Analysis of the order-implantation relationship for musculoskeletal tissue transplantation. / Análisis de la relación solicitud-implante de injerto para trasplante osteomuscular.

OBJECTIVE: To analyze orders requested from a musculoskeletal tissue bank and to evaluate the percentage of tissue implantation. MATERIAL AND METHODS: Two hundred and sixty-five orders for musculoskeletal tissue were analyzed over the course of a year. EXCLUSIONS: 5 duplications and 5 orders for which there was no availability to cover the need. We analyzed the number of surgeries in which the graft was finally used. RESULTS: Of a total of 255 orders, the graft was used in 178 (70%), and the graft was not used in 77 (30%). Of the 178 used, there was a partial refund in 23 (10%). Of the 77 orders not used, surgery was performed in 32 (13%) without the use of bank tissue, while surgery was discontinued in the remaining 45 (17%). DISCUSSION: A non-utilization rate of 30% was identified, of which 17% was from surgery that was not performed and 13% from surgery that was performed, but the tissue was returned to the tissue bank, because it was not required. In a further 10% there was partial return of the tissue. Based on this analysis, we consider that it is important to have direct confirmation of the surgery to avoid sending tissue for discontinued surgeries, since in addition to the economic impact, the bank must ensure adequate temperature maintenance during transportation and storage in the transplantation centre, to avoid discarding said tissue if it is returned.

Participation of membrane progesterone receptor α in the inhibitory effect of progesterone on prolactin secretion.

The membrane progesterone receptors (mPRα, mPRß, mPRγ, mPRδ and mPRε) are known to mediate rapid nongenomic progesterone functions in different cell types. However, the functions of these receptors in the pituitary have not been reported to date. In the present study, we show that the expression of mPRα was the highest among the mPRs in the rat anterior pituitary gland. Immunostaining of mPRα was detected in somatotrophs, gonadotrophs and lactotrophs. Interestingly, 63% of mPRα-positive cells within the pituitary were lactotrophs, suggesting that mPRα is involved in controlling prolactin (PRL) secretion in the pituitary. To test this hypothesis, rat pituitaries were incubated (1 hour) with either progesterone (P4) or the mPRα-specific agonist Org OD 02-0. PRL secretion was then measured by radioimmunoassay. The results of this experiment revealed that both P4 and Org OD 02-0 decreased PRL secretion. Moreover, the results from the GH3 cell line (CCL-82.1) showed that P4 and Org OD 02-0 inhibited PRL release, although the nuclear PR agonist R5020 was ineffective. Our investigation of the cellular mechanisms behind mPRα activity indicated that both P4 and Org OD 02-0 decreased cAMP accumulation, whereas R5020 was ineffective. In addition, the Org OD 02-0-effect on PRL release was blocked by pretreatment with pertussis toxin, an inhibitor of Go/Gi proteins. Because transforming growth factor (TGF)ß1 is a potent inhibitor of PRL secretion in lactotrophs, we lastly evaluated whether TGFß1 was activated by progesterone and whether this effect was mediated by mPRα. Our results showed that P4 and Org OD 02-0, but not R5020, increased active TGFß1 levels. This effect was not observed when cells were transfected with mPRα-small interfering RNA. Taken together, these data provide new evidence suggesting that mPRα mediates the progesterone inhibitory effect on PRL secretion through both decreases in cAMP levels and activation of TGFß1 in the lactotroph population.

Estradiol Upregulates c-FLIPlong Expression in Anterior Pituitary Cells.

Anterior pituitary cell turnover depends on a tight balance between proliferation and apoptosis. We have previously shown that estrogens sensitize anterior pituitary cells to pro-apoptotic stimuli. c-FLIP (cellular-FLICE-inhibitory-protein) isoforms are regulatory proteins of apoptosis triggered by death receptors. c-FLIPshort isoform competes with procaspase-8 inhibiting its activation. However, c-FLIPlong isoform may have a pro- or anti-apoptotic function depending on its expression level. In the present study, we explored whether estrogens modulate c-FLIP expression in anterior pituitary cells from ovariectomized (OVX) rats and in GH3 cells, a somatolactotrope cell line. Acute administration of 17ß-estradiol to OVX rats increased c-FLIPlong expression in the anterior pituitary gland without changing c-FLIPshort expression as assessed by Western blot. Estradiol in vitro also increased c-FLIPlong expression in anterior pituitary cells but not in GH3 cells. As determined by flow cytometry, the percentage of anterior pituitary cells expressing c-FLIP was higher than in GH3 cells. However, c-FLIP fluorescence intensity in GH3 cells was higher than in anterior pituitary cells. FasL increased the percentage of TUNEL-positive GH3 cells incubated either with or without estradiol suggesting that the pro-apoptotic action of Fas activation is estrogen-independent. Our results show that unlike what happens in nontumoral pituitary cells, estrogens do not modulate either c-FLIPlong expression or FasL-induced apoptosis in GH3 cells. The stimulatory effect of estradiol on c-FLIPlong expression could be involved in the sensitizing effect of this steroid to apoptosis in anterior pituitary cells. The absence of this estrogenic action in tumor pituitary cells could be involved in their tumor-like behavior.

