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Maser Laboratory: Research Projects

Polycystin-1 (PC-1) Membrane-Associated Structure. We have shown that PC-1 consists of 11 membrane-spanning or transmembrane (TM) domains (See Figure 1, Figure 1and Nims, N et al., 2003). This work was important to the PKD field since original models of PC-1 structure predicted from 9 to 13 TM domains, and multiple signaling studies have utilized expression constructs of the PC-1 carboxy terminal portion, or C-tail. To date, ours is the only experimental data confirming 11 TM domains which makes PC-1 and its related proteins an unusual family of heterotrimeric G protein coupled receptors (GPCRs).

We have also shown that the membrane topogenesis of PC-1 involves sequential insertion of the first 9 TM domains, and that membrane insertion of the final 2 TM domains is not sequential and may require cooperation between these TM domains 10 and 11. Since PC-1 has been shown to be a GPCR, with a G protein binding and activation site within its C-terminal, cytosolic tail, our data suggest that human mutations causing altered or aberrant membrane insertion/topology of PC-1 may affect its stability, cellular localization, and/or signaling functions. To investigate this hypothesis, we have produced mouse PC-1 expression constructs with reported human missense mutations within or near TM domains and assayed their membrane insertion and topology (Vassmer, D., et al., in preparation). One mutation was found to reduce translocation efficiency and reverse the membrane topology of TM domain 10. We plan to pursue the effects of this mutation on the structural and biological phenotype of PC-1.

Figure 2

Polycystin-1 and Nrf2/ARE Gene Regulation. We have found that stable or transient overexpression of a membrane-directed PC-1 cytosolic C-tail fusion construct is capable of constitutively activating the endogenous glutathione S-transferase (GST)-Ya gene or a GST-Ya ARE-luciferase reporter (see Figure 2). Activation of the ARE reporter has been shown to involve the transcription factor, Nrf2. Using the transient transfection system, we have examined multiple signaling pathways, and in addition to previously demonstrated PC-1-activated pathways, we have uncovered some unique signaling effectors in the PC-1 C-tail-mediated ARE activation pathway (Phe, S., et al., in preparation). Transient transfections using a full-length human PC-1 expression construct also activates the ARE luciferase reporter via the same major signaling effectors as the C-tail construct.

In support of these in vitro observations, we have determined that the expression of GST-Ya, and two other ARE-regulated genes, NAD(P)H-quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), are reduced in embryonic kidneys from homozygous Pkd1 null mice as compared to nonmutant kidneys. Altogether these data suggest that PC-1 functions in the modulation of (basal) Nrf2/ARE gene activation.

This work identifies a novel target of PC-1 signaling, whose aberrant regulation could play a role in PKD pathogenesis. We are working to determine whether Nrf2/ARE-regulated gene products play a direct role in cystogenesis

Fluid Shear Stress and Renal Nrf2/ARE Gene Expression. Nrf2/ARE gene expression was recently shown to be activated by laminar fluid flow in endothelial cells, and has been proposed to play an important role in protection against atherosclerosis. We proposed that ARE genes would also be regulated by fluid shear stress in renal epithelial cells. To investigate this hypothesis, we adapted a laminar fluid flow apparatus that enables us to subject renal epithelial cells to varying levels of fluid shear stress for different lengths of time in a 37˚C humidified, CO2 incubator (see Figure 3A).

Figure 3

Prototypical ARE gene expression was examined in wildtype M-1 (cortical collecting duct) cells by real time RT-PCR, which revealed that shear stress differentially activates ARE genes in a temporal- and magnitude-specific manner (v).

We have begun to investigate the mechanism of fluid shear stress-activated ARE gene expression in M-1 cells and are examining the involvement of calcium signaling, microtubule-based structures such as the primary cilium (Figure 3C), PC1, and Nrf2. For these studies we have utilized primary cultures of normal human kidney and autosomal dominant polycystic kidney disease (ADPKD), provided by our colleague, Darren Wallace (KUMC, Dept of Medicine), Director of the KUMC PKD Center Biomaterials Core, and conditionally immortalized distal nephron/collecting duct cell lines from Nrf2 knockout and Pkd1 null mice prepared in our laboratory (Ziemer, D., Chiselita, A., et al., in preparation).

This work shows for the first time that Nrf2/ARE-regulated genes are activated by fluid shear stress in renal epithelial cells. Some of our current projects involve examining the role of Nrf2, PC-1, and fluid shear stress in protection from injury, redox homeostasis, and cell cycle regulation using our Nrf2- and Pkd1-deficient cell lines.

Figure 4

Role of oxidant stress in pathogenesis of PKD. We propose that PC1 has a novel role in cellular defense by upregulating protective Nrf2/ARE gene expression (Figure 4). In PKD kidneys, deficiency of PC1 results in reduction of cellular defenses that could predispose renal cells to injury and/or elevated levels of reactive oxygen species (ROS). Excessive ROS can drive unscheduled cell cycle reentry, disrupt signaling pathways, and/or promote apoptosis.

To test this hypothesis, we are utilizing a cell-based approach to establish that cells deficient in PC1 have reduced antioxidant protection and therefore, a lowered threshold for oxidant-induced damage, proliferation and apoptosis. Using our conditionally immortalized Pkd1 null and Nrf2 KO renal epithelial cell lines, and primary cultures of normal human (NHK) and ADPKD kidneys supplied by the KUMC PKD Center Biomaterials Core, we have found that Nrf2-/- and Pkd1-/- cells are exquisitely sensitive to cell death when challenged with oxidant-promoting treatments.

Interestingly, Pkd1+/- cells have an intermediate level of stress susceptibility, which is consistent with in vivo “oxidant treatments” of Pkd1+/- mice and will be a focus in future studies. Human ADPKD cells respond to cellular stress treatments with increased levels of proliferation and apoptosis when compared to normal cells. In addition, we will determine if fluid shear stress protects (wildtype) renal epithelial cells from oxidant challenge. Additional studies will examine the mechanisms of increased susceptibility to cellular stress in these cells.

Antioxidant-based therapies for PKD. Most recently, we have begun investigating the ability of anti-oxidant-promoting compounds to ameliorate PKD using the metanephric organ culture system and genetic rodent models of PKD. Figure 5

Figure 5 illustrates the ability of 3 different anti-oxidant-promoting chemicals to reduce cAMP-mediated cystic tubule expansion in metanephric organ cultures.

Last modified: May 29, 2019