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Background: Sentinel lymph node (SLN) biopsy may improve nodal staging in cervical cancer. The aims of this study are to determine the rate of unusual patterns of cervical lymphatic drainage, to determine the rates of micrometastases and isolated tumor cells (ITCs) in SLNs, and to assess the clinical impact of SLN biopsy., Methods: Multicenter prospective study conducted between January 2005 and June 2007 in women undergoing laparoscopic surgery for early cervical cancer. Combined technetium/Patent Blue labeling was used. Lymphoscintigraphy was performed before surgery. SLN location was recorded, and factors associated with location were explored. SLNs underwent step sectioning ± immunohistochemistry., Results: 145 patients were enrolled and 139 included in a modified intention-to-diagnose analysis. Although 80.6 % of SLNs were in external iliac and interiliac areas, 38.2 % of patients had at least one SLN in an unexpected area and 5.1 % had SLNs only in unexpected areas. In unexpected areas, the number of SLNs per patient was not significantly different between lymphoscintigraphy and intraoperative detection (0.79 [0.62-1.02] versus 0.50 [0.37-0.68]; P = 0.096). In expected locations, there were significantly more blue and hot SLNs per patient than blue or hot SLNs (1.70 [1.45-1.99], 0.42 [0.30-0.57], 0.52 [0.39-0.69]). Of 28 metastatic SLNs, 17 contained micrometastases or ITCs. SLN involvement was found only by immunohistochemistry in 39.1 % of patients with positive nodes, and involved SLNs were located in unexpected areas in 17 % of those patients., Conclusions: Sentinel lymph node biopsy detects unusual drainage pathways and micrometastases in a substantial proportion of patients, thus improving nodal staging.

Chronic kidney disease, even at moderate stages, is characterized by a high incidence of cardiovascular events. Subclinical damage to large arteries, such as increased arterial stiffness and outward remodeling, is a classical hallmark of patients with chronic kidney disease. Whether large artery stiffness and remodeling influence the occurrence of cardiovascular events and the mortality of patients with chronic kidney disease (stages 2-5) is still debated. This prospective study included 439 patients with chronic kidney disease (mean age, 59.8 ± 14.5 years) with a mean measured glomerular filtration rate of 37 mL/min per 1.73 m(2). Baseline aortic stiffness was estimated through carotid-femoral pulse wave velocity measurements; carotid stiffness, diameter, and intima-media thickness were measured with a high-resolution echotracking system. For the overall group of patients, the 5-year estimated survival and cumulative incidence of cardiovascular events were 87% and 16%, respectively. In regression analyses adjusted on classical cardiovascular and renal risk factors, aortic stiffness remained significantly associated with all-cause mortality (for 1 SD, Cox model-derived relative risk [95% CI], 1.48 [1.09-2.02]) and with fatal and nonfatal cardiovascular events (for 1 SD, Fine and Gray competing risks model-derived relative risk [95% CI], 1.35 [1.05-1.75]). Net reclassification improvement index was significant (29.0% [2.3-42.0%]). Carotid internal diameter was also independently associated with all-cause mortality. This study shows that increased aortic stiffness and carotid internal diameter are independent predictors of mortality in patients with stages 2 to 5 chronic kidney disease and that aortic stiffness improves the prediction of the risk.

Aims: Fabry disease is a lysosomal storage disorder due to deficient alpha-galactosidase A activity, characterised by glycosphingolipids deposition in tissues. Patients have a common arterial involvement and contract progressive renal and cardiac disease. Although short-term effects of enzyme replacement therapy (ERT) on target organs have been established, no data are available on the long-term outcome., Methods and Results: We studied the effects of ERT (agalsidase beta, 1 mg/kg/14 days) on arterial and cardiac structure and function during a longitudinal study beginning in 1999, with 4.5 ± 0.4 years follow-up (four visits) in 30 patients (age: 33 ± 12 years). In addition, we studied 16 untreated Fabry patients during 2.6 ± 1.6 years (two visits). Aortic stiffness was determined by carotid-femoral pulse wave velocity, central pulse pressure by aplanation tonometry, and carotid and radial intima-media thickness and diameter by high definition echotracking device. Left ventricular mass was determined by MRI. A significant decrease in aortic stiffness (-0.56 ± 0.13 m/s/yr, p = 0.0002) was observed after ERT whereas central pulse pressure did not change. Carotid intima-media thickness (IMT) increased (+18 ± 6 µm/yr; p < 0.005) whereas radial IMT remained stable. Radial artery diameter decreased (-50 ± 20 µm/years, p < 0.05) whereas carotid diameter did not change. Carotid circumferential wall stress was reduced (-1.7 ± 0.6 kPa/yrs, p < 0.01). Left ventricular mass index significantly decreased (-7.8 ± 2.3 g/m(2)/yr, p < 0.005)., Conclusion: A sustained reduction in aortic stiffness and left ventricular hypertrophy, and a limited radial artery wall thickening were observed after long-term enzyme replacement therapy. There was no significant benefit of treatment on carotid hypertrophy.

Background and Objectives: Anemia in patients with CKD is highly related to impaired erythropoietin (EPO) response, the timing and determinants of which remain unknown., Design, Setting, Participants, & Measurements: This study measured EPO levels and studied their relation to GFR measured by 51Cr-EDTA renal clearance (mGFR) in 336 all-stage CKD patients not receiving any erythropoiesis-stimulating agent., Results: In patients with anemia defined by World Health Organization criteria (hemoglobin [Hb] <13 g/dl in men and 12 g/dl in women), EPO response to Hb level varied by mGFR level. EPO and Hb levels were negatively correlated (r=-0.22, P=0.04) when mGFR was >30 ml/min per 1.73 m(2), whereas they were not correlated when mGFR was <30 (r=0.09, P=0.3; P for interaction=0.01). In patients with anemia, the ratio of observed EPO to the level predicted by the equation for their Hb level decreased from 0.72 (interquartile range, 0.57-0.95) for mGFR ≥60 ml/min per 1.73 m(2) to 0.36 (interquartile range, 0.16-0.69) for mGFR <15. Obesity, diabetes with nephropathy other than diabetic glomerulopathy, absolute iron deficiency, and high C-reactive protein concentrations were associated with increased EPO levels, independent of Hb and mGFR., Conclusions: Anemia in CKD is marked by an early relative EPO deficiency, but several factors besides Hb may persistently stimulate EPO synthesis. Although EPO deficiency is likely the main determinant of anemia in patients with advanced CKD, the presence of anemia in those with mGFR >30 ml/min per 1.73 m(2) calls for other explanatory factors.