Inhibition of nuclear factor-kappa B sensitises anterior pituitary cells to tumour necrosis factor-α- and lipopolysaccharide-induced apoptosis.

Nuclear factor-kappa B (NF-κB), an important pro-inflammatory factor, is a crucial regulator of cell survival. Both lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-α activate NF-κB signalling. Oestrogens were shown to suppress NF-κB activation. Oestrogens exert a sensitising action to pro-apoptotic stimuli such as LPS and TNF-α in anterior pituitary cells. In the present study, we show by western blotting that 17ß-oestradiol (E(2)) decreases TNF-α-induced NF-κB/p65 and p50 nuclear translocation in primary cultures of anterior pituitary cells from ovariectomised (OVX) rats. Also, the in vivo administration of E(2) decreases LPS-induced NF-κB/p65 and p50 nuclear translocation. To investigate whether the inhibition of NF-κB pathway sensitises anterior pituitary cells to pro-apoptotic stimuli, we used an inhibitor of NF-κB activity, BAY 11-7082 (BAY). BAY, at a concentration that fails to induce apoptosis, has permissive action on TNF-α-induced apoptosis of lactotrophs and somatotrophs from OVX rats, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Pharmacological inhibition of NF-κB signalling enhances E(2)-sensitising effect to TNF-α-induced apoptosis in lactotrophs but not in somatotrophs. In vivo administration of BAY allowed LPS-induced apoptosis in anterior pituitary cells from OVX rats (determined by fluorescence activated cell sorting). Furthermore, LPS-induced expression of Bcl-xL in pituitaries of OVX rats is decreased by E(2) administration. Our results show that inhibition of the NF-κB signalling pathway sensitises anterior pituitary cells to the pro-apoptotic action of LPS and TNF-α. Because E(2) inhibits LPS- and TNF-α-activated NF-κB nuclear translocation, the present study suggests that E(2) sensitises anterior pituitary cells to TNF-α- and LPS-induced apoptosis by inhibiting NF-κB activity.

Estrogens exert a rapid apoptotic action in anterior pituitary cells.

It is now accepted that estrogens not only stimulate lactotrope proliferation but also sensitize anterior pituitary cells to proapoptotic stimuli. In addition to their classical mechanism of action through binding to intracellular estrogen receptors (ERs), there is increasing evidence that estrogens exert rapid actions mediated by cell membrane-localized ERs (mERs). In the present study, we examined the involvement of membrane-initiated steroid signaling in the proapoptotic action of estradiol in primary cultures of anterior pituitary cells from ovariectomized rats by using estren, a synthetic estrogen with no effect on classical transcription and a cell-impermeable 17beta-estradiol conjugate (E2-BSA). Both compounds induced cell death of anterior pituitary cells after 60 min of incubation as assessed by flow cytometry and the [3-(4,5-dimethylthiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Estren, E2, and E2-BSA induced apoptosis of lactotropes and somatotropes as evaluated by the deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and immunodetection of prolactin (PRL) and growth hormone (GH). The proapoptotic effect of E2-BSA was abrogated by ICI-182,780, an antagonist of ERs. The expression of membrane-associated ERalpha was observed in PRL- and GH-bearing cells. Our results indicate that estradiol is able to exert a rapid apoptotic action in anterior pituitary cells, especially lactotropes and somatotropes, by a mechanism triggered by mERs. This mechanism could be involved in anterior pituitary cell turnover.

