Kelly A. Hyndman

Comparison of Molecular and Histological Changes in Zebrafish Gills Exposed to Metallic Nanoparticles

Research has demonstrated that metallic nanoparticles produce toxicity in aquatic organisms that is due largely to effects of particulates as opposed to release of dissolved ions. The present research examined the interplay of nanoparticle composition and dissolution on response of the zebrafish gill following exposure to toxic (nanocopper or nanosilver) or nontoxic (nano-TiO2) nanometals. Female zebrafish were exposed to 48-h no observable effects concentration of nanocopper and nanosilver or to soluble Cu and Ag that matched the concentration of dissolved metals released during nanoparticle exposure. Both nanocopper and nanosilver exposures increased metal content associated with gill tissue, though silver concentrations were much higher following nanosilver exposures suggesting that intact silver nanoparticles are associated with the gill. Morphological and transcriptional responses of the gills differed among various nanomaterials and between nanoparticulate and soluble species. Nanocopper increased mean gill filament width by three to fourfold between 24 and 48 h, whereas nanosilver did not alter gill filament width at either time point. Global gene expression analysis demonstrates that the exposure to each nanometal or soluble metal produces a distinct gene expression profile at both 24 and 48 h, suggesting that each exposure is producing biological response by a different mechanism. The differences in responses among the exposures indicates that each particle is having a distinct biological effect that does not appear to be driven solely by release of soluble metal ions into the water column. Based on these results, care should be taken when inferring toxicity of nanomaterials from data on a different material.

Renal Collecting Duct NOS1 Maintains Fluid–Electrolyte Homeostasis and Blood Pressure

Nitric oxide is a pronatriuretic and prodiuretic factor. The highest renal NO synthase (NOS) activity is found in the inner medullary collecting duct. The collecting duct (CD) is the site of daily fine-tune regulation of sodium balance, and led us to hypothesize that a CD-specific deletion of NOS1 would result in an impaired ability to excrete a sodium load leading to a salt-sensitive blood pressure phenotype. We bred AQP2-CRE mice with NOS1 floxed mice to produce flox control and CD-specific NOS1 knockout (CDNOS1KO) littermates. CDs from CDNOS1KO mice produced 75% less nitrite, and urinary nitrite+nitrate (NOx) excretion was significantly blunted in the knockout genotype. When challenged with high dietary sodium, CDNOS1KO mice showed significantly reduced urine output, sodium, chloride, and NOx excretion, and increased mean arterial pressure relative to flox control mice. In humans, urinary NOx is a newly identified biomarker for the progression of hypertension. These findings reveal that NOS1 in the CD is critical in the regulation of fluid–electrolyte balance, and this new genetic model of CD NOS1 gene deletion will be a valuable tool to study salt-dependent blood pressure mechanisms.

Renal NOS activity, expression and localization in male and female spontaneously hypertensive rats.

The goal of this study was to examine the status of the renal nitric oxide (NO) system by determining NO synthase (NOS) isoform activity and expression within the three regions of the kidney in 14-wk-old male and female spontaneously hypertensive rats (SHR). NOS activity, and NOS1 and NOS3 protein expressions and localization were comparable in the renal cortex and outer medulla of male and female SHR. In contrast, male SHR had significantly less NOS1 and NOS3 enzymatic activity (0 +/- 5 and 53 +/- 7 pmol.mg(-1).30 min(-1), respectively) compared with female SHR (37 +/- 16 and 172 +/- 40 pmol.mg(-1).30 min(-1), respectively). Lower levels of inner medullary NOS1 activity in male SHR were associated with less NOS1 protein expression [45 +/- 7 relative densitometric units (RDU)] and fewer NOS1-positive cells in the renal inner medulla compared with female SHR (79 +/- 12 RDU). Phosphorylation of NOS3 is an important determinant of NOS activity. Male SHR had significantly greater phosphorylation of NOS3 on threonine 495 in the renal cortex compared with females (0.25 +/- 0.05 vs. 0.15 +/- 0.06 RDU). NOS3 phosphorylation was comparable in males and females in the other regions of the kidney. cGMP levels were measured as an indirect index of NO production. cGMP levels were significantly lower in the renal cortex (0.08 +/- 0.01 pmol/mg) and inner medulla (0.43 +/- 0.02 pmol/mg) of male SHR compared with females (cortex: 0.14 +/- 0.02 pmol/mg; inner medulla: 0.56 +/- 0.02 pmol/mg). Our data suggest that the effect of the sex of the animal on NOS activity and expression is different in the three regions of the SHR kidney and supports the hypothesis that male SHR have lower NO bioavailability compared with females.