Chronic kidney disease definition is based on glomerular filtration rate (GFR) estimations which are derived from creatinine-based equations. The accuracy of GFR estimation is thus largely dependent of those of serum creatinine assays. International recommendations highlight the need for traceable creatinine assays. The French Society of Clinical Biochemistry conducted a study for measuring accuracy of creatinine enzymatic methods. This evaluation involved 25 clinical laboratories. Creatinine was measured in serum pools ranging from 35.9±0.9 μmol/L to 174.5±3.1 μmol/L (IDMS determination) using 12 creatinine enzymatic methods. For all creatinine values greater than 74.4±1.4 μmol/L, the bias and imprecision did not exceed 5% and 5.9%, respectively. For the lowest value (35.9±0.9 μmol/L), the bias ranged from -1.8 to 9.9% (with one exception). At this level, the imprecision ranged from 1.9 to 7.8%. The true performances of the assays (couples of bias and relative standard deviation), were evaluated using Monte-Carlo simulations. Most of the assays fall within the maximum Total Error of 12% at all concentrations. This study demonstrates substantial improvements in the calibration, traceability and precision of the enzymatic methods, reaching the NKDEP recommendations. Moreover, most of these assays allowed accurate creatinine measurements for creatinine levels lower than 40 μmol/L., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Little is known about normal kidney function level and the prognostic significance of low estimated glomerular filtration rate (eGFR) in the elderly., Methods: We determined age and sex distribution of eGFR with both the Modification of Diet in Renal Disease (MDRD) study and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations in 8705 community-dwelling elderly aged ≥ 65 years and studied its relation to 6-year mortality. In a subsample of 1298 subjects examined at 4 years, we assessed annual eGFR decline and clinically relevant markers including microalbuminuria (3-30 mg/mmol creatinine) with diabetes, proteinuria ≥ 50 mg/mmol, haemoglobin <11 g/L or resistant hypertension despite three drugs., Results: Median (interquartile range) MDRD eGFR was 78 (68-89) mL/min/1.73 m(2) in men and 74 (65-83) in women; there were 79 (68-87) and 77 (67-85) for CKD-EPI eGFR, respectively. Prevalence of MDRD eGFR <60 mL/min/1.73 m(2) was 13.7% and of CKD-EPI eGFR was 12.9%. After adjustment for several confounders, only those with an eGFR <45 mL/min/1.73 m(2) had significantly higher all-cause and cardiovascular mortality than those with an eGFR of 75-89 mL/min/1.73 m(2) whatever the equation. In the subsample men and women with an MDRD eGFR of 45-59 mL/min/1.73 m(2), 15 and 13% had at least one clinical marker and 15 and 3% had microalbuminuria without diabetes, respectively; these percentages were 41 and 21% and 23 and 10% in men and women with eGFR <45, respectively. Mean MDRD eGFR decline rate was steeper in men than in women, 1.75 versus 1.41 mL/min/1.73 m(2)/year., Conclusions: Moderately decreased eGFR is more often associated with clinical markers in men than in women. In both sexes, eGFR <45 mL/min/1.73 m(2) is related to poor outcomes. The CKD-EPI and the MDRD equations provide very similar prevalence and long-term risk estimates in this elderly population.

Background: Vitamin D (25 hydroxyvitamin D [25(OH)D]) deficiency is common in patients with chronic kidney disease (CKD). Neither the relation of this deficiency to the decrease in glomerular filtration rate (GFR) nor the effects on CKD mineral and bone disorders (MBD) are clearly established., Study Design: Cross-sectional analysis of baseline data from a prospective cohort, the NephroTest Study., Setting & Participants: 1,026 adult patients with all-stage CKD not on dialysis therapy or receiving vitamin D supplementation., Predictors: For part 1, measured GFR (mGFR) using (51)Cr-EDTA renal clearance; for part 2, 25(OH)D deficiency at <15 ng/mL., Outcomes & Measurements: For part 1, 25(OH)D deficiency and several circulating MBD markers; for part 2, circulating MBD markers., Results: For part 1, the prevalence of 25(OH)D deficiency was associated inversely with mGFR, ranging from 28%-51% for mGFR ≥60-<15 mL/min/1.73 m(2). It was higher in patients of African origin; those with obesity, diabetes, hypertension, macroalbuminuria, and hypoalbuminemia; and during winter. After adjusting for these factors, ORs for 25(OH)D deficiency increased from 1.4 (95% CI, 0.9-2.3) to 1.4 (95% CI, 0.9-2.1), 1.7 (95% CI, 1.1-2.7), and 1.9 (95% CI, 1.1-3.6) as mGFR decreased from 45-59 to 30-44, 15-29, and <15 (reference, ≥60) mL/min/1.73 m(2) (P for trend = 0.02). For part 2, 25(OH)D deficiency was associated with higher age-, sex-, and mGFR-adjusted ORs of ionized calcium level <1.10 mmol/L (2.6; 95% CI, 1.2-5.9), 1,25 dihydroxyvitamin D concentration <16.7 pg/mL (1.8; 95% CI, 1.3-2.4), hyperparathyroidism (1.8; 95% CI, 1.3-2.4), and serum C-terminal cross-linked collagen type I telopeptides concentration >1,000 pg/mL (1.6; 95% CI, 1.0-2.6). It was not associated with hyperphosphatemia (phosphate >1.38 mmol/L)., Limitations: Cross-sectional analysis of the data prevents causal inferences., Conclusions: 25(OH)D deficiency is related independently to impaired mGFR. Both mGFR decrease and 25(OH)D deficiency are associated with abnormal levels of circulating MBD biomarkers., (Copyright © 2011 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

In CKD, large arteries remodel and become increasingly stiff. The greater pulsatile pressure reaching the glomerulus as a result of increased aortic stiffness could induce renal damage, suggesting that the stiffening and remodeling of large arteries could affect the progression of CKD. We measured carotid-femoral pulse wave velocity, aortic pressure and carotid remodeling and stiffness parameters in 180 patients with CKD (mean measured GFR, 32 ml/min per 1.73 m(2)) and followed them prospectively for a mean of 3.1 years. During follow-up, carotid stiffness significantly increased (+0.28 ± 0.05 m/s; P<0.0001) but aortic stiffness did not. Carotid intima-media thickness decreased significantly during follow-up and the internal diameter of the carotid increased, producing increased circumferential wall stress (+2.08 ± 0.43 kPa/yr; P<0.0001). In a linear mixed model, circumferential wall stress significantly associated with faster GFR decline after adjustment for risk factors of cardiovascular disease and progression of CKD. In a multivariable Cox model, carotid circumferential wall stress and pulse pressure independently associated with higher risk for ESRD. None of the arterial stiffness parameters associated with progression of CKD. In conclusion, maladaptive remodeling of the carotid artery and increased pulse pressure independently associate with faster decline of renal function and progression to ESRD., (Copyright © 2011 by the American Society of Nephrology)

Background and Objectives: Creatinine excretion rate (CER) indicates timed urine collection accuracy. Although equations to estimate CER exist, their bias and precision are untested and none simultaneously include age, sex, race, and weight., Design, Setting, Participants, & Measurements: Participants (n = 2466) from three kidney disease trials were randomly allocated into equation development (2/3) and internal validation (1/3) data sets. CER served as the dependent variable in linear regression to develop new equations. Their stability was assessed within the internal validation data set. Among 987 individuals from three additional studies the equations were externally validated and compared with existing equations., Results: Mean age was 46 years, 42% were women, and 9% were black. Age, sex, race, weight, and serum phosphorus improved model fit. Two equations were developed, with or without serum phosphorus. In external validation, the new equations showed little bias (mean difference [measured - estimated CER] -0.7% [95% confidence interval -2.5% to 1.0%] and 0.3% [95% confidence interval -2.6% to 3.1%], respectively) and moderate precision (estimated CER within 30% of measured CER among 79% [76% to 81%] and 81% [77% to 85%], respectively). Corresponding numbers within 15% were 51% [48% to 54%] and 54% [50% to 59%]). Compared with existing equations, the new equations had similar accuracy but showed less bias in individuals with high measured CER., Conclusions: CER can be estimated with commonly available variables with little bias and moderate precision, which may facilitate assessment of accuracy of timed urine collections.

In 2010, a working group from the French Society of Clinical Chemistry (Société française de biologie clinique - SFBC) and the French Society of Nephrologie (SN) make a proposal to up-date the guidelines for measuring plasma creatinine.

Background: Damages to large arteries are related to bone disease in end-stage renal disease and contribute to cardiovascular mortality. An outward remodeling and stiffening of carotid artery already exist at an earlier stage of chronic kidney disease (CKD). We made the hypothesis that bone disease could be associated with the carotid outward remodeling in parallel with the decline of renal function in this population., Methods: One hundred and seven patients (60.4 +/- 14.6 years) with CKD (mean glomerular filtration rate = 34 +/- 17 mL/min/1.73 m(2)) were included in this cross-sectional study. Common carotid artery diameter, intima-media thickness and carotid stiffness were determined with an echotracking system. Bone evaluation was performed by bone densitometry and the measurement of a bone-remodeling marker, bone-specific alkaline phosphatase (BSALP)., Results: After adjustment for age, sex, mean blood pressure, carotid pulse pressure and glomerular filtration rate, bone mineral densities measured at the radius, hip and lumbar spine were significantly and negatively correlated with carotid internal diameter (P = 0.0001, P = 0.0003, P = 0.01, respectively). This association exists only in patients with glomerular filtration rate < or =38 mL/min/ 1.73 m(2). BSALP was independently and positively correlated with carotid internal diameter and explained 13% of the variance., Conclusions: Bone mineral density and serum marker of bone remodeling are independently correlated with arterial remodeling in CKD patients suggesting a crosstalk between kidney, arterial wall and bone.

Background: We investigated whether a novel, synthetic, peptide-based erythropoietin-receptor agonist (Hematide, Affymax) can stimulate erythropoiesis in patients with anemia that is caused by antierythropoietin antibodies., Methods: In this open-label, single-group trial, we enrolled patients with chronic kidney disease who had pure red-cell aplasia or hypoplasia due to antierythropoietin antibodies and treated them with a synthetic peptide-based erythropoietin-receptor agonist. The agonist was administered by subcutaneous injection at an initial dose of 0.05 mg per kilogram of body weight every 4 weeks. The primary end point was a hemoglobin concentration above 11 g per deciliter without the need for transfusions., Results: We treated 14 patients with the peptide agonist for a median of 28 months. The median hemoglobin concentration increased from 9.0 g per deciliter (with transfusion support in the case of 12 patients) before treatment to 11.4 g per deciliter at the time of the last administration of the agonist; transfusion requirements diminished within 12 weeks after the first dose, after which 13 of the 14 patients no longer required regular transfusions. Peak reticulocyte counts increased from a median of 10x10(9) per liter before treatment to peak counts of greater than 100x10(9) per liter. The level of antierythropoietin antibodies declined over the course of the study and became undetectable in six patients. One patient who initially responded to treatment had a diminished hematologic response a few months later despite increased doses of the agonist and required transfusions again; this patient was found to have antibodies against the agonist. One patient died 4 months after the last dose of the agonist, and a grade 3 or 4 adverse event occurred in seven other patients during the study period., Conclusions: This novel agonist of the erythropoietin receptor can correct anemia in patients with pure red-cell aplasia caused by antierythropoietin antibodies. (ClinicalTrials.gov number, NCT00314795.)., (Copyright 2009 Massachusetts Medical Society.)

Cystatin C is an endogenous glomerular filtration marker hence its serum level is affected by the glomerular filtration rate (GFR). To study what other factors might affect it blood level we performed a cross-sectional analysis of 3418 patients which included a pooled dataset of clinical trial participants and a clinical population with chronic kidney disease. The serum cystatin C and creatinine levels were related to clinical and biochemical parameters and errors-in-variables models were used to account for errors in GFR measurements. The GFR was measured as the urinary clearance of 125I-iothalamate and 51Cr-EDTA. Cystatin C was determined at a single laboratory while creatinine was standardized to reference methods and these were 2.1+/-1.1 mg/dL and 1.8+/-0.8 mg/L, respectively. After adjustment for GFR, cystatin C was 4.3% lower for every 20 years of age, 9.2% lower for female gender but only 1.9% lower in blacks. Diabetes was associated with 8.5% higher levels of cystatin C and 3.9% lower levels of creatinine. Higher C-reactive protein and white blood cell count and lower serum albumin were associated with higher levels of cystatin C and lower levels of creatinine. Adjustment for age, gender and race had a greater effect on the association of factors with creatinine than cystatin C. Hence, we found that cystatin C is affected by factors other than GFR which should be considered when the GFR is estimated using serum levels of cystatin C.

Chronic kidney disease (CKD) guidelines recommend evaluating patients with GFR <60 ml/min per 1.73 m(2) for complications, but little evidence supports the use of a single GFR threshold for all metabolic disorders. We used data from the NephroTest cohort, including 1038 adult patients who had stages 2 through 5 CKD and were not on dialysis, to study the occurrence of metabolic complications. GFR was measured using renal clearance of (51)Cr-EDTA (mGFR) and estimated using two equations derived from the Modification of Diet in Renal Disease study. As mGFR decreased from 60 to 90 to <20 ml/min per 1.73 m(2), the prevalence of hyperparathyroidism increased from 17 to 85%, anemia from 8 to 41%, hyperphosphatemia from 1 to 30%, metabolic acidosis from 2 to 39%, and hyperkalemia from 2 to 42%. Factors most strongly associated with metabolic complications, independent of mGFR, were younger age for acidosis and hyperphosphatemia, presence of diabetes for acidosis, diabetic kidney disease for anemia, and both male gender and the use of inhibitors of the renin-angiotensin system for hyperkalemia. mGFR thresholds for detecting complications with 90% sensitivity were 50, 44, 40, 39, and 37 ml/min per 1.73 m(2) for hyperparathyroidism, anemia, acidosis, hyperkalemia, and hyperphosphatemia, respectively. Analysis using estimated GFR produced similar results. In summary, this study describes the onset of CKD-related complications at different levels of GFR; anemia and hyperparathyroidism occur earlier than acidosis, hyperkalemia, and hyperphosphatemia.

Background: The objective of this study was to use information from a donor to establish the lowest possible serum creatinine (SCr) of the recipient as a means of identifying early graft dysfunction., Methods: We analyzed retrospectively 58 pairs of living donors and recipients. The lowest possible SCr was calculated from four different formulae derived from Cockcroft-Gault formula: Ax(140-recipient age)xrecipient weight/donor GFR (A, women: 1.04, men: 1.23). Donor GFR was represented by the following values (a) mSCrD0: measured pretransplant GFR for both kidneys, (b) mSCrSKD0: single transplanted kidney GFR on day 0, (c) eSCrD0: estimated GFR based on SCr at day 0, or (d) eSCrD30: on day 30. These resulting estimated SCr were tested for correlation coefficient, bias, precision, and accuracy in predicting the lowest observed recipient SCr during the first year posttransplant., Results: The lowest possible SCr was 80+/-22 micromol/L for mSCrD0, 79.1+/-23 micromol/L for mSCrSKD0, 83+/-27 micromol/L for eSCrD0, and 115+/-22 micromol/L for eSCrD30. Mean values for lowest possible SCr correlated significantly with the lowest observed SCr for all four formulae. The eSCrD0 formula showed the best correlation (r=0.47), the smallest positive bias (20.5 micromol/L), the highest precision (21.9 micromol/L), and the second highest percentage of predicted values that fell within 30% of the observed SCr (69%)., Conclusion: The use of the Cockcroft-Gault derived formula with eSCrD0 may be a useful tool to detect early discrepancy between observed and lowest possible SCr value. This could help to identify patients who may require invasive investigations.

Disorders of mineral and bone metabolism are prevalent in patients with chronic kidney disease (CKD). The recent National Kidney Foundation Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines recommend that blood calcium (Ca) be regularly measured in patients with stages 3 to 5 CKD. The Kidney Disease: Improving Global Outcomes (KDIGO) position states that the measurement of ionized Ca (iCa) is preferred and that if total Ca (tCa) concentration is used instead, then it should be adjusted in the setting of hypoalbuminemia. In 691 consecutive patients with stages 3 to 5 CKD, we compared the ability of noncorrected and albumin-corrected tCa concentration to identify low, normal, or high iCa concentration. The agreement between noncorrected or albumin-corrected tCa and iCa was only fair. The risk for underestimating ionized calcium was independently increased by a low total CO(2) concentration when either noncorrected or albumin-corrected Ca was used and by a low albumin concentration only when noncorrected tCa was used. The risk for overestimating iCa was increased by a low albumin concentration only when albumin-corrected Ca was used. In conclusion, albumin-corrected tCa does not predict iCa better than noncorrected tCa. Moreover, both noncorrected and albumin-corrected tCa concentrations poorly predict hypo- or hypercalcemia in patients with CKD.

During the last years, GFR estimation has received substantial attention with a focus on comparing results of new formulas with GFR measurements, and standardization of creatinine assays. Calibration of creatinine should improve performances. However, frequently used equations have lower precision in high GFR populations. This is the reason why a continuous effort in improving predicting equations is still needed. The use of calibrated creatinine, the onset of new GFR markers such as cystatin C, and pooling data across many study populations are underway to develop better prediction.

Cystatin C is a low molecular weight-protein, which may replace creatinine for the evaluation of renal function, particularly in the clinical settings where the relationship between creatinine production and muscular mass impairs the clinical performance of creatinine. This paper intends to summarize the current knowledge about the physiology of cystatin C and about its use as a renal marker, alone or within formulas developed to estimate the glomerular filtration rate. Moreover, this paper reviews the recent data about potential other applications of cystatin C, especially in cardiology, in oncology and in clinical pharmacology.

Background: The long-term risk of chronic kidney disease (CKD) in lithium (Li)-treated patients has been well established in the recent years., Methods: We have evaluated GFR and serum calcium monitoring in 1179 Li-treated outpatients from an ambulatory laboratory database study. This has been performed in a single private laboratory in Paris from February 1997 to December 2004. Estimated GFR (eGFR) has been calculated using the abbreviated MDRD equation., Results: During an 8-year period, 695 patients (59%) had at least one serum creatinine measurement, whereas 484 had no creatinine measurement. The former group had also more frequent serum Li measurements. Mean serum lithium levels, were similar in both groups, 0.65 mmol/l vs 0.62 mmol/l. The percentage of patients with CKD stage 3 (eGFR 30-59 ml/min/1.73 m(2)) were 36%, 53%, 73% and 77%, and with CKD stage 4, 3%, 5%, 5%, 8% in patients aged 20-39, 40-59, 60-69, and > or = 70 years respectively. There was no significant rise in creatinine measurements (from 35% of the patients with at least one serum creatinine in 2003 to 39% in 2004; P = 0.66) despite intervention to intensify GFR monitoring by physicians. Serum calcium was tested at least once in 212 patients (18%) of whom 15 (7%) were found with hypercalcaemia., Conclusion: A very high percentage of Li-treated outpatients have low eGFR. GFR monitoring is neglected in these patients, the majority of whom are no longer attending specialized clinics. Hypercalcaemia is less common but serum calcium monitoring is even more neglected. Ambulatory laboratory database surveillance provides a powerful means to contribute to CKD screening and monitoring.

Cystatin C is a low-molecular-weight protein which has been proposed as a marker of renal function that could replace creatinine. Indeed, the concentration of cystatin C is mainly determined by glomerular filtration and is particularly of interest in clinical settings where the relationship between creatinine production and muscle mass impairs the clinical performance of creatinine. Since the last decade, numerous studies have evaluated its potential use in measuring renal function in various populations. More recently, other potential developments for its clinical use have emerged. This review summarises current knowledge about the physiology of cystatin C and about its use as a renal marker, either alone or in equations developed to estimate the glomerular filtration rate. This paper also reviews recent data about the other applications of cystatin C, particularly in cardiology, oncology and clinical pharmacology.

Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at under-standing the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

Progressive decline in kidney function is a cardinal manifestation of Fbry disease. Proteinuria is usually the earliest biological marker of renal involvement. Renoprotection should associate both effective Enzyme Replocement Therapy (ERT) and non-specific anti-proteinuric therapy as recommended for all chronic kidney diseases. ERT has demonstrated high efficiency in the early clearing of globotriaosylceromide deposits, however the functional protecting effect on GFR decrease remains to be validated, especially in patients with CKD stage 3 or higher Estimation of GFR should be very cautious using creatinine based formulas, leading to measure GFR whenever possible in order to characterize the progression of CKD in Fabry patients.

Anemia is a well-known consequence of chronic kidney disease (CKD), and its prevalence progressively increases when the estimated glomerular filtration rate decreases to less than 60 mL/min/1.73 m2. However, analyses of the consequences of anemia and of the mechanisms of progression of CKD suggest that anemia also could contribute to the deterioration of kidney function. This hypothesis is based mostly on experimental data that imply that hypoxia of tubular cells plays an important role in tubulointerstitial damage associated with CKD and, thus, in the progression of renal failure. It also is supported by the fact that red blood cells represent a major antioxidant component of blood and that oxidative stress appears to contribute to glomerulosclerosis and tubulointerstitial damage. In humans, post hoc analysis of the Reduction of End points in non insulin-dependent diabetes mellitus (NIDDM) with the Angiotensin II Antagonist Losartan study and analyses of smaller prospective cohorts of CKD patients have shown that anemia is an independent risk factor for progression of CKD. In addition, 3 small randomized studies have suggested that anemia correction could slow the progression of CKD. Thus, the existence of a relationship between anemia and progression of CKD is not only plausible biologically, but also is supported by observational studies and by small intervention studies. However, only a large, randomized, prospective trial will be able to establish if anemia correction can slow the progression of CKD effectively.

Kidney function is defined by the glomerular filtration rate (GFR). It has been clearly established that serum creatinine should not be used alone to estimate GFR, and that estimated GFR should be calculated using formulas that take into account various parameters including serum creatinine. In adults, two formulas are mostly used to estimate GFR: the Cockcroft-Gault formula and the MDRD formula. In elderly, GFR should only be estimated using the MDRD formula, since the Cockcroft-Gault formula systematically underestimates GFR. GFR tends to decrease with age, but it should be 60 mL/min/1,73 m2 or higher in all subjects who do not suffer from kidney disease, regardless of age.

Objectives: To evaluate detection rate, topography and false negatives of sentinel lymph node in endometrial cancer., Material and Methods: Twenty-six patients were included. Lymphoscintigraphy was performed the day before surgery. Preoperative detection of the sentinel lymph node was performed with cervical blue dye injection and a gamma probe. Separate pathology examinations were performed for sentinel and non-sentinel lymph nodes. Sentinel lymph nodes were examined with hematoxylin-eosin-safran stain, and immunohistochemistry if negative., Results: Twenty-six patients had a positive lymphoscintigraphy. Preoperative detection was successful in 21 patients (80.8%): the detection rate with isotopic method, 19 cases (73.1%), was superior to the dye detection, 15 cases (57.7%). No isolated lombo-aortic sentinel lymph nodes were observed, and all sentinel lymph nodes were in the ilio-obturator region. Seven patients presented lymphatic spread, and 4 of them had at least one sentinel node. There was one micrometastasis in sentinel node, associated with isolated tumoral cells in pelvic lymphadenectomy. There was no false negative of sentinel node., Conclusion: The biopsy of sentinel lymph node is a feasible procedure in endometrial cancer. There was one micrometastatic sentinel node. However there was no isolated lomboaortic sentinel lymph node in this study.

Analysis of the biologic effects of erythropoietin and pathophysiology of chronic kidney diseases (CKD) suggests that treatment with erythropoiesis-stimulating agents (ESA) could slow the progression of CKD. By decreasing hypoxia and oxidative stress, it could prevent the development of interstitial fibrosis and the destruction of tubular cells. It could have direct protective effects on tubular cells through its antiapoptotic properties. It could help maintain the integrity of the interstitial capillary network through its effects on endothelial cells. Thus, suggesting that correcting anemia with ESA could slow the progression of CKD is biologically plausible. In patients with CKD, three small prospective studies and a retrospective study have suggested that treatment with ESA may have protective effects. Post-hoc analysis of the Reduction in Endpoints in Noninsulin-dependent Diabetes Mellitus with the Angiotensin II Antagonist Losartan study has also shown that anemia was an independent risk factor for progression of nephropathy in patients with type 2 diabetes. In addition, a large clinical trial, which had to be stopped prematurely because of labeling change for subcutaneous administration of epoetin alfa, suggests that complete normalization of hemoglobin levels is safe in CKD patients not on dialysis and without severe cardiovascular disease. Thus, it seems reasonable to advocate starting a large randomized, prospective study to determine if normalization of hemoglobin concentration can effectively slow the progression of CKD.

Recent recommendations emphasize the need to assess kidney function using creatinine-based predictive equations to optimize the care of patients with chronic kidney disease. The most widely used equations are the Cockcroft-Gault (CG) and the simplified Modification of Diet in Renal Disease (MDRD) formulas. However, they still need to be validated in large samples of subjects, including large non-U.S. cohorts. Renal clearance of (51)Cr-EDTA was compared with GFR estimated using either the CG equation or the MDRD formula in a cohort of 2095 adult Europeans (863 female and 1232 male; median age, 53.2 yr; median measured GFR, 59.8 ml/min per 1.73 m(2)). When the entire study population was considered, the CG and MDRD equations showed very limited bias. They overestimated measured GFR by 1.94 ml/min per 1.73 m(2) and underestimated it by 0.99 ml/min per 1.73 m(2), respectively. However, analysis of subgroups defined by age, gender, body mass index, and GFR level showed that the biases of the two formulas could be much larger in selected populations. Furthermore, analysis of the SD of the mean difference between estimated and measured GFR showed that both formulas lacked precision; the CG formula was less precise than the MDRD one in most cases. In the whole study population, the SD was 15.1 and 13.5 ml/min per 1.73 m(2) for the CG and MDRD formulas, respectively. Finally, 29.2 and 32.4% of subjects were misclassified when the CG and MDRD formulas were used to categorize subjects according to the Kidney Disease Outcomes Quality Initiative chronic kidney disease classification, respectively.

The sentinel lymph node (SLN) biopsy has been proposed for the cancers of the uterus in order to optimize the diagnosis of lymphatic metastases and micrometastases in early stage tumors. Patients with early invasive cervical (n = 8) or endometrial (n = 15) cancers were enrolled. A lymphoscintigraphy was carried out before the intervention. Intraoperative SLN identification was performed with blue dye combined to a handheld gamma probe detection. Non-sentinel pelvic nodes were separately cleared out. SLNs were examined with frozen sections, permanent sections with hematoxylin-eosin staining and further serial sections with immunohistochemistry if negative. Six cervical cancer patients and 13 endometrial cancer patients had a positive lymphoscintigraphy, showing in 5 patients extra-iliac SLN(s). The intraoperative detection was successful in 6 cervical cancer patients and 14 endometrial cancer patients. The higher detection rate was obtained with the isotopic method. Most of the SLNs were ilio-obturator. Four endometrial cancer patients had a lymphatic spread, only involving the SLN in each case. No false negative SLN has been noted. SLN biopsy appears feasible in cervical and endometrial cancers. This procedure could improve the lymphatic evaluation of these cancers.

Studying the apical Na/H exchanger NHE2 is difficult in the intact thick ascending limb (TAL) because of its weak expression and transport activity compared with the co-expressed NHE3. From a mouse transgenic for a recombinant plasmid adeno-SV(40) (PK4), we developed an immortalized TAL cell line, referred to as MKTAL, which selectively expresses NHE2 protein and activity. The immortalized cells retain the main properties of TAL cells. They have a stable homogeneous epithelial-like phenotype, express SV(40) T antigen and exhibit polarity with an apical domain bearing few microvilli and separated from lateral domains by typical epithelial-type junctional complexes expressing ZO1 protein. Tamm-Horsfall protein is present on the apical membrane. MKTAL cells express NHE2 and NHE1 proteins but not NHE3 and NHE4, whereby NHE2 protein is expressed selectively in the apical domain of the plasma membrane. NHE2 contributed about half of the total Na/H exchange activity. mRNAs for the Na-K-2Cl cotransporter-2 (NKCC2) and the anion exchangers AE2 and AE3 were also present. While acute exposure to NO donors did not alter NHE2 activity, chronic exposure inhibited NHE2 activity selectively and down-regulated NHE2 mRNA abundance. In conclusion, MKTAL cells retain structural and functional properties of their in vivo TAL counterparts and express functional NHE2 protein in the apical membrane, which may be inhibited by NO. Thus, MKTAL cells may be an appropriate model for studying the cellular mechanisms of NHE2 regulation.

