Loss of primary cilia increases polycystin-2 and TRPV4 and the appearance of a nonselective cation channel in the mouse cortical collecting duct.

Flow-related bending of cilia results in Ca2+ influx through a polycystin-1 (Pkd1) and polycystin-2 (Pkd2) complex, both of which are members of the transient receptor potential (TRP) family (TRPP1 and TRPP2, respectively). Deletion of this complex as well as cilia result in polycystic kidney disease. The Ca2+ influx pathway has been previously characterized in immortalized collecting duct cells without cilia and found to be a 23-pS channel that was a multimere of TRPP2 and TRPV4. The purpose of the present study was to determine if this TRPP2 and TRPV4 multimere exists in vivo. Apical channel activity was measured using the patch-clamp technique from isolated split-open cortical collecting ducts from adult conditional knockout mice with (Ift88flox/flox) or without (Ift88-/-) cilia. Single tubules were isolated for measurements of mRNA for Pkd1, Pkd2, Trpv4, and epithelial Na+ channel subunits. The predominant channel activity from Ift88flox/flox mice was from epithelial Na+ channel [5-pS Na+-selective channels with long mean open times (475.7 ± 83.26 ms) and open probability > 0.2]. With the loss of cilia, the predominant conductance was a 23-pS nonselective cation channel (reversal potential near 0) with a short mean open time (72 ± 17 ms), open probability < 0.08, and a characteristic flickery opening. Loss of cilia increased mRNA levels for Pkd2 and Trpv4 from single isolated cortical collecting ducts. In conclusion, 23-pS channels exist in vivo, and activity of this channel is elevated with loss of cilia, consistent with previous finding of an elevated-unregulated Ca2+-permeable pathway at the apical membrane of collecting duct cells that lack cilia.

Attenuation of accelerated renal cystogenesis in Pkd1 mice by renin-angiotensin system blockade.

The intrarenal renin angiotensin system (RAS) is activated in polycystic kidney disease. We have recently shown in the Pkd1 mouse that Gen 2 antisense oligonucleotide (ASO), which suppresses angiotensinogen (Agt) synthesis, is efficacious in slowing kidney cyst formation compared with lisinopril. The aim of this current study was to determine 1) if unilateral nephrectomy accelerates cystogenesis in Pkd1 mice (as previously shown in cilia knockout mice) and 2) whether Agt ASO can slow the progression in this accelerated cystic mouse model. Adult Pkd1 conditional floxed allele mice expressing cre were administered tamoxifen, resulting in global knockout of Pkd1. Three weeks after tamoxifen injection, mice underwent left unilateral nephrectomy. Mice were then treated with Agt ASO (75 mg/kg per week) or aliskiren (20 mg/kg per day)+Agt ASO or control for 8 wk. Unilateral nephrectomy accelerated kidney cyst formation compared with nonnephrectomized mice. Both Agt ASO and Aliskiren+Agt ASO treatments significantly reduced plasma and urinary Agt levels. Blood pressure was lowest in Aliskiren+Agt ASO mice among all treatment groups, and the control group had the highest blood pressure. All mice developed significant kidney cysts at 8 wk after nephrectomy, but Agt ASO and Aliskiren+Agt ASO groups had fewer kidney cysts than controls. Renal pAkt, pS6 levels, and apoptosis were significantly suppressed in those receiving Agt ASO compared with controls. These results indicate that suppressing Agt using an ASO slowed the progression of accelerated cystic kidney disease induced by unilateral nephrectomy in Pkd1 mice by suppressing intrarenal RAS, mammalian target of rapamycin pathway, and cell proliferation.

Activation of the intrarenal renin-angiotensin-system in murine polycystic kidney disease.

