Lydie Cheval

Rat G Protein-Coupled Receptor Kinase GRK4: Identification, Functional Expression, and Differential Tissue Distribution of Two Splice Variants*

G protein-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G protein-coupled receptors, leading to the homologous mode of desensitization. We report here on the cloning of complementary DNAs that encode two rat GRK4 variants. Rat GRK4A (575 amino acids) displays 76% identity with the long human GRK4 splice variant. Rat GRK4B (545 amino acids) delineates a new variant that is identical to GRK4A except for a 31-amino acid deletion in the N-terminal domain, corresponding to exon VI in the human GRK4 gene. GRKs4A and B are likely produced by alternative splicing from a single gene, the partial characterization of which revealed a structural organization similar to that of the human GRK4 gene. GRK4A messenger RNA (mRNA) is abundant only in testis. A combination of in situ hybridization and quantitative RT-PCR studies demonstrated that GRK4A mRNA level increases during testicular development and predominates in leptotene to late pachytene primary spermatocytes and round sp...

Sodium retention and ascites formation in a cholestatic mice model: Role of aldosterone and mineralocorticoid receptor?

Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone‐sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal‐intact or corticosteroid‐clamped mice. Corticosteroid‐clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24‐hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium‐ and potassium‐dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4‐5 weeks, cirrhosis induced sodium retention in adrenal‐intact mice and formation of ascites in 50% of mice. At that time, sodium‐ and potassium‐dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum‐ and glucocorticoid‐regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid‐clamp prevented induction of sgk1 but not cirrhosis‐induced sodium retention, formation of ascites and stimulation of sodium‐ and potassium‐dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. Conclusion: Bile duct ligation in mice induces cirrhosis which, within 4‐5 weeks, leads to the induction of sodium‐ and potassium‐dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium‐ and potassium‐dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids. (HEPATOLOGY 2007;46:173–179.)

Expression Profile of Nuclear Receptors along Male Mouse Nephron Segments Reveals a Link between ERRβ and Thick Ascending Limb Function

The nuclear receptor family orchestrates many functions related to reproduction, development, metabolism, and adaptation to the circadian cycle. The majority of these receptors are expressed in the kidney, but their exact quantitative localization in this ultrastructured organ remains poorly described, making it difficult to elucidate the renal function of these receptors. In this report, using quantitative PCR on microdissected mouse renal tubules, we established a detailed quantitative expression map of nuclear receptors along the nephron. This map can serve to identify nuclear receptors with specific localization. Thus, we unexpectedly found that the estrogen-related receptor β (ERRβ) is expressed predominantly in the thick ascending limb (TAL) and, to a much lesser extent, in the distal convoluted tubules. In vivo treatment with an ERR inverse agonist (diethylstilbestrol) showed a link between this receptor family and the expression of the Na+,K+-2Cl− cotransporter type 2 (NKCC2), and resulted in phenotype presenting some similarities with the Bartter syndrom (hypokalemia, urinary Na+ loss and volume contraction). Conversely, stimulation of ERRβ with a selective agonist (GSK4716) in a TAL cell line stimulated NKCC2 expression. All together, these results provide broad information regarding the renal expression of all members of the nuclear receptor family and have allowed us to identify a new regulator of ion transport in the TAL segments.

Renal proteinase activated receptor 2: A new actor in the control of blood pressure and plasma potassium level

Background: The function of proteinase-activated receptor 2 (PAR2) in the distal nephron remains unknown. Results: PAR2 activation increases electroneutral sodium reabsorption and inhibits potassium secretion in collecting ducts and thereby controls blood pressure and plasma potassium. Conclusion: PAR2 controls sodium and potassium homeostasis. Significance: PAR2 is a new actor of aldosterone paradox but also an aldosterone-independent modulator of blood pressure and plasma potassium. Proteinase-activated receptor 2 (PAR2) is a G protein-coupled membrane receptor that is activated upon cleavage of its extracellular N-terminal domain by trypsin and related proteases. PAR2 is expressed in kidney collecting ducts, a main site of control of Na+ and K+ homeostasis, but its function remains unknown. We evaluated whether and how PAR2 might control electrolyte transport in collecting ducts, and thereby participate in the regulation of blood pressure and plasma K+ concentration. PAR2 is expressed at the basolateral border of principal and intercalated cells of the collecting duct where it inhibits K+ secretion and stimulates Na+ reabsorption, respectively. Invalidation of PAR2 gene impairs the ability of the kidney to control Na+ and K+ balance and promotes hypotension and hypokalemia in response to Na+ and K+ depletion, respectively. This study not only reveals a new role of proteases in the control of blood pressure and plasma potassium level, but it also identifies a second membrane receptor, after angiotensin 2 receptor, that differentially controls sodium reabsorption and potassium secretion in the late distal tubule. Conversely to angiotensin 2 receptor, PAR2 is involved in the regulation of sodium and potassium balance in the context of either stimulation or nonstimulation of the renin/angiotensin/aldosterone system. Therefore PAR2 appears not only as a new actor of the aldosterone paradox, but also as an aldosterone-independent modulator of blood pressure and plasma potassium.