COX2 in a euryhaline teleost, Fundulus heteroclitus: primary sequence, distribution, localization, and potential function in gills during salinity acclimation.

SUMMARY In the kidneys of mammals, cyclooxygenase type 2 (COX2) is expressed in medullary interstitial cells, the macula densa and epithelial cells of the cortical thick ascending limb where it generates prostaglandins that regulate hormone secretion, inhibit ion transport, and support cell survival during salt loading and dehydration. In teleosts, the gills are in direct contact with an aquatic environment and are the dominant site of osmoregulation. During transfers between salinities, specialized cells in the gills (chloride cells) rapidly regulate NaCl secretion for systemic osmoregulation while they simultaneously are exposed to acute osmotic shock. This study was conducted to determine if COX2 is expressed in the gills, and if so, to evaluate its function in cellular and systemic osmoregulation. Degenerate primers, reverse transcription–PCR and rapid amplification of cDNA ends were used to deduce the complete cDNA sequence of a putative COX2 enzyme from the gills of the euryhaline killifish (Fundulus heteroclitus). The 2738 base pair cDNA includes a coding region for a 610 amino acid protein that is over 70% identical to mammalian COX2. A purified antibody generated against a conserved region of mouse COX2 labeled chloride cells, suggesting that the enzyme may control NaCl secretion as an autocrine agent. Real-time PCR was then used to demonstrate that mRNA expression of the COX2 homologue was threefold greater in gills from chronic seawater killifish than in gills from chronic freshwater killifish. Expression of Na+/K+/2Cl– cotransporter and the cystic fibrosis transmembrane conductance regulator were also greater in seawater, suggesting that chronic COX2 expression in the gills is regulated in parallel to the key ion transporters that mediate NaCl secretion. Real-time PCR was also used to demonstrate that acute transfer from seawater to freshwater and from freshwater to seawater led to rapid, transient inductions of COX2 expression. Together with previous physiological evidence, the present molecular and immunological data suggest that constitutive branchial COX2 expression is enhanced in seawater, where prostaglandins can regulate NaCl secretion in chloride cells. Our data also suggest that branchial COX2 expression may play a role in cell survival during acute osmotic shock.

Endothelin and endothelin converting enzyme-1 in the fish gill: evolutionary and physiological perspectives

SUMMARY In euryhaline fishes like the killifish (Fundulus heteroclitus) that experience daily fluctuations in environmental salinity, endothelin 1 (EDN1) may be an important regulator molecule necessary to maintain ion homeostasis. The purpose of this study was to determine if EDN1 and the endothelin converting enzyme (ECE1; the enzyme necessary for cleaving the precursor proendothelin-1 to EDN1) are present in the killifish, to determine if environmental salinity regulates their expression, and to examine the phylogenetic relationships among the EDNs and among the ECEs. We sequenced killifish gill cDNA for two EDN1 orthologues, EDN1A and EDN1B, and also sequenced a portion of ECE1 cDNA. EDN1A and ECE1 mRNA are expressed ubiquitously in the killifish while EDN1B mRNA has little expression in the killifish opercular epithelium or gill. Using in situ hybridization and immunohistochemistry, EDN1 was localized to large round cells adjacent to the mitochondrion-rich cells of the killifish gill, and to lamellar pillar cells. In the gill, EDN1A and EDN1B mRNA levels did not differ with acute (<24 h) or chronic (30 days) acclimation to seawater (SW); however, EDN1B levels increased threefold post SW to freshwater (FW) transfer, and ECE1 mRNA levels significantly increased twofold over this period. ECE1 mRNA levels also increased sixfold over 24 h post FW to SW transfer. Chronic exposure to SW or FW had little effect on ECE1 mRNA levels. Based upon our cellular localization studies, we modeled EDN1 expression in the fish gill and conclude that it is positioned to act as a paracrine regulator of gill functions in euryhaline fishes. It also may function as an autocrine on pillar cells, where it is hypothesized to regulate local blood flow in the lamellae. From our phylogenetic analyses, ECE is predicted to have an ancient origin and may be a generalist endoprotease in non-vertebrate organisms, while EDNs are vertebrate-specific peptides and may be key characters in vertebrate evolution.