The mechanism for early hypertension in polycystic kidney disease (PKD) has not been elucidated. One potential pathway that may contribute to the elevation in blood pressure in PKD is the activation of the intrarenal renin-angiotensin-system (RAS). For example, it has been shown that kidney cyst and cystic fluid contains renin, angiotensin II (AngII), and angiotensinogen (Agt). Numerous studies suggest that ciliary dysfunction plays an important role in PKD pathogenesis. However, it is unknown whether the primary cilium affects the intrarenal RAS in PKD. The purpose of this study was to determine whether loss of cilia or polycystin 1 (PC1) increases intrarenal RAS in mouse model of PKD. Adult Ift88 and Pkd1 conditional floxed allele mice with or without cre were administered tamoxifen to induce global knockout of the gene. Three months after tamoxifen injection, kidney tissues were examined by histology, immunofluorescence, western blot, and mRNA to assess intrarenal RAS components. SV40 immortalized collecting duct cell lines from hypomorphic Ift88 mouse were used to assess intrarenal RAS components in collecting duct cells. Mice without cilia and PC1 demonstrated increased kidney cyst formation, systolic blood pressure, prorenin, and kidney and urinary angiotensinogen levels. Interestingly immunofluorescence study of the kidney revealed that the prorenin receptor was localized to the basolateral membrane of principal cells in cilia (-) but not in cilia (+) kidneys. Collecting duct cAMP responses to AngII administration was greater in cilia (-) vs. cilia (+) cells indicating enhanced intrarenal RAS activity in the absence of cilia. These data suggest that in the absence of cilia or PC1, there is an upregulation of intrarenal RAS components and activity, which may contribute to elevated blood pressure in PKD.

Hyperglycemia in the absence of cilia accelerates cystogenesis and induces renal damage.

In polycystic kidney disease (PKD), the rate of cyst formation and disease progression is highly variable. The lack of predictability in disease progression may be due to additional environmental factors or pathophysiological processes called "third hits." Diabetes is a growing epidemic, and recent studies suggest that PKD patients may be at an increased risk for this disease. We sought to determine if hyperglycemia enhances the initiation and rate of cystogenesis. Tamoxifen was administered to adult Ift88 conditional floxed allele mice to induce cilia loss in the presence of Cre. Subsequent administration of streptozotocin resulted in equivalent hyperglycemia in cilia(+) and cilia(-) mice. Hyperglycemia with loss of cilia increased the rate of cyst formation and cell proliferation. Structural and functional alterations in the kidney, including focal glomerular foot process effacement, interstitial inflammation, formation of primitive renal tubules, polyuria, and increased proteinuria, were also observed in hyperglycemic cilia(-) mice. Gene array analysis indicated enhanced Wnt and epithelial-to-mesenchymal transition signaling in the kidney of hyperglycemic cilia(-) mice. These data show that hyperglycemia, in the absence of cilia, results in renal structural and functional damage and accelerates cystogenesis, suggesting that diabetes is a risk factor in the progression of PKD.

Deletion of airway cilia results in noninflammatory bronchiectasis and hyperreactive airways.

The mechanisms for the development of bronchiectasis and airway hyperreactivity have not been fully elucidated. Although genetic, acquired diseases and environmental influences may play a role, it is also possible that motile cilia can influence this disease process. We hypothesized that deletion of a key intraflagellar transport molecule, IFT88, in mature mice causes loss of cilia, resulting in airway remodeling. Airway cilia were deleted by knockout of IFT88, and airway remodeling and pulmonary function were evaluated. In IFT88(-) mice there was a substantial loss of airway cilia on respiratory epithelium. Three months after the deletion of cilia, there was clear evidence for bronchial remodeling that was not associated with inflammation or apparent defects in mucus clearance. There was evidence for airway epithelial cell hypertrophy and hyperplasia. IFT88(-) mice exhibited increased airway reactivity to a methacholine challenge and decreased ciliary beat frequency in the few remaining cells that possessed cilia. With deletion of respiratory cilia there was a marked increase in the number of club cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of club cells, since these cells are involved in airway repair. Club cells may be prevented from differentiating into respiratory epithelial cells because of a lack of IFT88 protein that is necessary to form a single nonmotile cilium. This monocilium is a prerequisite for these progenitor cells to transition into respiratory epithelial cells. In conclusion, motile cilia may play an important role in controlling airway structure and function.

Reducing FLI1 levels in the MRL/lpr lupus mouse model impacts T cell function by modulating glycosphingolipid metabolism.

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1(+/+) or Fli1(+/-) T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1(+/-) lupus T cells compared to animals receiving Fli1(+/+) lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1(+/-) T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1(+/+) T cells. Moreover, the Fli1(+/-) T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1(+/+) T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus.

