X‐100‐soluble or –insoluble fraction content of occludin protein. Occludin protein staining at the tight junction region was diminished following H2O2 treatment. The most dramatic effect of H2O2 was on the dynamic mobility of GFP‐occludin into the tight junction region. H2O2 treatment slowed lateral movement of GFP‐occludin into the tight junction region but not on the apical membrane. Further, removal of the cytoplasmic C‐terminal region of occludin protein eliminated the effect of H2O2 on GFP‐occludin lateral movement into the tight junction region. An increase in the mobile fraction of GFP‐occludin was associated with a loss of response to H2O2. These data indicate that the H2O2‐induced increase in renal epithelial cell paracellular permeability is mediated, at least in part, through occludin protein, possibly through a slowing of the rate of occludin movement into the tight junction region. J. Cell. Biochem. 117: 769–779, 2016.

Correlation of occludin protein mobility with paracellular leak pathway permeability in renal epithelia

Studies have demonstrated regulation of the epithelial paracellular permeability barrier, the tight junction, by a variety of stimuli. Recent studies have reported a correlation between changes in paracellular permeability, particularly paracellular permeability to large solutes (leak pathway), and mobility of the tight junction protein, occludin, in the plane of the plasma membrane. This had led to the hypothesis that changes in occludin protein mobility are causative for changes in paracellular permeability. Using a renal epithelial cell model system, MDCK, we examined the effect of various manipulations on both leak pathway permeability, monitored as the paracellular movement of a fluorescent molecule (calcein), and occludin protein mobility, monitored through fluorescence recovery after photobleaching. Our results indicate that knockdown of the associated tight junction protein, ZO-1, increases baseline leak pathway permeability, whereas, knockdown of the related tight junction protein, ZO-2, does not alter baseline leak pathway permeability. Knockdown of either ZO-1 or ZO-2 decreases the rate of movement of occludin protein but only knockdown of ZO-2 protein alters the percent of occludin protein that is mobile. Further, treatment with hydrogen peroxide increases leak pathway permeability in wild type MDCK cells and in ZO-2 knockdown MDCK cells but not in ZO-1 knockdown MDCK cells. This treatment decreases the rate of occludin movement in all three cell lines but only alters the mobile fraction of occludin protein in ZO-1 knockdown MDCK cells. Finally, we examined the effect of renal ischemia/reperfusion injury on occludin protein mobility in vivo. Ischemia/reperfusion injury both increased the rate of occludin mobility and increased the fraction of occludin protein that is mobile. These results indicate that, at least in our cell culture and in vivo model systems, there is no consistent correlation between paracellular leak pathway permeability and occludin protein mobility.

ZO-1 protein is required for hydrogen peroxide to increase MDCK cell paracellular permeability in an ERK 1/2-dependent manner

Hydrogen peroxide (H2O2) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with H2O2 activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of H2O2 to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of H2O2 to increase paracellular permeability. H2O2 treatment did not, however, affect the total cell content or contents of the Triton X-100-soluble and -insoluble fractions for occludin, ZO-1, or ZO-2. H2O2 treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, H2O2 increased ERK 1/2 activation in ZO-1 knockdown and occludin knockdown cells. Inhibition of ERK 1/2 activation blocked the increase in paracellular permeability in occludin knockdown cells. ZO-1 knockdown cell paracellular permeability was regulated by PP1, an src inhibitor, indicating that the loss of response to H2O2 was not a general loss of the ability to regulate the paracellular barrier. Inhibition of myosin ATPase activity with blebbistatin increased paracellular permeability in ZO-1 knockdown cells but not in wild-type MDCK cells. H2O2 treatment sensitized wild-type MDCK cells to inhibition of myosin ATPase. Knockdown of TOCA-1 protein, which promotes formation of local branched actin networks, reproduced the effects of ZO-1 protein knockdown. These results demonstrate that H2O2 increases MDCK cell paracellular permeability through activation of ERK 1/2. This H2O2 action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.