Raymond Quigley

Effects of growth hormone and insulin-like growth factor I on rabbit proximal convoluted tubule transport.

This in vitro microperfusion study examined the effects of growth hormone and insulin-like growth factor I (IGF-I) on proximal convoluted tubule (PCT) transport. Tubules were perfused with an ultrafiltrate-like solution and bathed in a serum-like albumin solution. Neither a physiologic (5 x 10(-10) M), nor a pharmacologic (5 x 10(-8) M) dose of growth hormone had an effect on PCT phosphate or bicarbonate transport, or volume absorption. Addition of 5 x 10(-9) M and 5 x 10(-8) M IGF-I, but not 5 x 10(-10) M IGF-I, to the bathing solution resulted in an increase (12-15%) in phosphate transport, but no change in volume absorption or bicarbonate transport. Addition of IGF-I to the luminal perfusate also stimulated phosphate transport. The effect was noted at a concentration of 5 x 10(-11) M IGF-I (27% stimulation) and was maximal at a concentration of 5 x 10(-10) M IGF-I (46% stimulation). There was no effect of luminal IGF-I on volume absorption or bicarbonate transport. These data indicate that growth hormone has no direct effect on PCT transport. In the PCT, IGF-I stimulates phosphate transport specifically and acts via both basolateral and apical membranes. However, the magnitude of the maximal response to the luminal addition of IGF-I was threefold greater than that measured upon addition of the hormone to the bath, and the stimulation occurred at a 100-fold lower concentration. These data are consistent with IGF-I mediating the in vivo stimulation of phosphate transport by growth hormone.

Role of adenosine triphosphate (ATP) and NaK ATPase in the inhibition of proximal tubule transport with intracellular cystine loading.

Cellular cystine loading with cystine dimethyl ester inhibits volume absorption, transepithelial potential difference, glucose transport, and bicarbonate transport in proximal convoluted tubules perfused in vitro. This study examined the roles of ATP and NaK ATPase in this in vitro model of the Fanconi syndrome of cystinosis. Intracellular ATP was measured using the luciferin-luciferase assay. Intracellular ATP was reduced by 60% in proximal convoluted tubules incubated with 0.5 mM cystine dimethyl ester for 15 min at 37 degrees C (P less than 0.001). Incubation of cystine loaded tubules with 1 mM exogenous ATP increased intracellular ATP to levels not significantly different than that of controls. On the other hand, Vmax NaK ATPase activity was unchanged even though the incubation times and the concentration of cystine dimethyl ester were doubled to 30 min and 1 mM, respectively. In proximal convoluted tubules perfused in vitro, 0.5 mM cystine dimethyl ester resulted in an 89% inhibition in volume absorption (0.81 +/- 0.14 to 0.09 +/- 0.09 nl/mm.min), while there was only a 45% inhibition in volume absorption (P less than 0.01) due to cellular cystine loading in the presence of 1 mM lumen and bath ATP (0.94 +/- 0.05 to 0.52 +/- 0.11 nl/mm.min). These data demonstrate that proximal tubule cellular cystine loading decreases cellular ATP concentration, but does not directly inhibit NaK ATPase activity. The inhibition in transport and decrease in intracellular ATP due to cellular cystine loading was ameliorated by exogenous ATP. These data are consistent with cellular ATP depletion playing a major role in the inhibition of proximal tubule transport due to intracellular cystine loading.

Increased blood pressure in mice lacking cytochrome P450 2J5

The cytochrome P450 (CYP) enzymes participate in a wide range of biochemical functions, including metabolism of arachidonic acid and steroid hormones. Mouse CYP2J5 is abundant in the kidney where its products, the cis‐epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. To define the physiological role of CYP2J5 in the kidney, knockout mice were generated using a conventional gene targeting approach. Cyp2j5 (‐/‐) mice develop normally and exhibit no overt renal pathology. While renal EET biosynthesis was apparently unaffected by the absence of CYP2J5, deficiency of this CYP in female mice was associated with increased blood pressure, enhanced proximal tubular transport rates, and exaggerated afferent arteriolar responses to angiotensin II and endothelin I. Interestingly, plasma 17β‐estradiol levels were reduced in female Cyp2j5 (‐/‐) mice and estrogen replacement restored blood pressure and vascular responsiveness to normal levels. There was no evidence of enhanced estrogen metabolism, or altered expression or activities of steroidogenic enzymes in female Cyp2j5 (‐/‐) mice, but their plasma levels of luteinizing hormone and follicle stimulating hormone were inappropriately low. Together, our findings illustrate a sex‐specific role for CYP2J5 in regulation of blood pressure, proximal tubular transport, and afferent arteriolar responsiveness via an estrogen‐dependent mechanism.— Athirakul, K., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Ma, J., Zhao, Y., Couse, J. F., Quigley, R., Harder, D. R., Zhao, X., Imig, J. D., Pedersen, T. L., Newman, J. W., Hammock, B. D., Conley, A. J., Korach, K. S., Coffman, T. M., Zeldin, D. C. Increased blood pressure in mice lacking cytochrome P450 2J5. FASEB J. 22, 4096–4108 (2008)

Effect of luminal angiotensin II on rabbit proximal convoluted tubule bicarbonate absorption

The present in vitro microperfusion study examined the effect of luminal angiotensin II on proximal convoluted tubule (PCT) volume absorption and bicarbonate transport. Neither 10(-11) M, 10(-10) M, nor 2 x 10(-8) M luminal angiotensin II significantly affected PCT transport. When tubules were first perfused with enalaprilat to inhibit endogenous angiotensin II production, addition of 10(-10) M luminal angiotensin II increased volume absorption (0.72 +/- 0.08 vs. 0.86 +/- 0.07 nl x mm(-1) xmin(-1), P < 0.01) and bicarbonate transport (52.3 +/- 3.7 vs. 67.9 +/- 4.2 pmol x mm(-1) min(-1), P < 0.01). Addition of 10(-6) M losartan, an AT1 inhibitor, to the luminal perfusate inhibited volume absorption (0.95 +/- 0.14 vs. 0.72 +/- 0.11 nl x mm(-1) x min(-1), P < 0.05) and bicarbonate transport (65.0 +/- 7.3 vs. 54.7 +/- 9.2 pmol x mm(-1) x min(-1), P < 0.05). Addition of 10(-4) M luminal PD-123319, an AT2 inhibitor, was without effect. In tubules perfused with 10(-4) M luminal enalaprilat and 10(-4) M luminal PD-123319, addition of 10(-10) M luminal angiotensin II in the experimental period resulted in a stimulation in volume absorption (0.61 +/- 0.08 vs. 0.81 +/- 0.10 nl x mm(-1) x min(-1), P < 0.01) and bicarbonate transport (49.9 +/- 6.3 vs. 77.4 +/- 14.3 pmol x mm(-1) x min(-1), P < 0.01). In tubules perfused with 10(-6) M losartan and 10(-4) M enalaprilat, addition of luminal 10(-10) M angiotensin II resulted in no change in transport. These data are consistent with endogenous angiotensin II affecting PCT bicarbonate transport in vitro via luminal AT1 receptors.The present in vitro microperfusion study examined the effect of luminal angiotensin II on proximal convoluted tubule (PCT) volume absorption and bicarbonate transport. Neither 10-11 M, 10-10 M, nor 2 × 10-8 M luminal angiotensin II significantly affected PCT transport. When tubules were first perfused with enalaprilat to inhibit endogenous angiotensin II production, addition of 10-10 M luminal angiotensin II increased volume absorption (0.72 ± 0.08 vs. 0.86 ± 0.07 nl ⋅ mm-1 ⋅ min-1, P < 0.01) and bicarbonate transport (52.3 ± 3.7 vs. 67.9 ± 4.2 pmol ⋅ mm-1 ⋅ min-1, P < 0.01). Addition of 10-6 M losartan, an AT1 inhibitor, to the luminal perfusate inhibited volume absorption (0.95 ± 0.14 vs. 0.72 ± 0.11 nl ⋅ mm-1 ⋅ min-1, P < 0.05) and bicarbonate transport (65.0 ± 7.3 vs. 54.7 ± 9.2 pmol ⋅ mm-1 ⋅ min-1, P < 0.05). Addition of 10-4 M luminal PD-123319, an AT2 inhibitor, was without effect. In tubules perfused with 10-4 M luminal enalaprilat and 10-4 M luminal PD-123319, addition of 10-10M luminal angiotensin II in the experimental period resulted in a stimulation in volume absorption (0.61 ± 0.08 vs. 0.81 ± 0.10 nl ⋅ mm-1 ⋅ min-1, P < 0.01) and bicarbonate transport (49.9 ± 6.3 vs. 77.4 ± 14.3 pmol ⋅ mm-1 ⋅ min-1, P < 0.01). In tubules perfused with 10-6 M losartan and 10-4 M enalaprilat, addition of luminal 10-10 M angiotensin II resulted in no change in transport. These data are consistent with endogenous angiotensin II affecting PCT bicarbonate transport in vitro via luminal AT1receptors.