The bradykinin B(2) receptor induces multiple cellular responses leading to the proliferation of human renal carcinoma cell lines.

BACKGROUND: The vasoactive peptide bradykinin (BK) acts as a potent growth factor for normal kidney cells, but there have been few studies on the role of BK in renal cell carcinomas. PURPOSE: In this study, we tested the hypothesis that BK also acts as a mitogen in kidney carcinomas, and explored the effects of BK in human renal carcinoma A498 cells. METHODS: The presence of mRNAs for BK B(1) and BK B(2) receptors in A498 cells was demonstrated by reverse transcription-polymerase chain reaction. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca(2+), measured changes in extracellular pH as a reflection of Na(+)/H(+) exchange (NHE) with a Cytosensor microphysiometer, and assessed extracellular signal-regulated kinase (ERK) activation by Western blotting. RESULTS: Exposure to 100 nM of BK resulted in the rapid elevation of intracellular Ca(2+), caused a ≥30% increase in NHE activity, and a ≥300% increase in ERK phosphorylation. All BK signals were blocked by HOE140, a BK B(2) receptor antagonist, but not by a B(1) receptor antagonist. Inhibitor studies suggest that BK-induced ERK activation requires phospholipase C and protein kinase C activities, and is Ca(2+)/calmodulin-dependent. The amiloride analog 5-(N-methyl-N-isobutyl)-amiloride (MIA) blocked short-term NHE activation and inhibited ERK phosphorylation, suggesting that NHE is critical for ERK activation by BK. BK induced an approximately 40% increase in the proliferation of A498 cells as assessed by bromodeoxyuridine uptake. This effect was blocked by the ERK inhibitor PD98059, and was dependent on NHE activity. CONCLUSION: We conclude that BK exerts mitogenic effects in A498 cells via the BK B(2) receptor activation of growth-associated NHE and ERK.

Role of integrins in angiotensin II-induced proliferation of vascular smooth muscle cells.

Angiotensin II (AII) binds to G protein-coupled receptor AT(1) and stimulates extracellular signal-regulated kinase (ERK), leading to vascular smooth muscle cells (VSMC) proliferation. Proliferation of mammalian cells is tightly regulated by adhesion to the extracellular matrix, which occurs via integrins. To study cross-talk between G protein-coupled receptor- and integrin-induced signaling, we hypothesized that integrins are involved in AII-induced proliferation of VSMC. Using Oligo GEArray and quantitative RT-PCR, we established that messages for α(1)-, α(5)-, α(V)-, and ß(1)-integrins are predominant in VSMC. VSMC were cultured on plastic dishes or on plates coated with either extracellular matrix or poly-d-lysine (which promotes electrostatic cell attachment independent of integrins). AII significantly induced proliferation in VSMC grown on collagen I or fibronectin, and this effect was blocked by the ERK inhibitor PD-98059, suggesting that AII-induced proliferation requires ERK activity. VSMC grown on collagen I or on fibronectin demonstrated approximately three- and approximately sixfold increases in ERK phosphorylation after stimulation with 100 nM AII, respectively, whereas VSMC grown on poly-d-lysine demonstrated no significant ERK activation, supporting the importance of integrin-mediated adhesion. AII-induced ERK activation was reduced by >65% by synthetic peptides containing an RGD (arginine-glycine-aspartic acid) sequence that inhibit α(5)ß(1)-integrin, and by ∼60% by the KTS (lysine-threonine-serine)-containing peptides specific for integrin-α(1)ß(1). Furthermore, neutralizing antibody against ß(1)-integrin and silencing of α(1), α(5), and ß(1) expression by transfecting VSMC with short interfering RNAs resulted in decreased AII-induced ERK activation. This work demonstrates roles for specific integrins (most likely α(5)ß(1) and α(1)ß(1)) in AII-induced proliferation of VSMC.

Bradykinin B2 receptor interacts with integrin alpha5beta1 to transactivate epidermal growth factor receptor in kidney cells.