Citoquinas reguladoras de la respuesta al trasplante renal alogénico / Regulatory cytokines in the response to the allogeneic renal transplant

The outcome of the kidney allograft mainly depends on the immune response and on its complex regulation, where the cytokine network and other mediators play an important role. At present, kidney biopsy is the most useful tool for monitoring the transplant rejection and the diagnosis of the associated nephropathies, in spite of the invasiveness of the procedure. Thus, it is of great interest to find alternative tools for diagnosis. The evaluation of regulatory cytokines is a simple procedure of low cost that could be useful to increase the sensitivity of the detection of polymorphic differences, to predict the graft acceptance and for the early detection of rejection. Recent studies suggest that the high production of pro-inflammatory mediators, such as Th1 cytokines, could be detrimental, whereas the production of anti-inflammatory regulatory cytokines, such as interleukin (IL)-10 and tumor necrosis factor (TGF)-beta, could be beneficial for graft survival. In the early stages, the cellular cytotoxicity is activated by the Th1 response and the detection of cytotoxic molecules is associated to the acute rejection. Later, the balance between pro and anti-inflammatory mediators and the regulation of their levels could be more important. In this regard, TGF-beta is also fibrogenic and a high local production can contribute to kidney damage. On the other hand, the increased production of IL-10 in response to the allogeneic stimuli could be, in most cases, an important marker of long-term acceptance.

La aceptación o el rechazo del riñón alogénico dependen principalmente de la respuesta inmune y de su compleja regulación en la cual la red de citoquinas y otros mediadores juegan un importantepapel. Actualmente, la biopsia renal es, a pesar de lo invasor del procedimiento, la herramienta de mayor utilidadpara el control del rechazo al trasplante y el diagnóstico de las nefropatías asociadas. Por ello, es de graninterés encontrar métodos alternativos para el diagnóstico. La evaluación de citoquinas reguladoras de la respuestainmune es un procedimiento sencillo y de bajo costo que podría ser de utilidad para incrementar la sensibilidadde la detección de diferencias polimórficas, para pronosticar la aceptación del trasplante y para ladetección precoz del rechazo. Los estudios recientes sugieren que la producción exagerada de mediadores proinflamatorios, incluyendo a citoquinas Th1, sería desventajosa para la sobrevida del trasplante, mientras que la producción de citoquinas reguladoras anti-inflamatorias, como la interleuquina (IL)-10 y el factor de crecimiento tumoral (TGF)-β, sería beneficiosa. En las primeras etapas, la respuesta Th1 puede incrementar la actividad citotóxica y la detección de moléculas citotóxicas está asociada al rechazo agudo. Luego podría ser más importante considerar el balance entre la producción de mediadores pro- y anti-inflamatorios y la regulación desus niveles. Así, el TGF-β es también fibrogénico y su excesiva producción local puede contribuir al daño renal.Por otro lado, el incremento de la producción de IL-10 en respuesta al estímulo alogénico sería, en la mayoríade los casos, un marcador importante para pronosticar la aceptación prolongada.

Estrogens sensitize anterior pituitary gland to apoptosis.

Tissue homeostasis results from a balance between cell proliferation and cell death by apoptosis. Estradiol affects proliferation as well as apoptosis in hormone-dependent tissues. In the present study, we investigated the apoptotic response of the anterior pituitary gland to lipopolysaccharide (LPS) in cycling female rats, and the influence of estradiol in this response in ovariectomized (OVX) rats. The OVX rats were chronically estrogenized with implanted Silastic capsules containing 1 mg of 17beta-estradiol (E2). Cycling or OVX and E2-treated rats were injected with LPS (250 microg/rat ip). Apoptosis was determined by the terminal deoxynucleotidyl-mediated dUTP nick-end labeling (TUNEL) method in sections of the anterior pituitary gland and spleen. Chronic estrogenization induced apoptosis in the anterior pituitary gland. Acute endotoxemia triggered apoptosis of cells in the anterior pituitary gland of E2-treated rats but not of OVX rats. No differences were observed in the apoptotic response to LPS in spleen between OVX and E2-treated rats. The apoptotic response of the anterior pituitary to LPS was variable along the estrous cycle, being higher at proestrus than at estrus or diestrus I. Approximately 75% of the apoptotic cells were identified as lactotropes by immunofluorescence. In conclusion, our results indicate that estradiol induces apoptosis and enables the proapoptotic action of LPS in the anterior pituitary gland. Also, our study suggests that estrogens may be involved in anterior pituitary cell renewal during the estrous cycle, sensitizing lactotropes to proapoptotic stimuli.

Regulation of aquaporin-2 trafficking by vasopressin in the renal collecting duct. Roles of ryanodine-sensitive Ca2+ stores and calmodulin.

