Comparing United States and European Union academic animal programs: Organization, operation, and services offered.

The Yale Animal Resource Cost and Benchmarking survey©, conducted in US academic Animal Research/Resource Centers (ARC), was modified to capture similar thematic information in European Union (EU; including the non-EU countries Switzerland and the UK) academic ARCs, which are members of the League of European Research Universities (LERU). Participating institutions came from Denmark, England, Finland, France, Germany, Ireland, Italy, Netherlands, Scotland, Spain, and Switzerland. Survey data analysis suggests that: (a) in LERU programs, it is common to have more than one ARC under the umbrella of a single institution with organizational "lumping" of the financial, regulatory, and/or operational tasks under one administrative unit; (b) accreditation by an outside agency (e.g., the Association for the Assessment and Accreditation of Laboratory Animal Care) is more common in US than LERU ARCs; (c) LERU ARCs are responsible for murine breeding, which contrasts with US ARCs, where ∼40% of rodent breeding is managed by researchers; (d) cryopreservation is the most frequently requested fee-for-service offering among LERU participants (75% of participants) compared to 30% of US participants; (e) like US programs, almost all LERU programs have mice and rats, but fewer LERU programs have nonhuman primates (NHPs), and none have dogs on census; (f) LERU ARCs have about an equal amount of vivarium housing and procedure space, while US facilities have twice as much housing as procedure space; (g) a higher percentage of LERU colonies are free of Helicobacter and murine norovirus compared to US colonies; and (h) more LERU ARCs used environmental microbiologic monitoring of rodent colonies compared to US programs.

Comparing United States and European Union academic animal programs: Finances.

The Yale Animal Resource Cost and Benchmarking survey, conducted in United States (US) academic animal research/resource centres (ARC), was modified to capture similar information in European Union (EU) (including the non-EU countries Switzerland and the United Kingdom) academic ARCs, who are members of the League of European Research Universities (LERU). Participating institutions came from Denmark, England, Finland, France, Germany, Ireland, Italy, Netherlands, Scotland, Spain and Switzerland. Survey data analysis suggests that (a) per diem rates have similar compositions in LERU and US programs, with >50% of the rates dedicated to recovering salary and fringe, followed by supplies (∼25%), facility costs (∼10%) and other expenses (∼15%); (b) ∼60% of US and LERU programs under-recover mouse care costs; (c) on average, LERU programs have a small positive net-operating balance, while US programs average a large deficit; (d) in LERU programs <50% of institutions fund the animal program deficit, while in US programs almost 100% of such deficits are covered by the institution; and (e) when setting per diem rates, both US and LERU programs rank cost accounting as the most influential factor. Both US and LERU programs are reluctant to raise per diem rates to the extent required to recover costs and, thus, continue to under-recover costs, resulting in the animal program being 'caught in the middle' between the competing financial challenges of investigator 'affordability' and the animal program's fiduciary responsibility to the institution.

Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways.

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2-/-) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2-/- mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

The role of the IACUC in ensuring research reproducibility.

There is a "village" of people impacting research reproducibility, such as funding panels, the IACUC and its support staff, institutional leaders, investigators, veterinarians, animal facilities, and professional journals. IACUCs can contribute to research reproducibility by ensuring that reviews of animal use requests, program self-assessments and post-approval monitoring programs are sufficiently thorough, the animal model is appropriate for testing the hypothesis, animal care and use is conducted in a manner that is compliant with external and institutional requirements, and extraneous variables are minimized. The persons comprising the village also must have a shared vision that guards against reproducibility problems while simultaneously avoids being viewed as a burden to research. This review analyzes and discusses aspects of the IACUC's "must do" and "can do" activities that impact the ability of a study to be reproduced. We believe that the IACUC, with support from and when working synergistically with other entities in the village, can contribute to minimizing unintended research variables and strengthen research reproducibility.

Drosophila: a fruitful model for calcium oxalate nephrolithiasis?

Even though the prevalence of nephrolithiasis is increasing, our understanding of the pathophysiology has not kept pace and new therapeutic approaches have not emerged. The potential of a new physiological model (the fruitfly) is exciting. The model has strengths, namely the low cost of maintaining colonies and rapid deployment of new transgenic lines, but also weaknesses that may ultimately limit its usefulness, such as the mechanism of tubular fluid formation and difficulties in following plasma and urine biochemistries.

Low urinary citrate: an overview.

