Brian Siroky
University of Alabama at Birmingham
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Featured researches published by Brian Siroky.
Purinergic Signalling | 2008
Michael B. Hovater; Dragos Olteanu; Elizabeth L. Hanson; Nai Lin Cheng; Brian Siroky; Attila Fintha; Peter Komlosi; Wen Liu; Lisa M. Satlin; P. Darwin Bell; Bradley K. Yoder; Erik M. Schwiebert
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.
Journal of The American Society of Nephrology | 2009
Binli Tao; Su Bu; Zhihua Yang; Brian Siroky; John C. Kappes; Andreas Kispert; Lisa M. Guay-Woodford
Primary cilia are dynamic, complex structures that contain >500 proteins, including several related to polycystic kidney disease. How these proteins target to cilia and assemble is unknown. We previously identified Cys1 as the gene responsible for disease in Cys1(cpk) mice, a mouse model of autosomal recessive polycystic kidney disease; this gene encodes cystin, a 145-amino acid cilium-associated protein. Here, we characterized the localization of cystin in the embryonic kidney and liver, in isolated renal collecting ducts, and in an inner medullary collecting duct mouse cell line. Because endogenous levels of cystin expression are low, we generated inner medullary collecting duct cell lines that stably express enhanced green fluorescence protein-tagged constructs of wild-type cystin or various truncation mutants. We determined that cystin is myristoylated at its G2 residue and that N-myristoylated cystin fractionates with membrane microdomains. Furthermore, the N-myristoylation signal is necessary but not sufficient to target cystin to the primary cilium. Analysis of deletion and chimeric constructs identified an AxEGG motif that is necessary to target and retain cystin in the cilium. Derangement of these localization motifs may lead to cystic kidney disease.
American Journal of Physiology-renal Physiology | 2012
Takamitsu Saigusa; Ryan Reichert; Jennifer Guare; Brian Siroky; Monika Gooz; Stacy L. Steele; Robert A. Fenton; P. Darwin Bell; Robert J. Kolb
Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (-), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/Ift88 gene). Interestingly, only cilia (-) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (-) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (-) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current (I(sc)) in cilia (-) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of I(sc) in cilia (-) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.
Hypertension | 2003
M. Tino Unlap; Elizabeth Bates; Corey L. Williams; Peter Komlosi; Iantha Williams; Gergely Kovacs; Brian Siroky; P. Darwin Bell
Abstract—The Na+/Ca2+ exchanger regulates intracellular calcium ([Ca2+]i), and attenuation of Na+/Ca2+ exchange by oxidative stress might lead to dysregulation of [Ca2+]i. We have shown that the Na+/Ca2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na+/Ca2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45Ca2+ uptake (reverse mode) and [Ca2+]i elevation (forward mode) in the presence and absence of H2O2 and peroxynitrite. Our results showed that 45Ca2+ uptake in SNCX cells was attenuated at 500 and 750 &mgr;mol/L H2O2 (63±12% and 25±7%, respectively; n=16) and at 50 and 100 &mgr;mol/L peroxynitrite (47±9% and 22±9%, respectively; n=16). In RNCX cells, 45Ca2+ uptake was attenuated at only 750 and 100 &mgr;mol/L H2O2 and peroxynitrite (61±9% and 63±6%, respectively; n=16). In addition, the elevation in [Ca2+]i was greater in SNCX cells than in RNCX cells in response to 750 &mgr;mol/L H2O2 (58±5.5 vs 17±4.1 nmol/L; n=13) and 100 &mgr;mol/L peroxynitrite (33±5 vs 11±6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca2+]i that is found in this model of salt-sensitive hypertension.The Na + /Ca 2+ exchanger regulates intracellular calcium ([Ca 2+ ] i ), and attenuation of Na + /Ca 2+ exchange by oxidative stress might lead to dysregulation of [Ca 2+ ] i . We have shown that the Na + /Ca 2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na + /Ca 2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45 Ca 2+ uptake (reverse mode) and [Ca 2+ ] i elevation (forward mode) in the presence and absence of H 2 O 2 and peroxynitrite. Our results showed that 45 Ca 2+ uptake in SNCX cells was attenuated at 500 and 750 μmol/L H 2 O 2 (63±12% and 25±7%, respectively; n=16) and at 50 and 100 μmol/L peroxynitrite (47±9% and 22±9%, respectively; n=16). In RNCX cells, 45 Ca 2+ uptake was attenuated at only 750 and 100 μmol/L H 2 O 2 and peroxynitrite (61±9% and 63±6%, respectively; n=16). In addition, the elevation in [Ca 2+ ] i was greater in SNCX cells than in RNCX cells in response to 750 μmol/L H 2 O 2 (58±5.5 vs 17±4.1 nmol/L; n=13) and 100 μmol/L peroxynitrite (33±5 vs 11±6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca 2+ ] i that is found in this model of salt-sensitive hypertension.
Hypertension | 2003
M. Tino Unlap; Elizabeth Bates; Corey L. Williams; Peter Komlosi; Iantha Williams; Gergely Kovács; Brian Siroky; P. Darwin Bell
Abstract—The Na+/Ca2+ exchanger regulates intracellular calcium ([Ca2+]i), and attenuation of Na+/Ca2+ exchange by oxidative stress might lead to dysregulation of [Ca2+]i. We have shown that the Na+/Ca2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na+/Ca2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45Ca2+ uptake (reverse mode) and [Ca2+]i elevation (forward mode) in the presence and absence of H2O2 and peroxynitrite. Our results showed that 45Ca2+ uptake in SNCX cells was attenuated at 500 and 750 &mgr;mol/L H2O2 (63±12% and 25±7%, respectively; n=16) and at 50 and 100 &mgr;mol/L peroxynitrite (47±9% and 22±9%, respectively; n=16). In RNCX cells, 45Ca2+ uptake was attenuated at only 750 and 100 &mgr;mol/L H2O2 and peroxynitrite (61±9% and 63±6%, respectively; n=16). In addition, the elevation in [Ca2+]i was greater in SNCX cells than in RNCX cells in response to 750 &mgr;mol/L H2O2 (58±5.5 vs 17±4.1 nmol/L; n=13) and 100 &mgr;mol/L peroxynitrite (33±5 vs 11±6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca2+]i that is found in this model of salt-sensitive hypertension.The Na + /Ca 2+ exchanger regulates intracellular calcium ([Ca 2+ ] i ), and attenuation of Na + /Ca 2+ exchange by oxidative stress might lead to dysregulation of [Ca 2+ ] i . We have shown that the Na + /Ca 2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na + /Ca 2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45 Ca 2+ uptake (reverse mode) and [Ca 2+ ] i elevation (forward mode) in the presence and absence of H 2 O 2 and peroxynitrite. Our results showed that 45 Ca 2+ uptake in SNCX cells was attenuated at 500 and 750 μmol/L H 2 O 2 (63±12% and 25±7%, respectively; n=16) and at 50 and 100 μmol/L peroxynitrite (47±9% and 22±9%, respectively; n=16). In RNCX cells, 45 Ca 2+ uptake was attenuated at only 750 and 100 μmol/L H 2 O 2 and peroxynitrite (61±9% and 63±6%, respectively; n=16). In addition, the elevation in [Ca 2+ ] i was greater in SNCX cells than in RNCX cells in response to 750 μmol/L H 2 O 2 (58±5.5 vs 17±4.1 nmol/L; n=13) and 100 μmol/L peroxynitrite (33±5 vs 11±6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca 2+ ] i that is found in this model of salt-sensitive hypertension.
Current Neurovascular Research | 2005
Menjor Tino Unlap; Corey L. Williams; Darryl Morin; Brian Siroky; Attila Fintha; Amanda L. Fuson; Layla Dodgen; Gergely Kovacs; Peter Komlosi; William B. Ferguson; P. D. Bell
The Na+/Ca2+ exchangers, RNCX and SNCX, were cloned from mesangial cells of salt sensitive and salt resistant Dahl/Rapp rats, respectively, and differ at amino acid 218 (RNCXi/SNCXf) and in the exons expressed at the alternative splice site (RNCXB, D/SNCXB, D, F). These isoforms are also expressed in myocytes, neurons, and astrocytes where they maintain cytosolic calcium homeostasis. We demonstrated that cells expressing SNCX were more susceptible to oxidative stress than cells expressing RNCX. Others demonstrated that amyloid beta peptide (Abeta) augments the adverse effects of oxidative stress on calcium homeostasis. Therefore, we sought to assess the effect of Abeta 1-40 on the abilities of OK-PTH cells stably expressing RNCX and SNCX and human glioma cells, SKMG1, to regulate cytosolic calcium homeostasis. Our studies showed that Abeta 1-40 (1 microM) did not affect RNCX activity, as assessed by changes in [Ca2+]i (Delta[Ca2+]i, 260+/-10 nM to 267+/-8 nM), while stimulating exchange activity 2.4 and 3 fold in cells expressing SNCX (100+/-8 to 244+/-12 nM) and in SKMG1 cells (90+/-11 nM to 270+/-18 nM), respectively. Our results also showed that Abeta 1-40, while not affecting the rate of Mn2+ influx in cells expressing RNCX, stimulated the rate of Mn2+ influx 2.8 and 2.9 fold in cells expressing SNCX and in SKMG1 cells. Thus, our studies demonstrate that Abeta-induced cytosolic calcium increase is mediated through certain isoforms of the Na+/Ca2+ exchanger and reveals a possible mechanism by which Abeta 1-40 can alter cytosolic calcium homeostasis.
Journal of Cellular Physiology | 2004
Iantha Williams; Corey L. Williams; Brian Siroky; Elizabeth Bates; Gergely Kovacs; Janos Peti-Peterdi; Menjor Tino Unlap; P. D. Bell
An isoform of the Na+/Ca2+ exchanger (SDNCX1.10) was cloned from mesangial cells of Sprague–Dawley rat. Regulation of this isoform was compared to two other clones that were derived from the Dahl/Rapp salt sensitive (SNCX) and salt resistant rat (RNCX). All isoforms differ at the alternative splice site and at amino acid 218 for SNCX. PKC activates RNCX but not SNCX while SDNCX1.10 was also activated by PKC. Regulation of exchanger activities by intracellular calcium ([Ca2+]i), pH, and kinases was assessed using Na‐dependent 45Ca2+ uptake assays in OK‐PTH cells expressing the vector, RNCX, SNCX, or SDNCX1.10. [Ca2+]i was elevated from 50 to 125 nM (n = 4) with thapsigargin (40 nM) and reduced from 50 to 29 nM (n = 4) and 18 nM (n = 4) with 10 or 20 μM BAPTA, respectively. RNCX was active at all three [Ca2+]i while SNCX and SDNCX1.10 were only active at lower [Ca2+]i. Varying extracellular pH (pHe, without nigericin) or pHe and intracellular pH (pHi, with 10 μM nigericin) from pH 7.4 to 6.2, 6.8, or 8.0 showed that SNCX activity was attenuated at both low and high pHs. SDNCX1.10 activity was attenuated only at pH 6.2 and 6.8 (with or without nigericin) while RNCX activity was attenuated at pH 6.2 (with or without nigericin) and pH 6.8 (with nigericin). Finally, only SDNCX1.10 activity was stimulated by 250 μM CPT‐cAMP or 250 μM DB‐cGMP treatment. Thus the differential regulation of [Ca2+]i by these exchangers is dependent upon the pattern of cellular Na+/Ca2+ exchanger isoform expression. J. Cell. Physiol. 199: 181–193, 2004© 2003 Wiley‐Liss, Inc.
Hypertension | 2003
M. Tino Unlap; Elizabeth Bates; Corey L. Williams; Peter Komlosi; Iantha Williams; Gergely Kovács; Brian Siroky; P. Darwin Bell
Abstract—The Na+/Ca2+ exchanger regulates intracellular calcium ([Ca2+]i), and attenuation of Na+/Ca2+ exchange by oxidative stress might lead to dysregulation of [Ca2+]i. We have shown that the Na+/Ca2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na+/Ca2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45Ca2+ uptake (reverse mode) and [Ca2+]i elevation (forward mode) in the presence and absence of H2O2 and peroxynitrite. Our results showed that 45Ca2+ uptake in SNCX cells was attenuated at 500 and 750 &mgr;mol/L H2O2 (63±12% and 25±7%, respectively; n=16) and at 50 and 100 &mgr;mol/L peroxynitrite (47±9% and 22±9%, respectively; n=16). In RNCX cells, 45Ca2+ uptake was attenuated at only 750 and 100 &mgr;mol/L H2O2 and peroxynitrite (61±9% and 63±6%, respectively; n=16). In addition, the elevation in [Ca2+]i was greater in SNCX cells than in RNCX cells in response to 750 &mgr;mol/L H2O2 (58±5.5 vs 17±4.1 nmol/L; n=13) and 100 &mgr;mol/L peroxynitrite (33±5 vs 11±6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca2+]i that is found in this model of salt-sensitive hypertension.The Na + /Ca 2+ exchanger regulates intracellular calcium ([Ca 2+ ] i ), and attenuation of Na + /Ca 2+ exchange by oxidative stress might lead to dysregulation of [Ca 2+ ] i . We have shown that the Na + /Ca 2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na + /Ca 2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45 Ca 2+ uptake (reverse mode) and [Ca 2+ ] i elevation (forward mode) in the presence and absence of H 2 O 2 and peroxynitrite. Our results showed that 45 Ca 2+ uptake in SNCX cells was attenuated at 500 and 750 μmol/L H 2 O 2 (63±12% and 25±7%, respectively; n=16) and at 50 and 100 μmol/L peroxynitrite (47±9% and 22±9%, respectively; n=16). In RNCX cells, 45 Ca 2+ uptake was attenuated at only 750 and 100 μmol/L H 2 O 2 and peroxynitrite (61±9% and 63±6%, respectively; n=16). In addition, the elevation in [Ca 2+ ] i was greater in SNCX cells than in RNCX cells in response to 750 μmol/L H 2 O 2 (58±5.5 vs 17±4.1 nmol/L; n=13) and 100 μmol/L peroxynitrite (33±5 vs 11±6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca 2+ ] i that is found in this model of salt-sensitive hypertension.
American Journal of Physiology-renal Physiology | 2006
Brian Siroky; William B. Ferguson; Amanda L. Fuson; Yi Xie; Attila Fintha; Peter Komlosi; Bradley K. Yoder; Erik M. Schwiebert; Lisa M. Guay-Woodford; P. Darwin Bell
American Journal of Physiology-lung Cellular and Molecular Physiology | 2005
Veronica Swystun; Lan Chen; Phillip Factor; Brian Siroky; P. Darwin Bell; Sadis Matalon