RE-EVALUATION OF THE EXPRESSION OF THE GASTRIC H,K-ATPASE ALPHA SUBUNIT ALONG THE RAT NEPHRON

Abstractu2002Under normal conditions, the rat collecting duct displays an H,K-ATPase activity with kinetic and pharmacological properties very close to those of the gastric H,K-ATPase. However, whether the collecting duct H,K-ATPase and the gastric enzyme are identical remains controversial. Therefore, we re-evaluated the expression of the mRNAs encoding the gastric H,K-ATPase α subunit in the rat nephron. For this purpose, gastric H,K-ATPase mRNAs were quantitated by RT-PCR at the level of microdissected nephron segments using known amounts of gastric H,K-ATPase cRNA as external standards. Results indicate that gastric H,K-ATPase mRNAs are undetectable (<1 copy per cell) in the glomerulus and along the proximal tubule, thick ascending limb of Henle’s loop and collecting duct, although a faint expression (≈ 400 copies per μg total RNA) is measurable in whole–kidney preparations. Gastric H,K-ATPase mRNA is also absent along the nephron of K-depleted rats and of rats with chronic metabolic acidosis and alkalosis. Taken with other data from the literature, these results suggest that the collecting duct of normal rats might express an H,K-ATPase similar, but not identical, to the gastric isoform.

Tissue compartment analysis for biomarker discovery by gene expression profiling.

Background Although high throughput technologies for gene profiling are reliable tools, sample/tissue heterogeneity limits their outcomes when applied to identify molecular markers. Indeed, inter-sample differences in cell composition contribute to scatter the data, preventing detection of small but relevant changes in gene expression level. To date, attempts to circumvent this difficulty were based on isolation of the different cell structures constituting biological samples. As an alternate approach, we developed a tissue compartment analysis (TCA) method to assess the cell composition of tissue samples, and applied it to standardize data and to identify biomarkers. Methodology/Principal Findings TCA is based on the comparison of mRNA expression levels of specific markers of the different constitutive structures in pure isolated structures, on the one hand, and in the whole sample on the other. TCA method was here developed with human kidney samples, as an example of highly heterogeneous organ. It was validated by comparison of the data with those obtained by histo-morphometry. TCA demonstrated the extreme variety of composition of kidney samples, with abundance of specific structures varying from 5 to 95% of the whole sample. TCA permitted to accurately standardize gene expression level amongst >100 kidney biopsies, and to identify otherwise imperceptible molecular disease markers. Conclusions/Significance Because TCA does not require specific preparation of sample, it can be applied to all existing tissue or cDNA libraries or to published data sets, inasmuch specific operational compartments markers are available. In human, where the small size of tissue samples collected in clinical practice accounts for high structural diversity, TCA is well suited for the identification of molecular markers of diseases, and the follow up of identified markers in single patients for diagnosis/prognosis and evaluation of therapy efficiency. In laboratory animals, TCA will interestingly be applied to central nervous system where tissue heterogeneity is a limiting factor.

Renal Transcriptomes: Segmental Analysis of Differential Expression

Background/Aims: Progress accomplished by complete genomes and cDNA-sequencing projects calls for methods that fully use these resources to study gene expression patterns in characterized cell populations. However, since the number of functional genes cannot be readily inferred from the genomic sequence, it is highly desirable to make use of methods enabling to study both known and unknown genes. Methods: The method of serial analysis of gene expression provides short diagnostic cDNA tags without bias towards known genes. In addition, the frequency of each tag in the library conveys quantitative information on gene expression. A microassay was set-up to perform serial analysis of gene expression in minute samples such as those obtained by microdissecting nephron segments. Results: Studies carried out in the thick ascending limb of Henle’s loop and the collecting duct of the mouse kidney provided expression data for several thousand genes. Known markers were found appropriately enriched, and several of the thick ascending limb or collecting duct specific transcripts had no database match. Conclusions: The microassay for serial analysis of gene expression makes possible large-scale quantitative measurements of mRNA levels in nephron segments. The comprehensive picture generated by analyzing both known and unknown transcripts in defined cell populations should help to discover genes with dedicated functions.

Acute regulated expression of pendrin in human urinary exosomes

It is well known that pendrin, an apical Cl−/HCO3−exchanger in type B intercalated cells, is modulated by chronic acid-base disturbances and electrolyte intake. To study this adaptation further at the acute level, we analyzed urinary exosomes from individuals subjected to oral acute acid, alkali, and NaCl loading. Acute oral NH4Cl loading (nxa0=xa08) elicited systemic acidemia with a drop in urinary pH and an increase in urinary NH4 excretion. Nadir urinary pH was achieved 5xa0h after NH4Cl loading. Exosomal pendrin abundance was dramatically decreased at 3xa0h after acid loading. In contrast, after acute equimolar oral NaHCO3 loading (nxa0=xa08), urinary and venous blood pH rose rapidly with a significant attenuation of urinary NH4 excretion. Alkali loading caused rapid upregulation of exosomal pendrin abundance at 1xa0h and normalized within 3xa0h of treatment. Equimolar NaCl loading (nxa0=xa06) did not alter urinary or venous blood pH or urinary NH4 excretion. However, pendrin abundance in urinary exosomes was significantly reduced at 2xa0h of NaCl ingestion with lowest levels observed at 4xa0h after treatment. In patients with inherited distal renal tubular acidosis (dRTA), pendrin abundance in urinary exosomes was greatly reduced and did not change upon oral NH4Cl loading. In summary, pendrin can be detected and quantified in human urinary exosomes by immunoblotting. Acid, alkali, and NaCl loadings cause acute changes in pendrin abundance in urinary exosomes within a few hours. Our data suggest that exosomal pendrin is a promising urinary biomarker for acute acid-base and volume status changes in humans.