We have shown previously that the vasoactive peptide bradykinin (BK) stimulates proliferation of a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) via transactivation of epidermal growth factor receptor (EGFR) by a mechanism that involves matrix metalloproteinases (collagenase-2 and -3). Because collagenases lack an integral membrane domain, we hypothesized that receptors for extracellular matrix proteins, integrins, may play a role in BK-induced signaling by targeting collagenases to the membrane, thus forming a functional signaling complex. BK-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) in mIMCD-3 cells was reduced by approximately 65% by synthetic peptides containing an Arg-Gly-Asp sequence, supporting roles for integrins in BK-induced signaling. Neutralizing antibody against alpha5beta1 integrin partially (approximately 60%) blocked BK-induced ERK activation but did not affect EGF-induced ERK activation. Silencing of alpha5 and beta1 expression by transfecting cells with small interfering RNAs (siRNA) significantly decreased BK-induced ERK activation (approximately 80%) and EGFR phosphorylation (approximately 50%). This effect was even more pronounced in cells that were cotransfected with siRNAs directed against both collagenases and alpha5beta1 integrin. On the basis of our results, we suggested that integrin alpha5beta1 is involved in BK-induced signaling in mIMCD-3 cells. Using immunoprecipitation/Western blotting, we demonstrated association of BK B(2) receptor with alpha5beta1 integrin upon BK treatment. Furthermore, BK induced association of alpha5beta1 integrin with EGFR. These data provide the first evidence that specific integrins are involved in BK B(2) receptor-induced signaling in kidney cells, and ultimately might lead to development of new strategies for treatment of renal tubulointerstitial fibrosis.

Identification of functional bradykinin B(2) receptors endogenously expressed in HEK293 cells.

The human embryonic kidney (HEK) 293 cell line is widely used in cell biology research. Although HEK293 cells have been meticulously studied, our knowledge about endogenous G protein-coupled receptors (GPCR) in these cells is incomplete. While studying the effects of bradykinin (BK), a potent growth factor for renal cells, we unexpectedly discovered that BK activates extracellular signal-regulated protein kinase 1 and 2 (ERK) in HEK293 cells. Thus, we hypothesized that HEK293 cells possess endogenous BK receptors. RT-PCR demonstrated the presence of mRNAs for BK B(1) and BK B(2) receptors in HEK293 cells. Western blotting with BK B(1) and BK B(2) receptor antibodies confirmed this result at the protein level. To establish that BK receptors are functional, we employed fluorescent measurements of intracellular Ca(2+), measured changes in extracellular acidification rate (ECAR) as a reflection of the Na(+)/H(+) exchange (NHE) with a Cytosensortrade microphysiometer, and assessed ERK activation by Western blotting with a phospho-specific ERK antibody. Exposure of HEK293 cells to BK produced a concentration-dependent rise in intracellular Ca(2+) (EC(50)=36.5+/-8.0 x 10(-9)M), a rapid increase in tyrosine phosphorylation of ERK (EC(50)=9.8+/-0.4 x 10(-9)M), and elevation in ECAR by approximately 20%. All of these signals were blocked by HOE-140 (B(2) receptor antagonist) but not by des-Arg(10)-HOE-140 (B(1) receptor antagonist). We conclude that HEK293 cells express endogenous functional BK B(2) receptors, which couple to the mobilization of intracellular Ca(2+), increases in ECAR and increases in ERK phosphorylation.

The role of multidrug resistance proteins MRP1, MRP2 and MRP3 in cellular folate homeostasis.

Previously, we reported that the multidrug resistance proteins MRP1, MRP2 and MRP3 confer resistance to therapeutic antifolates by mediating their cellular extrusion. We now determined whether MRPs also play a role in controlling cellular homeostasis of natural folates. In MRP1, MRP2 and MRP3-transfected 2008 human ovarian carcinoma cells total cellular folate content was 32-38% lower than in 2008 cells (105+/-14pmolfolate/mgprotein) when grown in medium containing 2.3 microM folic acid (FA). Under these conditions cellular growth rates were not compromised. However, when cells were challenged under folate-depleted conditions with a short exposure (4 hr) to FA or leucovorin, MRP1 and MRP3 overexpressing cells were impaired in their growth. In contrast to wild-type cells, MRP1 transfected cells retained only 60% of the maximum growth when exposed to 500 nM leucovorin or 500 microM FA. For 2008/MRP1 and 2008/MRP3 cells FA growth stimulation capacity was dramatically decreased when, during a 4 hr exposure, metabolism into rapidly polyglutamatable and retainable dihydrofolate was blocked by the dihydrofolate reductase inhibitor trimetrexate. To retain growth under such conditions MRP1 overexpressing cells required much higher concentrations of FA (EC(50) > 500 microM) compared to 2008 cells (EC(50): 12 microM). These results suggest that down- and up-regulation of MRP1 (and MRP3) expression can influence cellular folate homeostasis, in particular when cellular retention by polyglutamylation of folates is attenuated.