Hyperphosphatemia in tumor lysis syndrome: the role of hemodialysis and continuous veno-venous hemofiltration.

We report a 4-year-old boy who developed tumor lysis syndrome complicated by severe hyperphosphatemia and acute renal failure, following chemotherapy for T-cell acute lymphoblastic leukemia. Despite successful treatment of hyperphosphatemia with hemodialysis, there was an immediate rebound in the high serum phosphorus level. The patient underwent a second treatment with hemodialysis which was then followed by continuous veno-venous hemofiltration (CVVH). CVVH maintained his serum phosphorus at a stable level until his renal function improved. CVVH can be used in conjunction with hemodialysis to successfully treat the hyperphosphatemia associated with tumor lysis syndrome.

Extubation failure due to post-extubation stridor is better correlated with neurologic impairment than with upper airway lesions in critically ill pediatric patients

The incidence of post-extubation stridor (PES) in a pediatric intensive care unit (PICU) and the need for reintubation is not known. Predictors of success on a subsequent extubation attempt and the efficacy of dexamethasone treatment prior to a subsequent extubation attempt are not established. In a prospective randomized double blind-controlled study in two PICUs in a university childrens hospital setting, of 5,566 admissions over 35-months, we identified 32 patients who failed primary extubation and were reintubated for PES. Twenty-six patients were enrolled in the study and three subsequently excluded. Twelve were randomized to receive dexamethasone and 11 received sodium chloride placebo. Fifteen patients succeeded study extubation and eight failed. Of those receiving dexamethasone, nine patients succeeded and three failed. Of those receiving placebo, six patients succeeded and five failed. There was a poor correlation between anatomical abnormalities of the airway and failure of study extubation. Extubation failure was better correlated with neurologic impairment in the patients. We present a stridor score and demonstrate that it is an excellent predictor of success versus failure for the study extubation. Dexamethasone pre-treatment did not reduce stridor score. We are unable to conclude if dexamethasone pre-treatment reduces extubation failure. We speculate that neurologic impairment leads to extubation failure in critically ill pediatric patients.

Timing of continuous renal replacement therapy and mortality in critically ill children

Objectives:Acute kidney injury and fluid overload frequently necessitate initiation of continuous renal replacement therapy in critically ill patients admitted to the ICU. In this study, our primary objective was to determine the effect of timing of initiation of continuous renal replacement therapy on ICU mortality in children requiring renal support for management of acute kidney injury and/or fluid overload. Design:Retrospective cohort study. Setting:Tertiary level, multidisciplinary PICU. Patients:Children who received continuous renal replacement therapy for management of acute kidney injury and/or fluid overload from January 2000 through July 2009 were included in the study. Patients requiring extracorporeal life support and patients initiated on continuous renal replacement therapy for indications other than acute kidney injury and/or fluid overload were excluded. Interventions:None. Measurements and Main Results:Timing of initiation was defined chronologically as time from ICU admission to continuous renal replacement therapy initiation. Three hundred eighty treatments were performed during the study period, of which 190 were eligible and included in the study. Overall ICU mortality was 47% among the study population. Median timing of initiation was higher among nonsurvivors compared with survivors (3.4 vs 2.0 d, p = 0.001). Multivariable logistic regression analysis identified timing of initiation as an independent predictor of mortality with an adjusted odds ratio of 1.05 (95% CI, 1.01, 1.11). Fluid overload, indication for continuous renal replacement therapy initiation, severity of illness at ICU admission, and active oncologic diagnosis were the other independent predictors of mortality that were identified in the final regression model. In the survival analysis, late initiators (> 5 d) had higher mortality than early initiators (⩽ 5 d) with a hazard ratio of 1.56 (95% CI, 1.02, 2.37). Conclusions:Earlier initiation of continuous renal replacement therapy was associated with lower mortality in this cohort of critically ill children. Future studies should focus on early identification of such children who may benefit from early continuous renal replacement therapy initiation.