In the renal collecting duct, vasopressin increases osmotic water permeability (P(f)) by triggering trafficking of aquaporin-2 vesicles to the apical plasma membrane. We investigated the role of vasopressin-induced intracellular Ca(2+) mobilization in this process. In isolated inner medullary collecting ducts (IMCDs), vasopressin (0.1 nm) and 8-(4-chlorophenylthio)-cAMP (0.1 mm) elicited marked increases in [Ca(2+)](i) (fluo-4). Vasopressin-induced Ca(2+) mobilization was completely blocked by preloading with the Ca(2+) chelator BAPTA. In parallel experiments, BAPTA completely blocked the vasopressin-induced increase in P(f) without affecting adenosine 3',5'-cyclic monophosphate (cAMP) production. Previously, we demonstrated the lack of activation of the phosphoinositide-signaling pathway by vasopressin in IMCD, suggesting an inositol 1,4,5-trisphosphate-independent mechanism of Ca(2+) release. Evidence for expression of the type 1 ryanodine receptor (RyR1) in IMCD was obtained by immunofluorescence, immunoblotting, and reverse transcription-polymerase chain reaction. Ryanodine (100 microm), a ryanodine receptor antagonist, blocked the arginine vasopressin-mediated increase in P(f) and blocked vasopressin-stimulated redistribution of aquaporin-2 to the plasma membrane domain in primary cultures of IMCD cells, as assessed by immunofluorescence immunocytochemistry. Calmodulin inhibitors (W7 and trifluoperazine) blocked the P(f) response to vasopressin and the vasopressin-stimulated redistribution of aquaporin-2. The results suggest that Ca(2+) release from ryanodine-sensitive stores plays an essential role in vasopressin-mediated aquaporin-2 trafficking via a calmodulin-dependent mechanism.

Conversion from azathioprine [correction of azathioprina] to mycophenolate mofetil in pediatric renal transplant recipients with chronic rejection.

BACKGROUND: Chronic rejection is the leading cause of graft failure. Both nonimmunological and immunological mechanisms contribute to this pathology. METHODS: We studied changes in kidney function, mixed lymphocyte culture, cell-mediated lympholysis, serum HLA class I antigens, cytotoxic antibodies, and lymphocyte population before and after 6 months of follow-up in 22 pediatric renal transplanted patients. The immunosuppressive protocol used was: cyclosporine, azathioprine, and corticosteroids. Eight patients demonstrated chronic graft rejection (by biopsy), group I; and eight patients had no clinical evidence of chronic and/or acute rejection, group II. Substitution of mycophenolate mofetil (MMF) (600 mg/m2 bid for azathioprine was done in patients of groups I and II. Another six patients with chronic rejection, did not receive MMF, group III. RESULTS: Creatinine clearance increased in group I (44+/-5 vs. 51.1+/- ml/min/1.73 m2, P<0.03) but it decreased in group III (30+/-3 vs. 25+/-2, P<0.01). Urinary protein excretion decreased only in group I (0.3+/-0.03 to 0.06+/-0.03 g/24 hr, P<0.03). During MMF therapy antidonor mixed lymphocyte culture decreased 62 and 70% (P<0.05) in group I and II. Cell-mediated lympholysis against lymphocyte of the donor decreased 65% (P<0.05) in group I. Cell-mediated lympholysis toward control cells decreased 54% (P<0.01) in group II. Serum HLA class I antigens, only decreased from 0.7+/-0.1 to 0.5+/-0.1 microl/ml, P<0.05, in group I. CD19+ decreased from 7.9+/-1.1 to 5.6+/-0.8%, P<0.05, and 7.8+/-1.2 to 5.5+/-0.9%, P<0.05, in groups I and II, respectively. CD16+ increased from 5.7+/-1.1 to 8.6+/-1.3 (P<0.05) only in group I. CONCLUSIONS: Our data suggest that substituting MMF for azathioprine therapy leads to an improvement in the immunosuppression and renal function in children with on-going chronic rejection.

A randomized, double-blind, placebo-controlled trial of the effects of prophylactic theophylline on renal function in term neonates with perinatal asphyxia.

BACKGROUND: The kidney is the most damaged organ in asphyxiated full-term infants. Experiments in rabbits and rats have shown that renal adenosine acts as a vasoconstrictive metabolite in the kidney after hypoxemia and/or ischemia, contributing to the fall in glomerular filtration rate (GFR) and filtration fraction. Vasoconstriction produced by adenosine can be inhibited by the nonspecific adenosine receptor antagonist, theophylline. Gouyon and Guignard performed studies in newborn and adult rabbits subjected to normocapnic hypoxemia. Their results clearly showed that the hypoxemia-induced drop in GFR could be avoided by the administration of low doses of theophylline. OBJECTIVE: This study was designed to determine whether theophylline could prevent and/or ameliorate renal dysfunction in term neonates with perinatal asphyxia. SETTING: Buenos Aires, Argentina. STUDY DESIGN: We randomized 51 severe asphyxiated term infants to receive intravenously a single dose of either theophylline (8 mg/kg; study group: n = 24) or placebo (control group: n = 27) during the first 60 minutes of life. The 24-hour fluid intake and the urine volumes formed were recorded during the first 5 days of life. Daily volume balances (water output/input ratio and weights) were determined. Severe renal dysfunction was defined as serum creatinine elevated above 1.50 mg/dL, for at least 2 consecutive days after a fluid challenge, or rising levels of serum creatinine (.3 mg/dL/day). The GFR was estimated during the second to third days of life by endogenous creatinine clearance (mL/minute/1.73 m2) and using Schwartz's formula: GFR (mL/minute/1.73 m2) =.45 x length (cm)/plasma creatinine (mg/100 mL) during the first 5 days of life. Tubular performance was assessed as the concentration of beta2-microglobulin (beta2M) determined by enzyme immunoassay, on the first voided urine 12 hours after theophylline administration. The statistical analysis for the evaluation of the differences between the groups was performed with Student's t and chi(2) tests as appropriate. RESULTS: During the first day of life, the 24-hour fluid balance was significantly more positive in the infants receiving placebo compared with the infants receiving theophyline. Over the next few days, the change in fluid balance favored the theophyline group. Significantly higher mean plasma values were recorded in the placebo group from the second to the fifth days of life. Severe renal dysfunction was present in 4 of 24 (17%) infants of the theophylline group and in 15 of 27 (55%) infants of the control group (relative risk:.30; 95% confidence interval:.12-.78). Mean endogenous creatinine clearance of the theophylline group was significantly increased compared with the creatinine clearance in infants receiving placebo (21.84 +/- 7.96 vs 6.42 +/- 4.16). The GFR (estimated by Schwartz's formula) was markedly decreased in the placebo group. Urinary beta2M concentrations were significantly reduced in the theophylline group (5.01 +/- 2.3 mg/L vs 11.5 +/- 7.1 mg/L). Moreover, 9 (33%) patients of the theophylline group versus 20 (63%) infants of the control group had urinary beta2M above the normal limit (<.018). There was no difference in the severity of the asphyxia between infants belonging to the theophylline and control groups in regards of Portman's score. Except for renal involvement, a similar frequency of multiorganic dysfunction, including neurologic impairment, was observed in both groups. The theophylline group achieved an average serum level of 12.7 microg/mL (range: 7.5-18.9 microg/mL) at 36 to 48 hours of live versus traces (an average serum level of .87 microg/mg) in the placebo group. CONCLUSIONS: Our data suggest that prophylactic theophylline, given early after birth, has beneficial effects on reducing the renal dysfunction in asphyxiated full-term infants. (ABSTRACT TRUNCATED)

Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation.

Acute hypertonicity causes cell cycle delay and apoptosis in mouse renal inner medullary collecting duct cells (mIMCD3) and increases GADD45 expression. Because the tumor suppressor protein p53 may be involved in these effects, we have investigated the role of p53 in mIMCD3 response to hyperosmotic stress. Acute elevation of osmolality with NaCl addition from the control level of 320 mosmol/kg to 500-600 mosmol/kg greatly increased the levels of total and Ser(15)-phosphorylated p53 within 15 min. However, similar elevation of osmolality with urea did not increase p53 levels. Our studies indicate that induced p53 is transcriptionally active because NaCl addition to 500-600 mosmol/kg stimulated transcription of a luciferase reporter containing a p53 consensus element and appropriately altered mRNA levels of known transcriptional targets of p53, i.e. increased MDM-2 and decreased BCL-2 levels. Elevating NaCl further to 700-800 mosmol/kg rapidly killed most of the cells by apoptosis. At these higher NaCl concentrations, p53 levels were further increased although Ser(15) phosphorylation and transcriptional activity were significantly lower than levels at 500-600 mosmol/kg. At NaCl-induced 500 mosmol/kg, apoptosis was rare in the presence of control, nonspecific oligonucleotide but highly prevalent upon addition of p53 antisense oligonucleotide that substantially reduced p53 levels. We conclude that induction of active p53 in mIMCD3 cells by hypertonic stress contributes to cell survival.

Syndrome of inappropriate secretion of antidiuretic hormone in nasopharynx carcinoma.

The syndrome of inappropriate secretion of antidiuretic hormone (ADH) or SIADH has been reported in various disorders. We report a pediatric patient with nasopharynx carcinoma who may have developed a clinical SIADH with severe hyponatremia and generalized seizure during the administration of intravenous hydration. We propose that the inappropriately high plasma level of ADH led to the inability to excrete sufficient amounts of free water during a hyperhydration protocol with a relatively hypotonic fluid, which resulted in acute hyponatremia and central nervous system involvement. To avoid this complication, intravenous hydration before chemotherapy in children with nasopharynx carcinoma should be performed at a slower infusion rate and with a sodium chloride concentration of more than half isotonic.

