Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel

The mechanistic target of rapamycin (mTOR), a serine-threonine-specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the collecting duct (CD) of the kidney, the epithelial sodium channel (ENaC) essential in the determination of final urine Na+ losses, has been demonstrated to be upregulated by mTOR, using cell culture and mTOR inhibition in ex vivo preparations. We tested whether CD-principal cell (PC) targeted deletion of mTOR using Cre-lox recombination would affect whole-body sodium homeostasis, blood pressure, and ENaC regulation in mice. Male and female CD-PC mTOR knockout (KO) mice and wild-type (WT) littermates (Cre-negative) were generated using aquaporin-2 (AQP2) promoter to drive Cre-recombinase. Under basal conditions, KO mice showed a reduced (∼30%) natriuretic response to benzamil (ENaC) antagonist, suggesting reduced in vivo ENaC activity. WT and KO mice were fed normal sodium (NS, 0.45% Na+) or a very low Na+ (LS, <0.02%) diet for 7-days. Switching from NS to LS resulted in significantly higher urine sodium losses (relative to WT) in the KO with adaptation occurring by day 2. Blood pressures were modestly (∼5–10 mm Hg) but significantly lower in KO mice under both diets. Western blotting showed KO mice had 20–40% reduced protein levels of all three subunits of ENaC under LS or NS diet. Immunohistochemistry (IHC) of kidney showed enhanced apical-vs.-cellular localization of all three subunits with LS, but a reduction in this ratio for γ-ENaC in the KO. Furthermore, the KO kidneys showed increased ubiquitination of α-ENaC and reduced phosphorylation of the serum and glucocorticoid regulated kinase, type 1 [serum glucocorticoid regulated kinase (SGK1)] on serine 422 (mTOR phosphorylation site). Taken together this suggests enhanced degradation as a consequence of reduced mTOR kinase activity and downstream upregulation of ubiquitination may have accounted for the reduction at least in α-ENaC. Overall, our data support a role for mTOR in ENaC activity likely via regulation of SGK1, ubiquitination, ENaC channel turnover and apical membrane residency. These data support a role for mTOR in the collecting duct in the maintenance of body sodium homeostasis.

CA1 pyramidal cell diversity is rootedin the time of neurogenesis

Cellular diversity supports the computational capacity and flexibility of cortical circuits. Accordingly, principal neurons at the CA1 output node of the murine hippocampus are increasingly recognized as a heterogeneous population. Their genes, molecular content, intrinsic morphophysiology, connectivity, and function seem to segregate along the main anatomical axes of the hippocampus. Since these axes reflect the temporal order of principal cell neurogenesis, we directly examined the relationship between birthdate and CA1 pyramidal neuron diversity, focusing on the ventral hippocampus. We used a genetic fate-mapping approach that allowed tagging three groups of age-matched principal neurons: pioneer, early- and late-born. Using a combination of neuroanatomy, slice physiology, connectivity tracing and cFos staining in mice, we show that birthdate is a strong predictor of CA1 principal cell diversity. We unravel a subpopulation of pioneer neurons recruited in familiar environments with remarkable positioning, morpho-physiological features, and connectivity. Therefore, despite the expected plasticity of hippocampal circuits, given their role in learning and memory, the diversity of their main components is also partly determined at the earliest steps of development.

Stereological Changes in Microvascular Parameters in Hippocampus of a Transgenic Rat Model of Alzheimer’s Disease

Background: Microcirculatory factors play an important role in amyloid-β (Aβ)-related neuropathology in Alzheimer’s disease (AD). Transgenic (Tg) rat models of mutant Aβ deposition can enhance our understanding of this microvascular pathology. Objective: Here we report stereology-based quantification and comparisons (between- and within-group) of microvessel length and number and associated parameters in hippocampal subregions in Tg model of AD in Fischer 344 rats and non-Tg littermates. Methods: Systematic-random samples of tissue sections were processed and laminin immunostained to visualize microvessels through the entire hippocampus in Tg and non-Tg rats. A computer-assisted stereology system was used to quantify microvessel parameters including total number, total length, and associated densities in dentate gyrus (DG) and cornu ammonis (CA) subregions. Results: Thin hair-like capillaries are common near Aβ plaques in hippocampal subregions of Tg rats. There are a 53% significant increase in average length per capillary across entire hippocampus (p≤0.04) in Tg compared to non-Tg rats; 49% reduction in capillary length in DG (p≤0.02); and, higher microvessel density in principal cell layers (p≤0.03). Furthermore, within-group comparisons confirm Tg but not non-Tg rats have significant increase in number density (p≤0.01) and potential diffusion distance (p≤0.04) of microvessels in principal cell layers of hippocampal subregions. Conclusion: We show the Tg deposition of human Aβ mutations in rats disrupts the wild-type microanatomy of hippocampal microvessels. Stereology-based microvascular parameters could promote the development of novel strategies for protection and the therapeutic management of AD.