Biventricular pacing (BiV) is emerging for patients with dilated cardiomyopathy (DCM) and asynchrony. We measured basal asynchrony and early resynchronization by radionuclide angioscintigraphy (RNA) in order to predict long-term evolution of ventricular function after BiV. Thirty-four patients (NYHA Class III-IV,65.4 +/- 11 years) with large QRS(179 +/- 18 ms)were implanted with BiV and studied by RNA before (D0), at day 8 (D8), and during follow-up(20 +/- 7 months). We calculated left and right ejection fractions, the interventricular dyssynchrony (TRVLV), and the apicobasal dyssynchrony (Tab). LVEF improved from 20.2 +/- 8.1%(D0) to27.1%+/- 12.6%(follow-up,P < 0.003 vs D0) and RVEF from 28.6%+/- 13%(D0) to 34.3 +/- 11.5%(follow-up,P < 0.03 vs D0). Inter- (DeltaTRVLV) and intraventricular resynchronization was immediate and remained stable: TRVLV decreased from 68.3 +/- 38 ms(D0) to 13.4 +/- 48.5 ms(D8) and1.8 +/- 39.2 ms(follow-up,P < 0.0001 vs D0); and Tab from 45.8 +/- 64.1 msto-18 +/- 68(D8) and-28.3 +/- 53.6 ms(follow-up,P < 0.0001 vs D0). Early inter- and intraventricular resynchronization (DeltaTab) at D8 were related to late LVEF and RVEF improvement. Together, an LVEF > 15% and a significant interventricular dyssynchrony (TRVLV > 60 ms) at D0 have a sensitivity of 79% and a positive predictive value of 83% to predict an improvement of LVEF superior to 5% at follow-up. In DCM patients, BiV resynchronizes ventricles early and in the long-term, while RVEF and LVEF improve progressively. Patients with large electromechanical dyssynchrony benefit most from BiV.

We studied the relationships of electrical and mechanical synchronization in patients with heart failure (CHF) and various degree of ventricular conduction delays. Ninety-two CHF patients (60 +/- 13 years old, LVEF < 45%), NYHA II-III-IV, and 35 age-matched control subjects were studied with angioscintigraphic phase analysis. We measured ejection fractions (LVEF, RVEF) and calculated the total activation time for the left (TtLV) and right ventricle (TtRV), and the synchronization time between right and left ventricle (TRVLV), and between LV apex and base (Tab). Patients were divided into three groups according to QRS duration: group 1 < 120 ms (n = 28), group 2 < 150 ms (n = 23), group 3 > or = 150 ms (n = 41). In group 1: LVEF = 31.1 +/- 10.9%, RVEF = 30.1 +/- 12.6%, TtLV = 204 +/- 70 ms, TtRV = 183 +/- 61 ms, TRVLV = 7 +/- 33 ms, Tab = 29 +/- 23 ms. In group 2, these were: 27.8 +/- 9.1%, 27.8 +/- 8.8%, 227 +/- 95 ms, 248 +/- 137 ms, 35 +/- 42 ms*, and 39 +/- 53 ms respectively. In group 3: LVEF = 20.5 +/- 9.5%t, RVEF = 28.4 +/- 16.1%, TtLV = 304 +/- 155 mst, TtRV = 234 +/- 106 mst, TRVLV = 64 +/- 42 mst, and Tab = 67 +/- 48 ms*, all P < 0.001 versus controls *P < 0.05 versus G1, tP < or = 0.01 versus G1. A significant relation links QRS to both inter- and intraventricular asynchrony (TRVLV: r = 0.65; TtLL: r = 0.70, Tab: r = 0.60), and to LV function (r = 0.72); while LVEF relates more closely to intraventricular asynchrony: TtLV (r = 0.52), TtLL (r = 0.67), than to interventricular asynchrony: TRVLV (r = 0.48); P < 0.01, P < or = 0.001. In CHF patients, electromechanical and contractile alterations are coupled; regional activation may be an early parameter allowing the detection of ventricular dyssynchronization.

Background: Regulation of renal transporter expression has been shown to support adaptation of transporter activities in several chronic situations. Basolateral and apical Na/H exchangers (NHE) in medullary thick ascending limb (MTAL) are involved in NH4+ and HCO3+ absorption, respectively. The NH4+ absorption rate in Henle's loop is increased in chronic metabolic acidosis (CMA) and potassium depletion (KD), which may be secondary to the increased NH4+ concentration in luminal fluid and/or to an increased NH4+ absorptive capacity of MTAL. HCO3- absorptive capacity in Henle's loop is increased in CMA and decreased in metabolic alkalosis, but is unchanged in KD despite the presence of metabolic alkalosis. The present study compared the effects of NH4Cl-induced CMA and KD on the expression of basolateral NHE-1 and the effect of KD on the expression of apical NHE-3 in MTAL., Methods: NHE-1 and NHE-3 mRNAs and proteins were assessed by a competitive reverse transcription-polymerase chain reaction (RT-PCR) method and semiquantitative immunoblots, respectively, in MTAL-purified suspensions from rats with CMA and KD., Results: NHE-1 protein abundance was similarly increased (approximately 90%) at two and five weeks of KD, while NHE-1 mRNA amount in MTAL cells was increased at two weeks of KD and returned to normal values by five weeks of KD. In contrast, NHE-1 mRNA and protein abundance did not change in CMA. NHE-3 protein abundance remained unchanged in both two and five weeks of KD, while NHE-3 mRNA was unchanged by two weeks of KD and reduced by approximately 50% at five weeks of KD., Conclusions: The results suggest the following: (1) in KD, where the increased NH4+ concentration of luminal fluid that favors NH4+ absorption is counterbalanced by a decrease in BSC1 expression and activity, the increased NHE-1 expression may support an increased MTAL NH4+ absorptive capacity in CMA, NHE-1 expression is not specifically regulated and remains unchanged, suggesting that the increase in NH4+ concentration in luminal fluid is the main determinant of increased NH4+ absorption in MTAL. (2) In KD, NHE-3 expression did not decrease despite the presence of metabolic alkalosis, in agreement with the unchanged HCO3- absorptive capacity of Henle's loop.