Distinct regulation of osmoprotective genes in yeast and mammals. Aldose reductase osmotic response element is induced independent of p38 and stress-activated protein kinase/Jun N-terminal kinase in rabbit kidney cells.

In yeast glycerol-3-phosphate dehydrogenase 1 is essential for synthesis of the osmoprotectant glycerol and is osmotically regulated via the high osmolarity glycerol (HOG1) kinase pathway. Homologous protein kinases, p38, and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) are hyperosmotically activated in some mammalian cell lines and complement HOG1 in yeast. In the present study we asked whether p38 or SAPK/JNK signal synthesis of the osmoprotectant sorbitol in rabbit renal medullary cells (PAP-HT25), analogous to the glycerol system in yeast. Sorbitol synthesis is catalyzed by aldose reductase (AR). Hyperosmolality increases AR transcription through an osmotic response element (ORE) in the 5'-flanking region of the AR gene, resulting in elevated sorbitol. We tested if AR-ORE is targeted by p38 or SAPK/JNK pathways in PAP-HT25 cells. Hyperosmolality (adding 150 mM NaCl) strongly induces phosphorylation of p38 and of c-Jun, a specific target of SAPK/JNK. Transient lipofection of a dominant negative mutant of SAPK kinase, SEK1-AL, into PAP-HT25 cells specifically inhibits hyperosmotically induced c-Jun phosphorylation. Transient lipofection of a dominant negative p38 kinase mutant, MKK3-AL, into PAP-HT25 cells specifically suppresses hyperosmotic induction of p38 phosphorylation. We cotransfected either one of these mutants or their empty vector with an AR-ORE luciferase reporter construct and compared the hyperosmotically induced increase in luciferase activity with that in cells lipofected with only the AR-ORE luciferase construct. Hyperosmolality increased luciferase activity equally (5-7-fold) under all conditions. We conclude that hyperosmolality induces p38 and SAPK/JNK cascades in mammalian renal cells, analogous to inducing the HOG1 cascade in yeast. However, activation of p38 or SAPK/JNK pathways is not necessary for transcriptional regulation of AR through the ORE. This finding stands in contrast to the requirement for the HOG1 pathway for hyperosmotically induced activation of yeast GPD1.

Urodecarboxilasa en sangre de pacientes hemodializados / Decarboxylase in blood in hemodialysed patients

En la porfiria cutánea tarda(PCT) hay una falla en la uroporfirinógeno decarboxilasa (Uro-D) hepática, como consecuencia se incrementa la concentración de porfirinas altamente carboxiladas. Enla PCT hereditaria la Uro-D está disminuída en sangre y es normal en la PCT adquirida. Parte de la población hemodializada presenta signos cutáneos que son histológica y morfológicamente semejantes a la PCT, asociado a niveles aumentados de porfirias plasmáticas. Se estudió el contenido de porfirinas plasmáticas y la actividad de la Uro-D eritrocitaria, en 12 pacientes hemodializados sin lesiones cutáneas, uno de ellos portador de PCT hereditaria. El contenido de porfirias en plasma estuvo aumentado(0,084 mas igual 0,,10 ug/ml; uroporfirina igual 80 por ciento, cproporfirina igual 20 por ciento) en 30 por ciento de los pacientes estudiados( valor normal: 0,048 mas igual 0,010 ug/ml; coproporfirina igual 100 por ciento). Las porfirinas plasmáticas del paciente PCT al inicio del tratamiento con S-adenosil-L-metionina fue: 1,71 ug/ml; uroporfirina igual 48 por ciento, firiaporfirina igual 41 por ciento hexaporfirina igual 7 por ciento, pentaporfirina igual 3 por ciento y coproporfirina igual 1 por ciento) y luego de 6 años, al final del tratamiento, los valores fueron: 0,089 ug/ml; uriporfirina igual 80 por ciento, firiaporfirina igual 20 por ciento. La remisión clínica se correlacionó con la bioquímica. En los pacientes hemodializados la actividad de URO_D estuvo dentro de los valores normales(12,45 mas igual 1,o U/ml GR), excepto en el portador de la PCT hereditaria en el cual la actividad estuvodisminuída al 50 por ciento. Estos resultados sugieren que la hemodialisis per se no modificaria a la actividad de URO-D eritrocitaria.

Betaine transporter cDNA cloning and effect of osmolytes on its mRNA induction.

