Renal handling of NH4+ in relation to the control of acid-base balance by the kidney.

The major component of urinary acid excretion is NH4+. To be appropriately excreted in urine, NH4+ must be synthesized by proximal tubular cells, secreted into the proximal tubular fluid, reabsorbed by the medullary thick ascending limb (MTAL) to be accumulated in the medullary interstitium, and finally secreted in medullary collecting ducts. Each step of this renal pathway is highly regulated and, in addition to acute events mediated by peptide hormones such as parathyroid hormone, the control of gene expression explains how the renal handling of NH4+ fully adapts to chronic changes in the acid-base status. Several targets have been identified at the gene expression level to account for the adaptation of renal NH4+ synthesis and transport in response to an acid load. These are the key enzymes of ammoniagenesis (mitochondrial glutaminase and glutamate dehydrogenase) and gluconeogenesis (phosphoenolpyruvate carboxykinase) in the proximal tubule, the apical Na(+)-K+(NH4+)-2Cl- cotransporter of the MTAL, and the basolateral Na(+)-K+(NH4+)-2Cl- cotransporter of medullary collecting ducts. At least two factors control the expression of these genes during metabolic acidosis: an acid pH and glucocorticoids, which appear to act in concert to coordinate the adaptation of various tubular cell types. The present review focuses on some aspects of these regulations that have been recently elucidated.

A TDG/CBP/RARα ternary complex mediates the retinoic acid-dependent expression of DNA methylation-sensitive genes.

The thymine DNA glycosylase (TDG) is a multifunctional enzyme, which is essential for embryonic development. It mediates the base excision repair (BER) of G:T and G:U DNA mismatches arising from the deamination of 5-methyl cytosine (5-MeC) and cytosine, respectively. Recent studies have pointed at a role of TDG during the active demethylation of 5-MeC within CpG islands. TDG interacts with the histone acetylase CREB-binding protein (CBP) to activate CBP-dependent transcription. In addition, TDG also interacts with the retinoic acid receptor α (RARα), resulting in the activation of RARα target genes. Here we provide evidence for the existence of a functional ternary complex containing TDG, CBP and activated RARα. Using global transcriptome profiling, we uncover a coupling of de novo methylation-sensitive and RA-dependent transcription, which coincides with a significant subset of CBP target genes. The introduction of a point mutation in TDG, which neither affects overall protein structure nor BER activity, leads to a significant loss in ternary complex stability, resulting in the deregulation of RA targets involved in cellular networks associated with DNA replication, recombination and repair. We thus demonstrate for the first time a direct coupling of TDG's epigenomic and transcription regulatory function through ternary complexes with CBP and RARα.

Exploring the role of galectin 3 in kidney function: a genetic approach.

Galectin 3 belongs to a family of glycoconjugate-binding proteins that participate in cellular homeostasis by modulating cell growth, adhesion, and signaling. We studied adult galectin 3 null mutant (Gal 3-/-) and wild-type (WT) mice to gain insights into the role of galectin 3 in the kidney. By immunofluorescence, galectin 3 was found in collecting duct (CD) principal and intercalated cells in some regions of the kidney, as well as in the thick ascending limbs at lower levels. Compared to WT mice, Gal 3-/- mice had approximately 11% fewer glomeruli (p < 0.04), associated with kidney hypertrophy (p < 0.006). In clearance experiments, urinary chloride excretion was found to be higher in Gal 3-/- than in WT mice (p < 0.04), but there was no difference in urinary bicarbonate excretion, in glomerular filtration, or urinary flow rates. Under chronic low sodium diet, Gal 3-/- mice had lower extracellular fluid (ECF) volume than WT mice (p < 0.05). Plasma aldosterone concentration was higher in Gal 3-/- than in WT mice (p < 0.04), which probably caused the observed increase in alpha-epithelial sodium channel (alpha-ENaC) protein abundance in the mutant mice (p < 0.001). Chronic high sodium diet resulted paradoxically in lower blood pressure (p < 0.01) in Gal 3-/- than in WT. We conclude that Gal 3-/- mice have mild renal chloride loss, which causes chronic ECF volume contraction and reduced blood pressure levels.

Acid pH increases the stability of BSC1/NKCC2 mRNA in the medullary thick ascending limb.

Chronic metabolic acidosis enhances the ability of the medullary thick ascending limb (MTAL) to absorb NH(4)(+) at least in part by stimulating the mRNA and protein expression of BSC1/NKCC2, the MTAL apical Na(+)-K(+)(NH(4)(+))-2Cl(-) co-transporter. For assessing the mechanism by which an acid pH enhances the BSC1 mRNA abundance, MTAL were harvested from adrenalectomized rats and incubated in control (pH 7.35) and acid (pH 7.10) 1:1 mixtures of Ham's nutrient mixture F-12 and DME. rBSC1 mRNA abundance and gene transcription rate were quantified by quantitative reverse transcription-PCR and run-off assay, respectively. Acid incubation enhanced mRNA abundance within 4 h in whole cell (P < 0.02) but not in nucleus. BSC1 gene transcription rate was not affected by acid incubation. In contrast, under conditions in which gene transcription was blocked, rBSC1 mRNA decreased within 6 h by 38 +/- 11% in control but only by 15 +/- 15% in acid medium (P < 0.02), which represented an increase in the BSC1 mRNA half-life from approximately 7 to approximately 17 h. Furthermore, in a mouse TAL cell line, acid incubation for 16 h significantly increased (P < 0.02) the amount of BSC1 mRNA in cells transfected with the full-length mBSC1 cDNA but not in cells transfected with a mBSC1 cDNA lacking the 3'-UTR. These results demonstrate that acid pH enhances the stability of BSC1 mRNA probably by activating pathways that act on the AU-rich 3'-UTR of BSC1 mRNA, which contributes to the renal response to metabolic acidosis.

Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb.

Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH(2)O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA (P < 0.04) and protein (P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA (P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium (P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats (P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors.