Peter Komlosi

Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus

Cilia are complex organelles involved in sensory perception and fluid or cell movement. They are constructed through a highly conserved process called intraflagellar transport (IFT). Mutations in IFT genes, such as Tg737, result in severe developmental defects and disease. In the case of the Tg737orpk mutants, these pathological alterations include cystic kidney disease, biliary and pancreatic duct abnormalities, skeletal patterning defects, and hydrocephalus. Here, we explore the connection between cilia dysfunction and the development of hydrocephalus by using the Tg737orpk mutants. Our analysis indicates that cilia on cells of the brain ventricles of Tg737orpk mutant mice are severely malformed. On the ependymal cells, these defects lead to disorganized beating and impaired cerebrospinal fluid (CSF) movement. However, the loss of the cilia beat and CSF flow is not the initiating factor, as the pathology is present prior to the development of motile cilia on these cells and CSF flow is not impaired at early stages of the disease. Rather, our results suggest that loss of cilia leads to altered function of the choroid plexus epithelum, as evidenced by elevated intracellular cAMP levels and increased chloride concentration in the CSF. These data suggest that cilia function is necessary for regulating ion transport and CSF production, as well as for CSF flow through the ventricles.

Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.

Macula densa (MD) cells express COX-2 and COX-2-derived PGs appear to signal the release of renin from the renal juxtaglomerular apparatus, especially during volume depletion. However, the synthetic machinery and identity of the specific prostanoid released from intact MD cells remains uncertain. In the present studies, a novel biosensor tool was engineered to directly determine whether MD cells release PGE2 in response to low luminal NaCl concentration ([NaCl]L). HEK293 cells were transfected with the Ca2+-coupled E-prostanoid receptor EP1 (HEK/EP1) and loaded with fura-2. HEK/EP1 cells produced a significant elevation in intracellular [Ca2+] ([Ca2+]i) by 29.6 +/- 12.8 nM (n = 6) when positioned at the basolateral surface of isolated perfused MD cells and [NaCl]L was reduced from 150 mM to zero. HEK/EP1 [Ca2+]i responses were observed mainly in preparations from rabbits on a low-salt diet and were completely inhibited by either a selective COX-2 inhibitor or an EP1 antagonist, and also by 100 microM luminal furosemide. Also, 20-mM graduated reductions in [NaCl]L between 80 and 0 mM caused step-by-step increases in HEK/EP1 [Ca2+]i. Low-salt diet greatly increased the expression of both COX-2 and microsome-associated PGE synthase (mPGES) in the MD. These studies provide the first direct evidence that intact MD cells synthesize and release PGE2 during reduced luminal salt content and suggest that this response is important in the control of renin release and renal vascular resistance during salt deprivation.

Angiotensin I Conversion to Angiotensin II Stimulates Cortical Collecting Duct Sodium Transport

Abstract—Angiotensin (Ang) II directly stimulates epithelial sodium channel activity in the rabbit cortical collecting duct. Because Ang I and converting enzyme analogues might be present in the distal nephron, this raises the possibility of intraluminal generation of Ang II. Conversion of Ang I to Ang II was monitored by Ang II–dependent changes in intracellular sodium concentration as a reflection of sodium transport across the apical membrane. This involved imaging-based fluorescence microscopy with sodium-binding benzofuran isophthalate in isolated, perfused, cortical collecting-duct segments from rabbit kidney. Principal and intercalated cells were differentiated by rhodamine-conjugated peanut lectin. Control principal cell intracellular sodium concentration, during perfusion with 25 mmol/L NaCl and zero sodium in the bath plus monensin (10−5 mol/L) averaged 5.8±0.14 mmol/L (n=156). The increase in intracellular sodium concentration, when luminal NaCl was increased from 25 to 150 mmol/L, was elevated by 3.5-fold in the presence of intraluminal Ang I (10−6 mol/L). Also, the effects of Ang I on sodium transport were not significantly different from the effects of Ang II (10−9 mol/L). Ang I was used in micromolar concentrations to ensure that there was sufficient substrate available for conversion to Ang II. Inhibition of the angiotensin-converting enzyme with captopril reduced the stimulatory effect of Ang I. These results suggest that intraluminal conversion of Ang I to Ang II can occur in the cortical collecting duct, resulting in enhanced apical sodium entry.

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.

Tubuloglomerular feedback mechanisms in nephron segments beyond the macula densa.

Purpose of reviewTo summarize recent evidence regarding the role of distal nephron segments other than the macula densa in sensing the tubular environment and transmitting this signal to the adjacent vasculature. Recent findingsIn addition to the classical contact site between the macula densa plaque and the afferent arteriole, there is accumulating evidence suggesting a functional association between the distal nephron and the vasculature at three distinct additional sites: at the terminal cortical thick ascending limb, at the early distal tubule and also at the connecting tubule segment. The epithelial cells around the macula densa also sense and respond to changes in tubular flow and salt content and may transmit this signal to the adjacent afferent arteriole. SummaryThere are multiple sites of anatomical and functional contact between the distal nephron and the vasculature supplying the glomerulus, and these may contribute to the regulation of glomerular filtration rate and renal hemodynamics.

Na+/Ca2+ Exchanger Target for Oxidative Stress in Salt-Sensitive Hypertension

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.

Na+/Ca2+ Exchanger

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.

Amyloid Beta Peptide 1-40 Stimulates the Na+ / Ca2+ Exchange Activity of SNCX

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.

Na + /Ca 2+ Exchanger

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.