Cells generally adapt to long-term hyperosmolality by accumulating compatible organic osmolytes, thereby helping to normalize both volume and intracellular inorganic ion concentration. When organic osmolytes are accumulated, as in renal inner medullary cells, it is the sum of their concentrations that is theoretically important. In effect, when one organic osmolyte rises, the others generally fall to maintain their sum approximately constant. The present study addresses the mechanism controlling betaine accumulation. Hypertonicity induces accumulation of betaine, sorbitol, inositol, and other organic osmolytes in PAP-HT25 cells, a line derived from rabbit renal papilla. Hypertonicity increases the betaine transporter expression in these cells. To obtain a specific probe for betaine transporter mRNA, we cloned from PAP-HT25 cells a cDNA that encodes the full protein. We then examined the effect of betaine, sorbitol, and inositol on betaine transporter mRNA abundance. Increased accumulation of any of these three organic osmolytes reduces betaine transporter mRNA. We previously observed similar results for aldose reductase, the enzyme responsible for osmotically regulated sorbitol accumulation. We conclude that the accumulation of organic osmolytes regulates betaine transporter gene expression. Because the aldose reductase gene is controlled in a similar fashion, we surmise that the two genes share a common signal for induction.

CD9 antigen mRNA is induced by hypertonicity in two renal epithelial cell lines.

In diverse organisms, cells adapt to hyperosmotic stress by accumulating organic osmolytes. Mammalian renal medullary cells are routinely under osmotic stress. Two renal cell lines, Madin-Darby canine kidney (MDCK) and PAP-HT25, have been widely used to study mammalian osmotic regulation. In these epithelial cells, extracellular hypertonicity induces gene transcription of proteins directly involved in the metabolism and transport of organic osmolytes. This induction is relatively specific and not part of a generalized stress response. Little is known about the signal transduction pathway between cellular detection of extracellular osmolality and increased specific gene transcription. Here, using differential mRNA display polymerase chain reaction on MDCK cells in isotonic vs. hypertonic medium, we identify a cDNA product corresponding to CD9 antigen mRNA. CD9 antigen is a cell surface glycoprotein originally found in cells of the immune system. Although CD9 antigen has been structurally characterized, its function is unclear. We further demonstrate that CD9 antigen mRNA is present in MDCK and PAP-HT25 cells and that its mRNA abundance is induced by extracellular hypertonicity, but not by heat stress. Also, we show that accumulation of organic osmolytes markedly attenuates the CD9 mRNA induction, as only recently demonstrated with genes involved in the hyperosmotic stress response. This suggests a role for CD9 antigen in this response.

Induction of gene expression by heat shock versus osmotic stress.

Elevated temperature rapidly increases expression of genes for heat shock proteins (HSP), including HSP-70. The response is presumably triggered by denaturation of cell proteins and helps in their renaturation. Hypertonicity may also denature proteins, but the protective response, which is accumulation of compatible organic osmolytes [including betaine and inositol in Madin-Darby canine kidney (MDCK) cells], apparently differs and is slow. Recently, hypertonicity was found also to increase expression of HSP-70 in MDCK cells, a response proposed to provide protection until organic osmolytes can accumulate. Our purpose was to examine whether 1) a gene involved in accumulation of organic osmolytes also responds to heat stress and 2) whether accumulation of organic osmolytes affects expression of HSP-70. We find that 1) the betaine transporter mRNA, which is greatly increased by hypertonicity (515 vs. 315 mosmol), is unaffected by high temperature (42 degrees C vs. 37 degrees C); 2) hypertonicity-induced increases in HSP-70 and betaine transporter mRNA are much greater when the medium (and cell) contain no betaine and no inositol than when high concentrations of these are present; and 3) high betaine greatly inhibits the increase in HSP-70 mRNA at high temperature. We conclude the following. 1) Although heat shock and betaine transporter genes both respond to hypertonicity, the betaine transporter is not a HSP. 2) Accumulation of organic osmolytes attenuates the HSP-70 response to hypertonicity, as it might if the HSP-70 expression were a temporizing response. 3) Betaine inhibits HSP-70 response to elevated temperature, presumably by its known effect of stabilizing proteins.

Measurement precision of an olfactory perception threshold test for use in field studies.