Dynamin activates NO production in rat renal inner medullary collecting ducts via protein-protein interaction with NOS1

We hypothesized that nitric oxide synthase (NOS) isoforms may be regulated by dynamin (DNM) in the inner medullary collecting duct (IMCD). The aims of this study were to determine which DNM isoforms (DNM1, DNM2, DNM3) are expressed in renal IMCDs, whether DNM interacts with NOS, whether a high-salt diet alters the interaction of DNM and NOS, and whether DNM activates NO production. DNM2 and DNM3 are highly expressed in the rat IMCD, while DNM1 is localized outside of the IMCD. We found that DNM1 interacts with NOS1α, NOS1β, and NOS3 in the inner medulla of male Sprague-Dawley rats on a 0.4% salt diet. DNM2 interacts with NOS1α, while DNM3 interacts with both NOS1α and NOS1β. DNM2 and DNM3 do not interact with NOS3 in the rat inner medulla. We did not observe any change in the DNM/NOS interactions with rats on a 4% salt diet after 7 days. Furthermore, NOS1α interacts with DNM2 in mIMCD3 and COS7 cells transfected with NOS1α and DNM2-GFP constructs and the NOS1 reductase domain is necessary for the interaction. Finally, COS7 cells expressing NOS1α or NOS1α/DNM2-GFP had significantly higher nitrite production compared with DNM2-GFP only. Nitrite production was blocked by the DNM inhibitor dynasore or the dominant negative DNM2K44A. Ionomycin stimulation further increased nitrite production in the NOS1α/DNM2-GFP cells compared with NOS1α only. In conclusion, DNM and NOS1 interact in the rat renal IMCD and this interaction leads to increased NO production, which may influence NO production in the renal medulla.

NOS1-dependent negative feedback regulation of the epithelial sodium channel in the collecting duct

With an increase in urine flow there is a significant increase in shear stress against the renal epithelium including the inner medullary collecting duct, resulting in an increase in nitric oxide (NO) production. The mechanisms of the shear stress-mediated increases in NO are undetermined. Previous studies found that shear stress increases epithelial sodium channel (ENaC) open probability and endothelin (ET)-1 production in an ENaC-dependent mechanism in the collecting duct (CD). Given that ET-1 stimulates NO production in the CD, we hypothesized that shear stress-induced NO production is downstream of shear stress-induced ENaC activation and ET-1 production in a negative feedback loop. We determined that nitric oxide synthase 1 (NOS1) and NOS3 contribute to shear stress-mediated NO production in the CD, that is attenuated by low doses of the ENaC inhibitors amiloride and benzamil. Moreover, ETB receptor blockade significantly blunted the shear stress-mediated NO production. We further elucidated whether mice lacking NOS1 in the collecting duct (CDNOS1KO) have an impaired renal ET-1 system in the CD. Although urinary ET-1 production and inner medullary ET receptor expression were similar between flox control and CDNOS1KO mice, acute ET-1 treatment significantly reduced ENaC open probability in CDs from flox mice but not CDNOS1KO mice compared with basal. Basal ENaC activity in CDs was similar between the genotypes. We conclude that during acute shear stress across the CD, ENaC acts in a negative feedback loop to stimulate NO production in an ETB/NOS1-dependent manner resulting in a decrease in ENaC open probability and promoting natriuresis.

Endothelin-1 as a master regulator of whole-body Na+ homeostasis.