Abstract 06: Mechanisms And Consequences Of Casein Kinase II And Ankyrin 3 Regulation Of The Epithelial Sodium Channel

Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical “anchor” ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in β-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a “switch” that favors Ank-3 binding to increase channel activity.

Mechanisms and consequences of casein kinase II and ankyrin-3 regulation of the epithelial Na+ channel

Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical “anchor” ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in β-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a “switch” that favors Ank-3 binding to increase channel activity.

CA1 pyramidal cell diversity is rooted in the time of neurogenesis

Cellular diversity supports the computational capacity and flexibility of cortical circuits. Accordingly, principal neurons at the CA1 output node of the hippocampus are increasingly recognized as a heterogeneous population. Their genes, molecular content, intrinsic morpho-physiology, connectivity, and function seem to segregate along the main anatomical axes of the hippocampus. Since these axes reflect the temporal order of principal cell neurogenesis, we directly examined the relationship between birthdate and CA1 pyramidal neuron diversity, focusing on the ventral hippocampus. We used a genetic fate-mapping approach that allowed tagging three groups of age-matched principal neurons: pioneer, early-and late-born. Using a combination of neuroanatomy, slice physiology, connectivity tracing and cFos staining, we show that birthdate is a strong predictor of CA1 principal cell diversity. We unravel a subpopulation of pioneer neurons recruited in familiar environments with remarkable positioning, morpho-physiological features, and connectivity. Therefore, despite the expected plasticity of hippocampal circuits, given their role in learning and memory, the diversity of their main components is significantly predetermined at the earliest steps of development.

AQP2: Mutations Associated with Congenital Nephrogenic Diabetes Insipidus and Regulation by Post-Translational Modifications and Protein-Protein Interactions

As a rare hereditary disease, congenital nephrogenic diabetes insipidus (NDI) is clinically characterized by polyuria with hyposthenuria and polydipsia. NDI results from collecting duct principal cell hyporesponsiveness or insensitivity to the antidiuretic action of arginine vasopressin (AVP). The principal cell-specific water channel aquaporin-2 (AQP2) plays an essential role in water reabsorption along osmotic gradients. The capacity to accumulate AQP2 in the apical plasma membrane in response to decreased fluid volume or increased plasma osmolality is critically regulated by the antidiuretic hormone AVP and its receptor 2 (AVPR2). Mutations in AVPR2 result in X-linked recessive NDI, the most common form of inherited NDI. Genetic defects in AQP2 cause autosomal recessive or dominant NDI. In this review, we provide an updated overview of the genetic and molecular mechanisms of congenital NDI, with a focus on the potential disease-causing mutations in AVPR2 and AQP2, the molecular defects in the AVPR2 and AQP2 mutants, post-translational modifications (i.e., phosphorylation, ubiquitination, and glycosylation) and various protein-protein interactions that regulate phosphorylation, ubiquitination, tetramerization, trafficking, stability, and degradation of AQP2.

Aldosterone Regulates Pendrin and Epithelial Sodium Channel Activity through Intercalated Cell Mineralocorticoid Receptor–Dependent and –Independent Mechanisms over a Wide Range in Serum Potassium

BackgroundAldosterone activates the intercalated cell mineralocorticoid receptor, which is enhanced with hypokalemia. Whether this receptor directly regulates the intercalated cell chloride/bicarbonate exchanger pendrin is unclear, as are potassium’s role in this response and the receptor’s effect on intercalated and principal cell function in the cortical collecting duct (CCD).MethodsWe measured CCD chloride absorption, transepithelial voltage, epithelial sodium channel activity, and pendrin abundance and subcellular distribution in wild-type and intercalated cell–specific mineralocorticoid receptor knockout mice. To determine if the receptor directly regulates pendrin, as well as the effect of serum aldosterone and potassium on this response, we measured pendrin label intensity and subcellular distribution in wild-type mice, knockout mice, and receptor-positive and receptor-negative intercalated cells from the same knockout mice.ResultsAblation of the intercalated cell mineralocorticoid receptor in CCDs from aldosterone-treated mice reduced chloride absorption and epithelial sodium channel activity, despite principal cell mineralocorticoid receptor expression in the knockout mice. With high circulating aldosterone, intercalated cell mineralocorticoid receptor gene ablation directly reduced pendrin’s relative abundance in the apical membrane region and pendrin abundance per cell whether serum potassium was high or low. Intercalated cell mineralocorticoid receptor ablation blunted, but did not eliminate, aldosterone’s effect on pendrin total and apical abundance and subcellular distribution.ConclusionsWith high circulating aldosterone, intercalated cell mineralocorticoid receptor ablation reduces chloride absorption in the CCD and indirectly reduces principal cell epithelial sodium channel abundance and function. This receptor directly regulates pendrin’s total abundance and its relative abundance in the apical membrane region over a wide range in serum potassium concentration. Aldosterone regulates pendrin through mechanisms both dependent and independent of the IC MR receptor.