Hypocitraturia is a known risk factor for kidney stone formation. By forming soluble complexes with calcium, citrate prevents crystal nucleation, aggregation and growth; therefore, the presence of citrate in the urine reduces the risk for calcium stone formation. Ingested citrate is rapidly metabolized, and plasma citrate levels vary little, so changes in filtered load do not significantly influence urinary citrate excretion. Changes in urinary citrate excretion are predominantly influenced by the rate of citrate absorption from the glomerular filtrate and metabolism by the proximal tubule cell. The former is mediated by the apical membrane cotransporter NaDC1, and the latter is mediated by both cytoplasmic and mitochondrial metabolism. Acid-base status is the most important physiological determinant of urinary citrate excretion, by modulating the activities of NaDC1 and cytoplasmic (ATP citrate lyase) and mitochondrial (m-aconitase) enzymes involved in citrate metabolism. Following an acid load, both the transport and metabolic processes are up-regulated leading to hypocitraturia; in contrast, an alkaline load increases citrate excretion, by regulating only the mitochondrial metabolic process.

Acid regulation of NaDC-1 requires a functional endothelin B receptor.

Metabolically generated acid is the major physiological stimulus for increasing proximal tubule citrate reabsorption, which leads to a decrease in citrate excretion. The activity of the Na-citrate cotransporter, NaDC-1, is increased in vivo by acid ingestion and in vitro by an acidic pH medium. In opossum kidney cells the acid stimulatory effect and the ability of endothelin-1 (ET-1) to stimulate NaDC-1 activity are both blocked by the endothelin B (ET(B)) receptor antagonist, BQ788. Acid feeding had no effect on brush border membrane NaDC-1 activity in mice in which ET(B) receptor expression was knocked out, whereas a stimulatory effect was found in wild-type mice. Using ET(A)/ET(B) chimeric and ET(B) C-terminal tail truncated constructs, ET-1 stimulation of NaDC-1 required a receptor C-terminal tail from either ET(A) or ET(B). The ET-1 effect was greatest when either the ET(B) transmembrane domain and C-terminal tail were present or the ET(B) C-terminal tail was linked to the ET(A) transmembrane domain. This effect was smaller when the ET(B) transmembrane domain was linked to the ET(A) C-terminal tail. Thus, the acid-activated pathway mediating stimulation of NaDC-1 activity requires a functional ET(B) receptor in vivo and in vitro, as does acid stimulation of NHE3 activity. Since increased NaDC-1 and NHE3 activities constitute part of the proximal tubule adaptation to an acid load, these studies indicate that there are similarities in the signaling pathway mediating these responses.

Biochemical and histological assessment of alkali therapy during high animal protein intake in the rat.

The Westernized diet is acidogenic due to the high content of sulfur-containing amino acids and relative deficiency of potassium organic anions. Chronic acid loads result in hypercalciuria and negative calcium balance often associated with loss of bone mineral. Alkali therapy tends to reverse the hypercalciuria but little is known regarding its effect on bone as assessed by bone histomorphometry. The present study utilized dynamic bone histomorphometry to evaluate the effects of alkali therapy on acid-induced changes in bone turnover. Serum and urine analyses and bone histomorphometry were assessed in adult rats after 2 months of either a low casein (LC) or high casein (HC) diet supplemented with either potassium chloride (KCl) or potassium citrate (KCit). Compared to animals on LC-KCl diet, HC-KCl diet delivered a substantial acid load as shown by significant increases in urinary sulfate, ammonium, and net acid excretion, and a lower urinary pH and citrate excretion without detectable changes in serum parameters. The acid load also resulted in hypercalciuria. Dynamic and static bone histomorphometry disclosed a significant reduction in cancellous bone volume and trabecular number associated with a 2.5-fold increase in eroded and a 3.5-fold increase in osteoclastic surfaces. There was also a near 2-fold increase in bone formation rate in rats on the HC-KCl diet. When animals on the HC diet were given KCit instead of KCl, all of the aforementioned changes in urine biochemistry and bone turnover were significantly attenuated or entirely prevented. These findings underscore the deleterious effects of high animal protein intake in promoting hypercalciuria and increasing bone turnover. Co-administration of potassium alkali attenuates or prevents these changes. In this animal model of high dietary animal protein intake, the major skeletal effect of alkali therapy is to reduce bone resorption, with little or no effect on bone formation.

RhoA required for acid-induced stress fiber formation and trafficking and activation of NHE3.

Exposure to an acid load increases apical membrane Na(+)/H(+) antiporter (NHE3) activity, a process that involves exocytic trafficking of the transporter to the apical membrane. We have previously shown that an intact microfilament structure is required for this exocytic process (Yang X, Amemiya M, Peng Y, Moe OW, Preisig PA, Alpern RJ. Am J Physiol Cell Physiol 279: C410-C419, 2000). The present studies demonstrate that acid-induced stress fiber formation is required for stimulation of NHE3 activity. Formation of stress fibers is associated with acid-induced tyrosine phosphorylation and increases in protein abundance of two focal adhesion proteins, p125(FAK) and paxillin. The Rho kinase inhibitor Y27632 completely blocks acid-induced stress fiber formation and the increases in apical membrane NHE3 abundance and activity, but it has no effect on acid-induced tyrosine phosphorylation of p125(FAK) or paxillin. Herbimycin A completely blocks acid-induced tyrosine phosphorylation of p125(FAK) and paxillin but only partially blocks stress fiber formation and NHE3 activation. These studies demonstrate that Rho kinase mediates acid-induced stress fiber formation, which is required for NHE3 exocytosis, and increases in NHE3 activity. Acid-induced tyrosine phosphorylation of the focal adhesion proteins p125(FAK) and paxillin is not Rho kinase dependent. Thus these two acid-mediated effects are associated, yet independent processes.

A consensus sequence in the endothelin-B receptor second intracellular loop is required for NHE3 activation by endothelin-1.

Endothelin-1 (ET-1) increases the activity of Na(+)/H(+) exchanger 3 (NHE3), the major proximal tubule apical membrane Na(+)/H(+) antiporter. This effect is seen in opossum kidney (OKP) cells expressing the endothelin-B (ET(B)) and not in cells expressing the endothelin-A (ET(A)) receptor. However, ET-1 causes similar patterns of protein tyrosine phosphorylation, adenylyl cyclase inhibition, and increases in cell [Ca(2+)] in ET(A)- and ET(B)-expressing OKP cells, implying that an additional mechanism is required for NHE3 stimulation by the ET(B) receptor. The present studies used ET(A) and ET(B) receptor chimeras and site-directed mutagenesis to identify the ET receptor domains that mediate ET-1 regulation of NHE3 activity. We found that binding of ET-1 to the ET(A) receptor inhibits NHE3 activity, an effect for which the COOH-terminal tail is necessary and sufficient. ET-1 stimulation of NHE3 activity requires the COOH-terminal tail and the second intracellular loop of the ET(B) receptor. Within the second intracellular loop, a consensus sequence was identified, KXXXVPKXXXV, that is required for ET-1 stimulation of NHE3 activity. This sequence suggests binding of a homodimeric protein that mediates NHE3 stimulation.

Pyk2 activation is integral to acid stimulation of sodium/hydrogen exchanger 3.

The present study examines the role of Pyk2 in acid regulation of sodium/hydrogen exchanger 3 (NHE3) activity in OKP cells, a kidney proximal tubule epithelial cell line. Incubation of OKP cells in acid media caused a transient increase in Pyk2 phosphorylation that peaked at 30 seconds and increased Pyk2/c-Src binding at 90 seconds. Pyk2 isolated by immunoprecipitation and studied in a cell-free system was activated and phosphorylated at acidic pH. Acid activation of Pyk2 (a) was specific for Pyk2 in that acid did not activate focal adhesion kinase, (b) required calcium, and (c) was associated with increased affinity for ATP. Transfection of OKP cells with dominant-negative pyk2(K457A) or small interfering pyk2 duplex RNA blocked acid activation of NHE3, while neither had an effect on glucocorticoid activation of NHE3. In addition, pyk2(K457A) blocked acid activation of c-Src kinase, which is also required for acid regulation of NHE3. The present results demonstrate that Pyk2 is directly activated by acidic pH and that Pyk2 activation is required for acid activation of c-Src kinase and NHE3. Given that partially purified Pyk2 can be activated by acid in a cell-free system, Pyk2 may serve as the pH sensor that initiates the acid-regulated signaling cascade involved in NHE3 regulation.

An autocrine role for endothelin-1 in the regulation of proximal tubule NHE3.