Loss of folylpoly-gamma-glutamate synthetase activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates in multiple human leukemia sublines.

We have studied the molecular basis of drug resistance in human CCRF-CEM leukemia cells exposed to high dose intermittent pulses of novel polyglutamatable antifolates that target various folate-dependent enzymes. These include the dihydrofolate reductase (DHFR) inhibitors edatrexate, methotrexate and aminopterin, the thymidylate synthase (TS) inhibitors ZD1694 and GW1843, the glycinamide ribonucleotide formyltransferase (GARTF) inhibitor DDATHF as well as the multitargeted antifolate LY231514 inhibiting both TS, DHFR and GARTF. Fourteen antifolate-resistant sublines were isolated, 11 of which displayed a drug resistance phenotype that was based on impaired folylpoly-gamma-glutamate synthetase (FPGS) activity as these cell lines: 1) typically lost 90-99% of parental FPGS activity; 2) expressed 1.4-3.3-fold less FPGS mRNA (only 4 cell lines); 3) displayed up to 10(5)-fold resistance to polyglutamylation-dependent antifolates including ZD1694 and MTA; 4) retained sensitivity to polyglutamylation-independent antifolates including ZD9331 and PT523; 5) were up to 19-fold hypersensitive to the lipid-soluble antifolates trimetrexate and AG377; 6) had a normal or a small decrease in [(3)H]MTX transport; and 7) had a 2.1-8.3-fold decreased cellular folate pools and a consequently increased folate growth requirement. The remaining 3 antifolate-resistant sublines lost 94-97% of parental [(3)H]MTX transport and thus displayed a high level resistance to all hydrophilic antifolates. To screen for mutations in the hFPGS gene, we devised an RT-PCR single strand conformational polymorphism (SSCP) assay. RT-PCR-SSCP analysis and DNA sequencing showed that only a single FPGS-deficient subline harbored an FPGS mutation (Cys346Phe). Three-dimensional modeling of the human FPGS based on the crystal structure of Lactobacillus casei FPGS suggested that this mutation maps to the active site and interferes with the catalytic activity of the enzyme due to a putative bulky clash between the mutant Phe346 and a native Phe350 within alpha-helix A10 in a highly conserved C-terminal hydrophobic core. This was consistent with a 23-fold decreased affinity of the mutant Cys346Phe FPGS for L-glutamate. We conclude that decreased FPGS activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates upon a high-dose intermittent exposure schedule. The finding that cells may exhibit 5 orders of magnitude of resistance to polyglutamylation-dependent antifolates but in the same time retain parental sensitivity or hypersensitivity to polyglutamylation-independent antifolates or lipophilic antifolates offers a potentially promising treatment strategy in the overcoming of FPGS-based anticancer drug resistance.

Response to 5-fluorouracil chemotherapy is modified by dietary folic acid deficiency in Apc(Min/+) mice.

5-Fluorouracil (5-FU) has been the foundation of advanced colorectal cancer treatment for over 40 years. The Apc(Min/+) mouse, which is genetically predisposed to intestinal neoplasia, was used to examine the effects of 5-FU in this system and the impact of dietary folic acid on those effects. 5-FU treatment resulted in a 60-80% reduction in tumor number. Clinically relevant toxicities, including myelosuppression and mucositis, are a part of this response. Tumor numbers rebounded completely following termination of 5-FU therapy, indicating that the drug inhibits tumor growth but does not eradicate them. In mice that were fed with a defined diet containing no folic acid (0 ppm), 5-FU not only induced regression of pre-existing tumors, but also inhibited tumor recovery following drug withdrawal. Our data indicate that a dietary folic acid deficiency, in promoting tumor regression and inhibiting tumor recovery, may enhance the therapeutic effects of 5-FU.

Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis.

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.