Sophie de Seigneux

Calcium-sensing Receptor Attenuates AVP-induced Aquaporin-2 Expression via a Calmodulin-dependent Mechanism

Recent evidence suggests that arginine vasopressin (AVP)-dependent aquaporin-2 expression is modulated by the extracellular calcium-sensing receptor (CaSR) in principal cells of the collecting duct, but the signaling pathways mediating this effect are unknown. Using a mouse cortical collecting duct cell line (mpkCCD(cl4)), we found that increasing the concentration of apical extracellular calcium or treating with the CaSR agonists neomycin or Gd(3+) attenuated AVP-dependent accumulation of aquaporin-2 mRNA and protein; CaSR gene-silencing prevented this effect. Calcium reduced the AVP-induced accumulation of cAMP, but this did not occur by increased degradation of cAMP by phosphodiesterases or by direct inhibition of adenylate cyclase. Notably, the effect of extracellular calcium on AVP-dependent aquaporin-2 expression was prevented by inhibition of calmodulin. In summary, our results show that high concentrations of extracellular calcium attenuate AVP-induced aquaporin-2 expression by activating the CaSR and reducing coupling efficiency between V(2) receptor and adenylate cyclase via a calmodulin-dependent mechanism in cultured cortical collecting duct cells.

NADPH-Oxidase 4 Protects against Kidney Fibrosis during Chronic Renal Injury

NADPH oxidases synthesize reactive oxygen species that may participate in fibrosis progression. NOX4 and NOX2 are NADPH oxidases expressed in the kidneys, with the former being the major renal isoform, but their contribution to renal disease is not well understood. Here, we used the unilateral urinary obstruction model of chronic renal injury to decipher the role of these enzymes using wild-type, NOX4-, NOX2-, and NOX4/NOX2-deficient mice. Compared with wild-type mice, NOX4-deficient mice exhibited more interstitial fibrosis and tubular apoptosis after obstruction, with lower interstitial capillary density and reduced expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in obstructed kidneys. Furthermore, NOX4-deficient kidneys exhibited increased oxidative stress. With NOX4 deficiency, renal expression of other NOX isoforms was not altered but NRF2 protein expression was reduced under both basal and obstructed conditions. Concomitant deficiency of NOX2 did not modify the phenotype exhibited by NOX4-deficient mice after obstruction. NOX4 silencing in a mouse collecting duct (mCCD(cl1)) cell line increased TGF-β1-induced apoptosis and decreased NRF2 protein along with expression of its target genes. In addition, NOX4 silencing decreased hypoxia-inducible factor-1α and expression of its target genes in response to hypoxia. In summary, these results demonstrate that the absence of NOX4 promotes kidney fibrosis, independent of NOX2, through enhanced tubular cell apoptosis, decreased microvascularization, and enhanced oxidative stress. Thus, NOX4 is crucial for the survival of kidney tubular cells under injurious conditions.

Determination of the best method to estimate glomerular filtration rate from serum creatinine in adult patients with sickle cell disease: a prospective observational cohort study.

BackgroundSickle cell disease (SCD) leads to tissue hypoxia resulting in chronic organ dysfunction including SCD associated nephropathy. The goal of our study was to determine the best equation to estimate glomerular filtration rate (GFR) in SCD adult patients.MethodsWe conducted a prospective observational cohort study. Since 2007, all adult SCD patients in steady state, followed in two medical departments, have had their GFR measured using iohexol plasma clearance (gold standard). The Cockcroft-Gault, MDRD-v4, CKP-EPI and finally, MDRD and CKD-EPI equations without adjustment for ethnicity were tested to estimate GFR from serum creatinine. Estimated GFRs were compared to measured GFRs according to the graphical Bland and Altman method.ResultsSixty-four SCD patients (16 men, median age 27.5 years [range 18.0-67.5], 41 with SS-genotype were studied. They were Sub-Saharan Africa and French West Indies natives and predominantly lean (median body mass index: 22 kg/m2 [16-33]). Hyperfiltration (defined as measured GFR >110 mL/min/1.73 m2) was detected in 53.1% of patients. Urinary albumin/creatinine ratio was higher in patients with hyperfiltration than in patients with normal GFR (4.05 mg/mmol [0.14-60] versus 0.4 mg/mmol [0.7-81], p = 0.01). The CKD-EPI equation without adjustment for ethnicity had both the lowest bias and the greatest precision. Differences between estimated GFRs using the CKP-EPI equation and measured GFRs decreased with increasing GFR values, whereas it increased with the Cockcroft-Gault and MDRD-v4 equations.ConclusionsWe confirm that SCD patients have a high rate of glomerular hyperfiltration, which is frequently associated with microalbuminuria or macroalbuminuria. In non-Afro-American SCD patients, the best method for estimating GFR from serum creatinine is the CKD-EPI equation without adjustment for ethnicity. This equation is particularly accurate to estimate high GFR values, including glomerular hyperfiltration, and thus should be recommended to screen SCD adult patients at high risk for SCD nephropathy.