Effective removal of methotrexate by high-flux hemodialysis

Abstract. The purpose of the present study was to examine the clearance of methotrexate (MTX) by high-flux hemodialysis (HD) in pediatric oncology patients. We present three patients who experienced nephrotoxicity and prolonged exposure to toxic MTX concentrations following high-dose infusions during treatment for osteogenic sarcomas. Each patient was successfully treated with high-flux HD, followed by carboxypeptidase G2 (CPDG2) in two cases. Minimal systemic toxicity occurred. We review the literature and discuss guidelines for early and aggressive treatment for this complication of high-dose MTX therapy. Clinically important removal of MTX depends upon prompt initiation of HD after detection of nephrotoxicity and delayed clearance of MTX. Therapy is indicated in cases where compassionate use of CPDG2 may not be available, or while awaiting its delivery.

Glucocorticoids stimulate rabbit proximal convoluted tubule acidification.

Glucocorticoids have an important role in renal acidification; however, a direct effect of glucocorticoids on proximal convoluted tubule (PCT) acidification has not been directly demonstrated. In the present in vitro microperfusion study PCT from animals receiving dexamethasone (600 micrograms/kg twice daily for 2 d and 2 h before killing) had a significantly higher rate of bicarbonate absorption than did controls (92.0 +/- 13.3 vs 59.9 +/- 3.2 pmol/mm.min, P < 0.01). To examine if glucocorticoids had a direct epithelial action, dexamethasone was added to the bath of PCT perfused in vitro. After 3 h of incubation in paired experiments 10(-6) M and 10(-5) M dexamethasone resulted in an approximately 30% stimulation in the rate of bicarbonate absorption. 10(-7) M dexamethasone and 10(-6) M aldosterone had no effect on bicarbonate absorption. The stimulation of acidification by 10(-5) M dexamethasone was blocked by actinomycin D and cycloheximide. These data are consistent with a direct effect of glucocorticoids on PCT acidification, and this effect is dependent upon protein synthesis.

Diagnosis of urinary tract infections in children

Purpose of review Urinary tract infections remain a significant cause of serious bacterial infections in children and can result in chronic kidney disease. Thus, prompt diagnosis and initiation of treatment of urinary tract infections are paramount objectives. Recent findings A number of advances in technology have allowed expeditious examination of the urine. Recent meta-analyses evaluated the ability of these tests to determine the presence or absence of urinary tract infection in children. In addition, understanding the prevalence of urinary tract infection in various populations will help guide the clinician to the appropriate level of suspicion and the appropriate work-up for urinary tract infection. Summary Although culture of the urine remains the gold standard for diagnosing and treating urinary tract infections, technical considerations including method of collection of the urine as well as the time necessary for culture results remain problematic. More rapid techniques include dipstick analyses for the presence of leukocyte esterase or nitrites, microscopic analysis for white blood cells or bacteria, and automated urine cell analyzer to determine bacterial and white blood cell counts in the urine. Recent results indicate it is possible to limit the number of urine cultures performed by eliminating those that have a low probability of being positive. In addition, recent studies reexamining the prevalence of urinary tract infections in various populations indicate that diagnostic testing can be aimed at those patients who are in the higher-risk groups.