The current study was designed to determine whether vascular endothelial‐derived endothelin‐1 (ET‐1) is important for skin Na+ buffering. In control mice (C57BL/6J), plasma Na+ and osmolarity were significantly elevated in animals on high‐ vs. low‐salt (HS and LS, respectively) intake. The increased plasma Na+ and osmolarity were associated with increased ET‐1 mRNA in vascular tissue. There was no detectable difference in skin Na+:H2O in HS fed mice (0.119 ± 0.005 mM vs. 0.127 ± 0.007 mM; LS vs. HS); however, skin Na+:H2O was significantly increased by blockade of the endothelin type A receptor with ABT‐627 (0.116 ± 0.006 mM vs. 0.137 ± 0.007 mM; LS vs. HS; half‐maximal inhibitory concentration, 0.055 nM). ET‐1 peptide content in skin tissue was increased in floxed control animals on HS (85.9 ± 0.9pg/mg vs. 106.4 ± 6.8 pg/mg; P< 0.05), but not in vascular endothelial cell endothelin‐1 knockout (VEET KO) mice (76.4 ± 5.7 pg/mg vs. 65.7 ± 7.9 pg/mg; LS vs. HS). VEET KO mice also had a significantly elevated skin Na+:H2O (0.113 ± 0.007 mM vs. 0.137 ± 0.005 mM; LS vs. HS; P < 0.05). Finally, ET‐1 production was elevated in response to increasing extracellular osmolarity in cultured human endothelial cells. These data support the hypothesis that increased extrarenal vascular ET‐1 production in response to HS intake is mediated by increased extracellular osmolarity and plays a critical role in regulating skin storage of Na+.—Speed, J. S., Heimlich, J. B., Hyndman, K. A., Fox, B. M., Patel, V., Yanagisawa, M., Pollock, J. S., Titze, J. M., Pollock, D. M. Endothelin‐1 as a master regulator of whole‐body Na+ homeostasis. FASEB J. 29, 4937–4944 (2015). www.fasebj.org

Sex differences in ET-1 receptor expression and Ca2+ signaling in the IMCD

The inner medullary collecting duct (IMCD) is the nephron segment with the highest production of endothelin-1 (ET-1) and the greatest expression of ET-1 receptors that function to adjust Na(+) and water balance. We have reported that male rats have reduced natriuresis in response to direct intramedullary infusion of ET-1 compared with female rats. Our aim was to determine whether alterations of ET-1 receptor expression and downstream intracellular Ca(2+) signaling within the IMCD could account for these sex differences. IMCDs from male and female rats were isolated for radioligand binding or microdissected for intracellular Ca(2+) ([Ca(2+)]i) measurement by fluorescence imaging of fura-2 AM. IMCD from male and female rats had similar ETB expression (655 ± 201 vs. 567 ± 39 fmol/mg protein, respectively), whereas male rats had significantly higher ETA expression (436 ± 162 vs. 47 ± 29 fmol/mg protein, respectively; P < 0.05). The [Ca(2+)]i response to ET-1 was significantly greater in IMCDs from male compared with female rats (288 ± 52 vs. 118 ± 32 AUC, nM × 3 min, respectively; P < 0.05). In IMCDs from male rats, the [Ca(2+)]i response to ET-1 was significantly blunted by the ETA antagonist BQ-123 but not by the ETB antagonist BQ-788 (control: 137 ± 27; BQ-123: 53 ± 11; BQ-788: 84 ± 25 AUC, nM × 3 min; P < 0.05), consistent with greater ETA receptor function in male rats. These data demonstrate a sex difference in ETA receptor expression that results in differences in ET-1 Ca(2+) signaling in IMCD. Since activation of ETA receptors is thought to oppose ETB receptor activation, enhanced ETA function in male rats could limit the natriuretic effects of ETB receptor activation.

Investigation of acute nanoparticulate aluminum toxicity in zebrafish.

In freshwater fish, aluminum is a well‐recognized gill toxicant, although responses are influenced by pH. Aluminum nanomaterials are being used in diverse applications that are likely to lead to environmental release and exposure. However, it is unclear if the effects of nanoparticulate aluminum are similar to those of other forms of aluminum or require special consideration. To examine the acute toxicological effects of exposure to aluminum nanoparticle (Al‐NP)s, adult female zebrafish were exposed to either Al‐NPs or aluminum chloride for up to 48 hours in moderately hard fresh water. Al‐NPs introduced into test water rapidly aggregated and up to 80% sedimented from the water column during exposures. No mortality was caused by concentrations of Al‐NP up to 12.5 mg/L. After exposure, tissue concentrations of aluminum, effects on gill morphology, Na+, K+ ‐ATPase (NKA) activity, and global gene expression patterns were examined. Exposure to both aluminum chloride and nanoparticulate aluminum resulted in a concentration dependent decrease in sodium potassium ATPase activity, although Al‐NP exposure did not alter gill morphology as measured by filament widths. Decreased ATPase activity coincided with decreases in filamental NKA staining and mucous cell counts. Analysis of gill transcriptional responses demonstrated that exposure to 5 mg/L Al‐NP only resulted in significant changes in expression of two genes, whereas aluminum chloride exposure significantly affected the expression of 105 genes. Taken together, these results indicate that nanoparticulate aluminum has little acute toxicity for zebrafish in moderately hard freshwater.