Aldosterone Activates NF-κB in the Collecting Duct

Besides its classical effects on salt homeostasis in renal epithelial cells, aldosterone promotes inflammation and fibrosis and modulates cell proliferation. The proinflammatory transcription factor NF-kappaB has been implicated in cell proliferation, apoptosis, and regulation of transepithelial sodium transport. The effect of aldosterone on the NF-kappaB pathway in principal cells of the cortical collecting duct, a major physiologic target of aldosterone, is unknown. Here, in both cultured cells and freshly isolated rat cortical collecting duct, aldosterone activated the canonical NF-kappaB signaling pathway, leading to increased expression of several NF-kappaB-targeted genes (IkappaBalpha, plasminogen activator inhibitor 1, monocyte chemoattractant protein 1, IL-1beta, and IL-6). Small interfering RNA-mediated knockdown of the serum and glucocorticoid-inducible kinase SGK1, a gene induced early in the response to aldosterone, but not pharmacologic inhibition of extracellular signal-regulated kinase and p38 kinase, attenuated aldosterone-induced NF-kappaB activation. Pharmacologic antagonism or knockdown of the mineralocorticoid receptor prevented aldosterone-induced NF-kappaB activity. In addition, activation of the glucocorticoid receptor inhibited the transactivation of NF-kappaB by aldosterone. In agreement with these in vitro findings, spironolactone prevented NF-kappaB-induced transcriptional activation observed in cortical collecting ducts of salt-restricted rats. In summary, aldosterone activates the canonical NF-kappaB pathway in principal cells of the cortical collecting duct by activating the mineralocorticoid receptor and by inducing SGK1.

Increased Apical Targeting of Renal Epithelial Sodium Channel Subunits and Decreased Expression of Type 2 11β-Hydroxysteroid Dehydrogenase in Rats with CCl4-Induced Decompensated Liver Cirrhosis

It was hypothesized that dysregulation of renal epithelial sodium channel (ENaC) subunits and/or 11beta-hydroxysteroid dehydrogenase (11betaHSD2) may play a role in the increased sodium retention in liver cirrhosis (LC). Experimental LC was induced in rats by CCl(4) (1 ml/kg, intraperitoneally, twice a week) for 12 wk (protocol 1) or for 11 wk (protocol 2). In both protocols, one group of rats with cirrhosis showed significantly decreased urinary sodium excretion and urinary Na/K ratio (group A), whereas a second group exhibited normal urinary sodium excretion (group B) compared with controls, even though extensive ascites was seen in both groups of rats with cirrhosis. In group A, protein abundance of alpha-ENaC was unchanged, whereas beta-ENaC abundance was decreased in the cortex/outer stripe of outer medulla compared with controls. The gamma-ENaC underwent a complex change associated with increased abundance of the 70-kD band with a concomitant decrease in the main 85-kD band, corresponding to an aldosterone effect. In contrast, no changes in the abundance of ENaC subunit were observed in group B. Immunoperoxidase microscopy revealed an increased apical targeting of alpha-, beta-, and gamma-ENaC subunits in distal convoluted tubule (DCT2), connecting tubule (CNT), and cortical and medullary collecting duct segments in group A but not in group B. Immunolabeling intensity of 11betaHSD2 in the DCT2, CNT, and cortical collecting duct was significantly reduced in group A but not in group B, and this was confirmed by immunoblotting. In conclusion, increased apical targeting of ENaC subunits combined with diminished abundance of 11betaHSD2 in the DCT2, CNT, and cortical collecting duct is likely to play a role in the sodium retaining stage of liver cirrhosis.