The general use of bilateral rather than separate renal function evaluation has led to the publication of conflicting results concerning the effect of percutaneous transluminal renal angioplasty (PTRA) on renal function, especially in patients with atherosclerotic renal artery stenosis. The aim of this study was to evaluate prospectively, in standardized conditions, split renal function (SRF) and GFR outcome after successful PTRA, by measuring single kidney GFR with synchronous inulin or (51)Cr-ethylenediaminetetraacetic acid clearance and (99m)Tc-diethylenetriamine pentaacetic acid scintigraphy, in a well-defined population of patients with unilateral renal artery stenosis. Thirty-two consecutive hypertensive patients (18 with atherosclerotic and 14 with dysplastic disease) with significant unilateral stenosis of the main native renal artery (> or = 60%) and normal renal function were included in the study. Renal and angiographic follow-up evaluations were performed 6 mo after PTRA. PTRA alone or combined with stenting (n = 2) was technically successful in all patients. Repeat PTRA was necessary in two patients, evaluated 6 mo after the second PTRA. Six mo after PTRA, total GFR had increased slightly but significantly in the 29 patients with positive lateralization indices. SRF and single-kidney GFR of the stenotic kidney increased significantly, whereas concurrently the GFR and SRF of the nonstenotic kidney decreased significantly. Six mo after successful PTRA reducing renal ischemia, a reversal of both the hypoperfusion of the stenotic side and the hyperperfusion of the nonstenotic side was observed, which was accompanied by a slight increase in total GFR.

Background: A new protein, named paracellin 1 (PCLN-1), expressed in human thick ascending limb (TAL) tight junctions, possibly plays a critical role in the control of magnesium and calcium reabsorption, since mutations of PCLN-1 are present in the hypomagnesemia hypercalciuria syndrome (HHS). However, no functional experiments have demonstrated that TAL magnesium and calcium reabsorption were actually impaired in patients with HHS., Methods: Genetic studies were performed in the kindred of two unrelated patients with HHS. Renal magnesium and calcium reabsorption in TAL were analyzed in one homozygous affected patient of each family, one patient with extrarenal hypomagnesemia (ERH), and two control subjects (CSs)., Results: We found two yet undescribed mutations of PCLN-1 (Gly 162 Val, Ala 139 Val). In patients with HHS, renal magnesium and calcium reabsorptions were impaired as expected; NaCl renal conservation during NaCl deprivation and NaCl tubular reabsorption in diluting segment were intact. Furosemide infusion in CS markedly increased NaCl, Mg, and Ca urinary excretion rates. In HHS patients, furosemide similarly increased NaCl excretion, but failed to increase Mg and Ca excretion. Acute MgCl(2) infusion in CS and ERH patient provoked a dramatic increase in urinary calcium excretion without change in NaCl excretion. When combined with MgCl(2) infusion, furosemide infusion remained able to induce normal natriuretic response, but was unable to increase urinary magnesium and calcium excretion further. In HHS patients, calciuric response to MgCl(2) infusion was blunted., Conclusion: This study is the first to our knowledge to demonstrate that homozygous mutations of PCLN-1 result in a selective defect in paracellular Mg and Ca reabsorption in the TAL, with intact NaCl reabsorption ability at this site. In addition, the study supports a selective physiological effect of basolateral Mg(2+) and Ca(2+) concentration on TAL divalent cation paracellular permeability, that is, PCLN-1 activity.

Biventricular (BV) pacing is an emerging treatment for patients with severe dilated cardiomyopathy and ventricular asynchrony. Radionuclide angioscintigraphy has shown that BV can reduce activation delays between left (LV) and right ventricles (RV), but alterations of electromechanical asynchrony between the LV base and apex have not been previously described. Radionuclide angioscintigraphy with Tc99m red cell labeling was performed in 21 patients, 64 +/- 17 years of age, in NYHA functional Class III or IV, and with a mean QRS duration of 180 +/- 15 ms. Right (RVEF) and LV ejection fraction (LVEF), and the synchronization between LV apex and base (Tab) in the left lateral view, were measured by a phase analysis program (1) at baseline, (2) on day 8 after BV pacemaker implantation (D8), and (3) at 12-month follow-up in BV (M12). BV pacing reversed Tab from 42 +/- 47 ms at baseline to -57 +/- 75** on D8, and -37 +/- 76** on M12. LVEF increased from 17.8 +/- 6.3% to 19.9 +/- 8.3 on D8, and 24.2 +/- 10.8* on M12, and RVEF increased from 27.6 +/- 16% to 29.9 +/- 16 on D8 and 34.1 +/- 12.1* on M12 (*P < 0.05, **P < 0.001). A close correlation was found between early LV apex-to-base resynchronization induced by BV and late increase in LVEF (r = 0.59**). In parallel with its known interventricular resynchronization effect, BV pacing reverses the apex-to-base ventricular activation sequence, causing early contraction of the LV base followed by the apex. These changes persisted up to 12 months along with an improvement in LV systolic function.

Background: We have developed a nontransformed immortalized mice kidney cortex epithelial cell (MKCC) culture from a mouse transgenic for a recombinant plasmid adeno-SV40 (PK4). Methods and Results. After 12 months in culture, the immortalized cells had a stable homogeneous epithelial-like phenotype, expressed simian virus 40 (SV40) T-antigen, but failed to induce tumors after injection in nude mice. Epithelium exhibited polarity with an apical domain bearing many microvilli separated from lateral domains by junctional complexes with ZO1 protein. The transepithelial resistance was low. A Na-dependent glucose uptake sensitive to phlorizin and a Na-dependent phosphate uptake sensitive to arsenate were present. Western blot analysis of membrane fractions showed that anti-Na-Pi antiserum reacted with a 87 kD protein. The Na/H antiporters NHE-1, NHE-2, and NHE-3 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). The corresponding proteins with molecular weights of 111, 81, and 75 kD, respectively, could be detected by Western blot and were shown to be functional. Parathyroid hormone (PTH) induced a tenfold increase in cAMP and reduced the Na-dependent phosphate uptake and NHE-3 activity, as observed in proximal tubule cells. Isoforms alpha, delta, epsilon, and zeta of protein kinase C (PKC) were present in the cells. Angiotensin II (Ang II) elicited a translocation of the PKC-alpha toward the basolateral and apical domains., Conclusion: Thus, the MKCC culture retains the structural and functional properties of proximal tubular cells. To our knowledge, it is the first cell culture obtained from transgenic mice that exhibits the NHE-3 antiporter and type II Na-Pi cotransporter. MKCCs also display functional receptors for PTH and Ang II. Thus, MKCCs offer a powerful in vitro system to study the cellular mechanisms of ion transport regulation in proximal epithelium.

The present studies examined the effects of chronic NaCl administration and metabolic alkalosis on NHE-3, an apical Na+/H+ exchanger of the rat medullary thick ascending limb of Henle (MTAL). NaCl administration had no effect on NHE-3 mRNA abundance as assessed by competitive RT-PCR, as well as on NHE-3 transport activity estimated from the Na+-dependent cell pH recovery of Na+-depleted acidified MTAL cells, in the presence of 50 microM Hoe-694, which specifically blocks NHE-1 and NHE-2. Two models of metabolic alkalosis were studied, one associated with high sodium intake, i.e., NaHCO3 administration, and one not associated with high sodium intake, i.e., chloride depletion alkalosis (CDA). In both cases, the treatment induced a significant metabolic alkalosis that was associated with a decrease in NHE-3 transport activity (-27% and -25%, respectively). Negative linear relationships were observed between NHE-3 activity and plasma pH or bicarbonate concentration. NHE-3 mRNA abundance and NHE-3 protein abundance, assessed by Western blot analysis, also decreased by 35 and 25%, respectively, during NaHCO3-induced alkalosis, and by 47 and 33%, respectively, during CDA. These studies demonstrate that high sodium intake has per se no effect on MTAL NHE-3. In contrast, chronic metabolic alkalosis, regardless of whether it is associated with high sodium intake or not, leads to an appropriate adaptation of NHE-3 activity, which involves a decrease in NHE-3 protein and mRNA abundance.