BACKGROUND: Chronic metabolic acidosis leads to an increase in NHE3 activity that is mediated by endothelin-1 (ET-1) expression and activation of the proximal tubule endothelin B receptor. Chronic metabolic acidosis increases preproET-1 mRNA abundance in kidney cortex, but the cell responsible has not been identified. METHODS: PreproET-1 mRNA abundance was quantified by competitive reverse transcription-polymerase chain reaction (RT-PCR) on tissue harvested from control rats or rats in which chronic metabolic acidosis was induced by addition of NH(4)Cl to the drinking water. RESULTS: Chronic metabolic acidosis leads to an increase in preproET-1 mRNA expression in kidney cortex, proximal tubules, and glomeruli. The increase in preproET-1 expression correlates with the decrease in blood [HCO3(-)]. ET-1 expression is also increased by acidosis in abdominal aorta, but not in cardiac muscle. CONCLUSION: In the renal proximal tubule, chronic metabolic acidosis induces an increase in preproET-1 expression, providing a mechanism for autocrine regulation of proximal tubule NHE3 activity. This response is not unique to the proximal tubule cell, but is also not ubiquitous.

OKP cells express the Na-dicarboxylate cotransporter NaDC-1.

Urinary citrate concentration, a major factor in the formation of kidney stones, is primarily determined by its rate of reabsorption in the proximal tubule. Citrate reabsorption is mediated by the Na-dicarboxylate cotransporter-1 (NaDC-1). The opossum kidney (OKP) cell line possesses many characteristics of the renal proximal tubule. The OKP NaDC-1 (oNaDC-1) cDNA was cloned and encodes a 2.4-kb mRNA. When injected into Xenopus oocytes, the cotransporter is expressed and demonstrates Na-coupled citrate transport with a stoichiometry of >or=3 Na:1 citrate, specificity for di- and tricarboxylates, pH-dependent citrate transport, and pH-independent succinate transport, all characteristics of the other NaDC-1 orthologs. Chronic metabolic acidosis increases proximal tubule citrate reabsorption, leading to profound hypocitraturia and an increased risk for stone formation. Under the conditions studied, endogenous OKP NaDC-1 mRNA abundance is not regulated by changes in media pH. In OKP cells transfected with a green fluorescent protein-oNaDC-1 construct, however, media acidification increases Na-dependent citrate uptake, demonstrating posttranscriptional acid regulation of NaDC-1 activity.

Dietary acid, endothelins, and sleep.

Acid addition to the body activates a series of homeostatic responses, one example of which is activation of NHE3, the proximal tubule Na(+)/H(+) antiporter. Feeding acid to rats increases apical membrane NHE3 abundance. Similarly, addition of acid to the media of OKP cells, a proximal tubule cell line, leads to an increase in apical membrane NHE3 activity that is due to increased trafficking of NHE3 to the apical membrane, and increased NHE3 mRNA and protein expression. Endothelins also increase NHE3 activity by inducing trafficking of NHE3 to the apical membrane, an effect mediated by the ET(B), but not the ET(A) receptor. Receptor specificity resides in the C-terminal loop and the second intracellular loop of the ET(B) receptor. In addition, the ET(B) receptor is required for acid signaling. An acid-induced signaling cascade has been defined that includes Pyk2, c-Src, ERK, c-fos, c-jun, and endothelin expression.

Effect of high protein diet on stone-forming propensity and bone loss in rats.

BACKGROUND: High protein diets are believed to cause kidney stone formation and bone loss, but the mechanisms mediating these changes are unknown. The purpose of this study was to create an animal model of animal protein excess and to evaluate the response of kidney and bone to the dietary protein load. METHODS: Rats (12 per group) were pair-fed with a high (48%) and low (12%) casein diets that were otherwise identical in their content of sodium, potassium, calcium, phosphorus, and magnesium. RESULTS: Compared with the low casein group, the high casein group delivered a substantial acid load during 59 days of study, since it significantly decreased urinary pH, and increased urinary ammonium, titratable acidity, and net acid excretion. Animals on high casein diet also had higher urinary volumes. On the high casein diet, urinary calcium excretion was significantly higher and urinary citrate excretion and concentration was significantly lower. On the high casein diet, urinary saturation of calcium phosphate was higher. Serum calcitriol concentration did not significantly differ between the two groups. Histomorphometric analysis of femur procured after 59 days on the diet showed marked increase in bone resorption in the high casein group. Hypocitraturia was associated with increased activity of sodium-citrate cotransporter in renal cortical brush-border membranes (BBM) in the high casein group. CONCLUSION: Both the kidney and bone contribute to the pathogenesis of hypercalciuria during high casein diet in rats. Hypocitraturia is probably renal in origin. This rat model will be useful in elucidating the mechanisms by which high protein intake increases the risk of nephrolithiasis and bone loss in human beings.