Chronic potassium depletion increases adrenal progesterone production that is necessary for efficient renal retention of potassium.

Modern dietary habits are characterized by high-sodium and low-potassium intakes, each of which was correlated with a higher risk for hypertension. In this study, we examined whether long-term variations in the intake of sodium and potassium induce lasting changes in the plasma concentration of circulating steroids by developing a mathematical model of steroidogenesis in mice. One finding of this model was that mice increase their plasma progesterone levels specifically in response to potassium depletion. This prediction was confirmed by measurements in both male mice and men. Further investigation showed that progesterone regulates renal potassium handling both in males and females under potassium restriction, independent of its role in reproduction. The increase in progesterone production by male mice was time dependent and correlated with decreased urinary potassium content. The progesterone-dependent ability to efficiently retain potassium was because of an RU486 (a progesterone receptor antagonist)-sensitive stimulation of the colonic hydrogen, potassium-ATPase (known as the non-gastric or hydrogen, potassium-ATPase type 2) in the kidney. Thus, in males, a specific progesterone concentration profile induced by chronic potassium restriction regulates potassium balance

Renal Intercalated Cells Sense and Mediate Inflammation via the P2Y14 Receptor

Uncontrolled inflammation is one of the leading causes of kidney failure. Pro-inflammatory responses can occur in the absence of infection, a process called sterile inflammation. Here we show that the purinergic receptor P2Y14 (GPR105) is specifically and highly expressed in collecting duct intercalated cells (ICs) and mediates sterile inflammation in the kidney. P2Y14 is activated by UDP-glucose, a damage-associated molecular pattern molecule (DAMP) released by injured cells. We found that UDP-glucose increases pro-inflammatory chemokine expression in ICs as well as MDCK-C11 cells, and UDP-glucose activates the MEK1/2-ERK1/2 pathway in MDCK-C11 cells. These effects were prevented following inhibition of P2Y14 with the small molecule PPTN. Tail vein injection of mice with UDP-glucose induced the recruitment of neutrophils to the renal medulla. This study identifies ICs as novel sensors, mediators and effectors of inflammation in the kidney via P2Y14

Altered V-ATPase expression in renal intercalated cells isolated from B1-subunit deficient mice by fluorescence activated cell sorting

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1(-/-)) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1(-/-) and wild type mice. However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild type and Atp6v1b1(-/-) mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1(+/+) mice) with Atp6v1b1(-/-) mice to generate novel EGFP-B1(-/-) mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1(+/+) and EGFP-B1(-/-) mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly down-regulated in EGFP-B1(-/-) mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions the B2 up-regulation compensates for the lack of B1, and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are down-regulated

P2Y₁₄ activation by UDP-glucose increases ERK1/2-phosphorylation in MDCK-C11 cells.

<p>Representative immunoblots showing triplicates of ERK1/2 phosphorylation (upper lane) versus total ERK1/2 (lower lane) in cells pretreated with vehicle or the P2Y₁₄ antagonist PPTN (10 μM), in the absence (CTRL) or presence of 100 μM UDP-glucose (UDP-glu). Quantification of the ratio of p-ERK/total ERK showed that UDP-glucose induced a significant increase in ERK1/2 phosphorylation (lower left panel, n = 7) and that PPTN prevented the increase in ERK1/2 phosphorylation induced by UDP-glucose (lower right panel, n = 5). Values are represented, relative to either control or PPTN alone, as means ± SEM, * p < 0.005.</p

Immunofluorescence localization of P2Y₁₄ in mouse kidney.

<p>Cortical (A) and medullary (B) sections double-labeled for P2Y₁₄ (green) and the V-ATPase B1 subunit (red). P2Y₁₄ was detected in ICs identified by their positive labeling for the V-ATPase (yellow in the merge panels shown in A and B). No P2Y₁₄ was detected in distal tubule cells, which also express the V-ATPase (red in the merge panel shown in A). The P2Y₁₄ staining was abolished after pre-incubation of the P2Y₁₄ antibody with its immunizing peptide in the cortex (C) and medulla (D). Scale bars = 25 μm.</p

Quantitative PCR detection of pro-inflammatory mediators in EGFP(+) cells.

<p>EGFP(+) cells were isolated by FACS from B1-EGFP mice 4h after an i.v. injection with saline (sham) or with saline containing 100 μM UDP-glucose (UDP-glu). All values are normalized to GAPDH. Data are represented as % changes relative to control. Values are mean ± SEM (n = 4), *P<0.05, ** P<0.001.</p

Expression of P2Y₁₄ in EGFP(+) cells.

<p>(A) Representative immunoblot profile of P2Y₁₄ in two EGFP(+) cell samples isolated by FACS. (B) Binding of [³H]UDP-glucose to total membranes prepared from FACS isolated EGFP(+) and EGFP(−) cells in the presence or absence of a saturating concentration (10^–5 M) of unlabeled UDP-glucose or ATP. Data are represented as fold changes compared to the binding measured in the presence of unlabeled UDP-glucose. Each bar represent the average of 3 independent experiments each performed in triplicate. Values are expressed as mean ± SEM, * P<0.05.</p

P2Y₁₄ expression in MDCK-C11 cells.

<p>(a) RT-PCR analysis of IC markers including the V-ATPase a4 subunit (V0A4), the V-ATPase B1 subunit (V1B1) and AE1, and the principal cell marker aquaporin 2 (AQP2), as well as P2Y₁₄ in MDCK-C11 cells. (b) Representative immunoblots following plasma membrane biotinylation showing cell surface versus total protein expression of the V-ATPase B1 and A subunits, and actin. (c) RT-PCR detection of P2 receptors in MDCK-C11 cells. (d) Immunoblot profile of P2Y₁₄ expression in MDCK-C11. Plasma membrane (left) and total cell expression (right) are represented under control conditions (C) and after treatment with endoglycosydase H (H) and PNGase F (F). (e) X-Z confocal microscopy representation of MDCK-C11 cells grown on filter, showing P2Y₁₄ expression (red). Plasma membrane is labeled with biotin-streptavidin FITC (green). The merge panel shows partial co-localization of P2Y₁₄ with biotin in the apical membrane (orange/yellow) as well as sub-apical localization (red). Scale bars = 4 μm. (f) Concentration-dependent inhibition of [³H]UDP-glucose binding to MDCK-C11 membranes by unlabeled ligands. Membranes (15 μg protein) were incubated for 3 hours at 22C with [³H]UDP-glucose (3 nM) and increasing concentrations of UDP-glucose or ATP. Each point represents the average of 4 independent experiments performed in triplicate. The data are expressed as values relative to the total binding observed in the absence of unlabeled ligand and are corrected for non specific binding determined in the presence of a saturating concentration of UDP-glucose (10 μM).</p