Chronic metabolic acidosis (CMA) is associated with an adaptive increase in the bicarbonate absorptive capacity of the rat medullary thick ascending limb (MTAL). To specify whether NHE-3, the apical MTAL Na/H exchanger, is involved in this adaptation, NHE-3 mRNA was quantified by a competitive RT-PCR using an internal standard which differed from the wild-type NHE-3 mRNA by an 80-bp deletion. CMA increased NHE-3 mRNA from 0.025+/-0.003 to 0.042+/-0.009 amol/ng total RNA (P < 0.005). NHE-3 transport activity was measured as the initial proton flux rate calculated from the Na-dependent cell pH recovery of Na-depleted acidified MTAL cells in the presence of 50 microM HOE694 which specifically blocks NHE-1, the basolateral MTAL NHE isoform. CMA caused a 68% increase in NHE-3 transport activity (P < 0.001). In addition, CMA was associated with a 71% increase in NHE-3 protein abundance (P < 0.05) as determined by Western blot analysis on MTAL membranes using a polyclonal antiserum directed against a cytoplasmic epitope of rat NHE-3. Thus, NHE-3 adapts to CMA in the rat MTAL via an increase in the mRNA transcript that enhances NHE-3 protein abundance and transport activity.

Low concentrations of angiotensin II (Ang II) increase, whereas high concentrations inhibit the apical Na/H antiporter activity in the proximal tubule, but the respective roles of the different signaling pathways in mediating these effects remains unsettled. We studied the effects of both low and high doses of Ang II in the presence of selective signaling pathway inhibitors, on the apical Na/H antiport activity of rat proximal tubule. Experiments were carried out in intact cells of freshly prepared tubule fragments obtained from the outer third of cortex, that is, devoid of basolateral Na/H antiport activity in the absence of bicarbonate transport and H(+)-ATPase activity. In tubules acid-loaded by an NH4Cl prepulse, Na/H antiport activity was assessed by the initial rate of intracellular pH recovery (dpHi/dt), measured with BCECF. When tubules were preincubated with low dose Ang II (10(-11) M for 3 min), dpHi/dt increased by 25 +/- 8%, whereas incubation with high dose Ang II (10(-7) M for 3 min) decreased dpHi/dt by 30 +/- 4%, compared to control (P < 0.01 in both cases). Both effects were abolished in the presence of 2.10(-3) M amiloride. Low dose Ang II-induced increase in dpHi/dt was not affected by preincubation with a specific PKA inhibitor, Rp-CPT-cAMP 10(-4) M, and was completely abolished by preincubation with PKC inhibitors, staurosporine 10(-7) M, sphingosine 5.10(-6) M, or calphostin 10(-6) M. In addition, pretreatment of rats with pertussis toxin led to a partial inhibition of the effect of low dose Ang II. The high dose-Ang II-induced decrease in dpHi/dt was not affected by pretreatment with a calcium-calmodulin kinase inhibitor W-7 10(-4) M. Conversely, pretreatment with the cytochrome P-450 inhibitor econazole 10(-5) M reversed the inhibitory effect of high dose Ang II to a stimulatory effect (24 +/- 8%, P < 0.01), quantitatively similar to the effect of low dose Ang II. In addition, arachidonate was found to exert an econazole-sensitive dose-dependent inhibitory effect on dpHi/dt, and 5,6-EET 10(-6) M, a cytochrome P-450 derived-arachidonic acid metabolite, induced a 38 +/- 9% inhibition, similar to that observed with high dose Ang II alone. There was no additive effect of 5,6-EET and high dose Ang II. Finally, pretreatment with two PLA2 inhibitors (BromoPhenacylBromide, 6.10(-6) M, and oleyloxyethyl phosphorylcholine, 5.10(-6) M) reversed the inhibitory effect of high dose Ang II to a stimulatory effect (32 +/- 11% and 25 +/- 11%, respectively, P < 0.05 for both inhibitors). We conclude that, in intact rat proximal cells, low dose Ang II stimulates the apical Na/H antiport through a pertussis toxin-sensitive G protein-dependent PKC pathway, whereas high dose Ang II inhibits the Na/H antiport activity through the PLA2- and cytochrome P-450-dependent metabolites of arachidonate.

Excessive animal protein consumption is associated with a greater risk of occurrence of renal calcium stone, presumably because of the attendant endogenous acid production. Indeed, chronic acid load enhances urinary calcium excretion possibly through an increased bone calcium release. Because acute studies are best designed to elucidate the mechanism, renal or extra renal, underlying hypercalciuria in the setting of enhanced acid load, we examined the response of 9 healthy adults (8 males, 1 female, aged 38 +/- 3 years, weight 67 +/- 2 kg) and 34 hypercalciuric recurrent calcium stone formers (31 males, 3 females, aged 44 +/- 2 years, weight 72 +/- 2 kg), without any associated disease, to an oral acid load (NH4Cl 2 mmol/kg body wt). After an overnight fast, each patient and control was studied during one one-hour period before and three two-hour periods after their intake of the acid load. An additional group of four time-control subjects (4 males, aged 33 +/- 2 years, weight 66 +/- 2 kg) was studied as the experimental groups except that they did not receive the acid load. On baseline, the three groups exhibited similar glomerular filtration rates, net acid excretions, and plasma calcium and magnesium concentrations. However, fasting urine calcium and magnesium excretions were higher in hypercalciuric calcium stone formers than in healthy control or time-control subjects. In time-control subjects, plasma acid base status, net acid excretion, filtered loads of calcium and magnesium, and urinary calcium and magnesium excretions remained unchanged all over the study. By contrast, after the oral acute acid load, net acid excretion increased and urinary pH decreased similarly in patient and control groups; glomerular filtration rate did not change, as well as plasma calcium and magnesium concentrations. Nevertheless, urinary calcium and magnesium excretions markedly increased, in both groups, independently of changes in tubular sodium handling and in plasma parathyroid hormone concentration. The increase in urinary calcium and magnesium excretions that occurred in the absence of any change in the filtered load of calcium and magnesium was therefore mediated by a decrease in tubular calcium and magnesium reabsorption, independent of PTH, but dependent on changes in net acid excretion. A positive linear relationship between urinary calcium and magnesium excretions suggested that the target tubular site for acid load was the thick ascending limb of Henle's loop. Finally, a negative linear relationship was demonstrated between the acid load-induced increase in urinary calcium excretion and fasting urinary calcium excretion; indeed, the lowest calciuric responses were observed in patients with the highest fasting urinary calcium excretion. Thus there was no additional effect of the acid load-induced inhibition on intrinsic defect in tubular calcium reabsorption which suggests that the tubular target site for acid load and the site of calcium transport defect in idiopathic hypercalciuria may be the same.