NF-kappaB inhibits sodium transport via down-regulation of SGK1 in renal collecting duct principal cells.

Tubulointerstitial inflammation is a common feature of renal diseases. We have investigated the relationship between inflammation and Na+ transport in the collecting duct (CD) using the mCCDcl1 and mpkCDDcl4 principal cell models. Lipopolysaccharide (LPS) decreased basal and aldosterone-stimulated amiloride-sensitive transepithelial current in a time-dependent manner. This effect was associated with a decrease in serum and gucocorticoid-regulated kinase 1 (SGK1) mRNA and protein levels followed by a decrease in epithelial sodium channel (ENaC) α-subunit mRNA levels. The LPS-induced decrease in SGK1 expression was confirmed in isolated rat CD. This decreased expression of either SGK1 or the ENaC α-subunit was not due to enhanced degradation of mRNA. In contrast, LPS inhibited transcriptional activity of the SGK1 promoter measured by luciferase-reporter gene assay. The effect of LPS was not mediated by inhibition of mineralocorticoid or glucocorticoid receptor, because expression of both receptors was unchanged and blockade of either receptor by spironolactone or RU486, respectively, did not prevent the down-regulation of SGK1. The effect of LPS was mediated by the canonical NF-κB pathway, as overexpression of a constitutively active mutant, IKKβ (inhibitor of nuclear factor κB kinase-β) decreased SGK1 mRNA levels, and knockdown of p65 NF-κB subunit by small interfering RNA increased SGK1 mRNA levels. Chromatin immunoprecipitation showed that LPS increased p65 binding to two NF-κB sites along the SGK1 promoter. In conclusion, we show that activation of the NF-κB pathway down-regulates SGK1 expression, which might lead to decreased ENaC α-subunit expression, ultimately resulting in decreased Na+ transport.

Epoetin administrated after cardiac surgery: effects on renal function and inflammation in a randomized controlled study

BackgroundExperimentally, erythropoietin (EPO) has nephroprotective as well as immunomodulatory properties when administered after ischemic renal injury. We tested the hypothesis that different doses of recombinant human EPO administered to patients after cardiac surgery would minimize kidney lesions and the systemic inflammatory response, thereby decreasing acute kidney injury (AKI) incidence.MethodsIn this double-blinded randomized control study, 80 patients admitted to the ICU post-cardiac surgery were randomized by computer to receive intravenously isotonic saline (n = 40) versus α-Epoetin (n = 40): either 40000 IU (n = 20) or 20000 IU (n = 20). The study lasted one year. The primary outcome was the change in urinary NGAL concentration from baseline and 48 h after EPO injection. Creatinine, cystatine C and urinary NGAL levels were measured on the day of randomization and 2–4 days after EPO injection. To assess acute inflammatory response, serum cytokines (IL6 and IL8) were measured at randomization and four days after r-HuEPO injection. Patients and care-takers were blinded for the assignment.ResultsNo patient was excluded after randomization. Patient groups did not differ in terms of age, gender, comorbidities and renal function at randomization. The rate of AKI assessed by AKIN criteria was 22.5% in our population. EPO treatment did not significantly modify the difference in uNGAl between 48 hours and randomization compared to placebo [2.5 ng/ml (−17.3; 22.5) vs 0.7 ng/ml (−31.77; 25.15), p = 0.77] and the incidence of AKI was similar. Inflammatory cytokines levels were not influenced by EPO treatment. Mortality and hospital stays were similar between the groups and no adverse event was recorded.ConclusionIn this randomized-controlled trial, α-Epoetin administrated after cardiac surgery, although safe, demonstrated neither nephroprotective nor anti-inflammatory properties.Trial registration numberNCT00676234

Rapid and segmental specific dysregulation of AQP2, S256-pAQP2 and renal sodium transporters in rats with LPS-induced endotoxaemia