Changes in olfactory function have been associated with workplace exposure to a variety of substances. In the workplace, smell can be particularly important, since it is commonly used to detect potentially hazardous situations or as an indicator of mask cartridge breakthrough. Sensitive quantitative measures of olfactory loss would be useful in epidemiological studies and workplace surveillance. The objective of the present study was to determine the reproducibility of an olfactory perception threshold test and variations with age, gender, and smoking status. The test was a standard olfactory kit (Olfactolab No. 11), including 18 serial dilutions of PM-carbinol, with an equal number of blanks. The forced choice method was used, with both tester and subject blinded as to which bottle contained the odorant. Olfactory perception threshold was recorded when the subject identified the same dilution three times. To assess reproducibility, testing was repeated four times over a period of 4 weeks, on the same weekday and the same time of day. Subjects (n = 63) ranged in age from 20 to 60 years (mean age: 39.7 +/- 12.5 years), 47.6% were women, 29.5% currently smoked, and 27.9% were former smokers. Results showed no inter-week differences in olfactory perception threshold (Analysis of variance for repeated measures: F = 0.59; p much greater than 0.05). Inter-class correlation for assessment of agreement of continuous variables was 0.76. Inter-week concordance of hyposmia showed fair to good agreement (0.55 greater than or equal to kappa less than or equal to 0.66). Three-way analysis of variance (ANOVA) revealed significant differences with respect to age category (F = 7.36; p less than 0.001) and current smoking status (F = 4.54; p less than 0.05), but not for gender (F = 2.32; p greater than 0.05). The multiple regression model with age and smoking as independent variables was highly significant (F = 13.03; p less than 0.001), explaining 28% of the variance; olfactory threshold increased 0.47 ds/year (t = 4.01; p less than 0.001) and 0.27 ds/cigarettes/day (t = 2.46; p less than 0.05). The findings of this study indicate that this test is reproducible and sensitive to expected changes with age and smoking status. It corresponds well to criteria for testing in the field and should be considered for studies characterizing olfactory functions and sensory loss among working populations.

[Postoperative hyperuricemia of cyanotic and acyanotic congenital cardiopathies]. / Hiperuricemia en el post-quirúrgico de cardiopatías congénitas cianóticas y acianóticas.

In a prospective study, 10 children with congenital heart disease were studied before and after surgery (24-48 h). Mean age and weight, type of disease and surgery performed are described in Table 1. Six patients had acyanotic disease and 4 were cyanotic. Before surgery, the acyanotic group (AG) showed hyperuricemia compared to normal children of the same chronological age (mean +/- SE: 5.53 +/- 0.42 vs 4.27 +/- 0.22, p less than 0.02). Initial seric creatinine (sCr), increased in 3 patients of the AC and in the 4 patients of the cyanotic group (CG) compared to normal values of sCr for height (AG: 0.47 +/- 0.05 vs 0.34 +/- 0.03, p less than 0.05; CG: 0.63 +/- 0.05 vs 0.38 +/- 0.05, p less than 0.01). Post-surgery, sCr and serum uric acid (sUA) increased significantly at 24 and 48 h in both groups (Fig 1); at 24 h the increment in sUA in the AG was higher than that in the CG (p less than 0.05). There was a direct and significant correlation between the increment in sUA and sCr in the AG (Fig. 2). The urine excretion of uric acid paralleled the increment of sUA in the CG (Table 2). Fractional excretion of, sodium (FENa) was less than 1% and greater than 1% in the AG and the CG, respectively, being the basal FENa of the AG significantly lower (Table 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor I (IGF-I) and growth in children undergoing hemodialysis or after successful renal transplantation

Los niños con insuficiencia renal crónica frecuentemente presentan retardo de crecimiento. Para analizar la relación entre IGF-I y crecimiento en esta situación clínica, se estudiaron 7 pacientes hemodializados y 7 pacientes transplantados con función renal normal. El IGF-I total del suero se midió por RIA luego de cromatografía ácida en columnas de OFDS silica. Los pacientes hemdializados crecían a muy baja velocidad (X1.5c,/a), y aunque los transplantados lo hacían a una velocidad significativamente mayor (X3.3 cm/a, p<0.025), 4/7 presentaban velocidades por debajo de lo normal. Todos los pacientes de ambos grupos tuvieron niveles de IGF-I dentro de los límites normales, pero la media de los transplantados fue significativamente mayor que la de los hemodializados (39.2 ñ 13.6 nM/I vs 13.4 ñ 3.0 nM/I --predialisis-- p<0.005). La medida de la desviación logarítmica de los valores de IGF-I de los pacientes hemodializados no difirió significativamente de lo normal, mientras que la media de los transplantados fue signifdicativamente mayor que lo normal (1.027 ñ 0.500, p<0.005). Estos últimos presentaban por lo tanto, como grupo, un aumento pequeño pero real de los valores de IGF-I.. Sin embargo, no se encontró una correlación estadísticamente significativa entre IGF-I y velocidad de crecimiento. Esto, junto a la ausencia de los valores de IGF-I por debajo de lo normal, indicaría que los niveles séricos de IGF-I no desempeñan un papel importante en el retardo de crecimiento de los pacientes con insuficiencia renal crónica