BACKGROUND Acute renal failure (ARF) is a frequent complication of sepsis. Characteristics of ARF in sepsis are impaired urinary concentration, increased natriuresis and decreased glomerular filtration rate (GFR), in which inducible nitric oxide synthase (iNOS) has been revealed to play a role. Aims. We aimed to investigate renal water and sodium excretion and in parallel the segmental regulation of renal AQP2 and major sodium transporters in rats with acute LPS-induced endotoxaemia. Next, we aimed to examine the changes of iNOS expression and activated macrophage infiltration in the kidney and the effects of iNOS inhibition on AQP2 and NKCC2 expression in LPS rats. METHODS Rats were treated with LPS (i.p.) or with LPS + iNOS inhibitor L-NIL, and 6 h later kidneys were subjected to semiquantitative immunoblotting and immunohistochemistry. RESULTS Polyuria and increased natriuresis were seen 6 h after LPS injection alongside downregulation of both AQP2 and S256-phosphorylated AQP2 in CTX/OSOM and ISOM but not in inner medulla (IM). Thick ascending limb sodium transporters NHE3 and NKCC2 were downregulated in ISOM and NaPi2 was decreased in CTX/OSOM, whereas NCC and ENaC were not consistently downregulated. Immunolabelling intensity of iNOS was increased in vascular structures and transitional epithelium, and an infiltration of activated macrophages was seen in CTX and ISOM. L-NIL co-treatment prevented the downregulation of NKCC2 but not AQP2 in LPS rats. CONCLUSIONS Early downregulation of AQP2 and sodium transporters takes place segmentally in the kidney after LPS administration. In addition, an infiltration of activated macrophages and increased iNOS expression may play a role in the urinary concentrating defect in acute LPS-induced entotoxaemia.

Evidence of follicular T-cell implication in a case of IgG4-related systemic disease with interstitial nephritis

IgG4-related systemic disease is a protean disorder that covers a wide variety of lesions. We report on a patient with tubulointerstitial nephritis, lymphadenopathies, sialadenitis and retroperitoneal fibrosis. The salivary gland and kidney interstitium were infiltrated with B lymphocytes and T lymphocytes and IgG3(+) and IgG4(+) plasma cells. The overexpression of IgG1 and IgG3, in addition to IgG4, the unusual abundance of interfollicular plasma cells and CD4(+) T cells in germinal centres of lymph nodes, and the dramatic response to rituximab point to possible roles of follicular helper T cells in enhancing a skewed B-cell terminal maturation and of CD20(+) B cells in disease progression.

Proteinuria Increases Plasma Phosphate by Altering Its Tubular Handling

Proteinuria and hyperphosphatemia are cardiovascular risk factors independent of GFR. We hypothesized that proteinuria induces relative phosphate retention via increased proximal tubule phosphate reabsorption. To test the clinical relevance of this hypothesis, we studied phosphate handling in nephrotic children and patients with CKD. Plasma fibroblast growth factor 23 (FGF-23) concentration, plasma phosphate concentration, and tubular reabsorption of phosphate increased during the proteinuric phase compared with the remission phase in nephrotic children. Cross-sectional analysis of a cohort of 1738 patients with CKD showed that albuminuria≥300 mg/24 hours is predictive of higher phosphate levels, independent of GFR and other confounding factors. Albuminuric patients also displayed higher plasma FGF-23 and parathyroid hormone levels. To understand the molecular mechanisms underlying these observations, we induced glomerular proteinuria in two animal models. Rats with puromycin-aminonucleoside-induced nephrotic proteinuria displayed higher renal protein expression of the sodium-phosphate co-transporter NaPi-IIa, lower renal Klotho protein expression, and decreased phosphorylation of FGF receptor substrate 2α, a major FGF-23 receptor substrate. These findings were confirmed in transgenic mice that develop nephrotic-range proteinuria resulting from podocyte depletion. In vitro, albumin did not directly alter phosphate uptake in cultured proximal tubule OK cells. In conclusion, we show that proteinuria increases plasma phosphate concentration independent of GFR. This effect relies on increased proximal tubule NaPi-IIa expression secondary to decreased FGF-23 biologic activity. Proteinuria induces elevation of both plasma phosphate and FGF-23 concentrations, potentially contributing to cardiovascular disease.