Hypertonic stress and cell death. Focus on "Multiple cell death pathways are independently activated by lethal hypertonicity in renal epithelial cells"

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Isoforms of Spectrin and Ankyrin Reflect the Functional Topography of the Mouse Kidney.

The kidney displays specialized regions devoted to filtration, selective reabsorption, and electrolyte and metabolite trafficking. The polarized membrane pumps, channels, and transporters responsible for these functions have been exhaustively studied. Less examined are the contributions of spectrin and its adapter ankyrin to this exquisite functional topography, despite their established contributions in other tissues to cellular organization. We have examined in the rodent kidney the expression and distribution of all spectrins and ankyrins by qPCR, Western blotting, immunofluorescent and immuno electron microscopy. Four of the seven spectrins (αΙΙ, βΙ, βΙΙ, and βΙΙΙ) are expressed in the kidney, as are two of the three ankyrins (G and B). The levels and distribution of these proteins vary widely over the nephron. αΙΙ/βΙΙ is the most abundant spectrin, found in glomerular endothelial cells; on the basolateral membrane and cytoplasmic vesicles in proximal tubule cells and in the thick ascending loop of Henle; and less so in the distal nephron. βΙΙΙ spectrin largely replaces βΙΙ spectrin in podocytes, Bowman's capsule, and throughout the distal tubule and collecting ducts. βΙ spectrin is only marginally expressed; its low abundance hinders a reliable determination of its distribution. Ankyrin G is the most abundant ankyrin, found in capillary endothelial cells and all tubular segments. Ankyrin B populates Bowman's capsule, podocytes, the ascending thick loop of Henle, and the distal convoluted tubule. Comparison to the distribution of renal protein 4.1 isoforms and various membrane proteins indicates a complex relationship between the spectrin scaffold, its adapters, and various membrane proteins. While some proteins (e.g. ankyrin B, βΙΙΙ spectrin, and aquaporin 2) tend to share a similar distribution, there is no simple mapping of different spectrins or ankyrins to most membrane proteins. The implications of this data are discussed

Distinct Roles for Basal and Induced COX-2 in Podocyte Injury

Transgenic mice that overexpress cyclooxygenase-2 (COX-2) selectively in podocytes are more susceptible to glomerular injury by adriamycin and puromycin (PAN). To investigate the potential roles of COX-2 metabolites, we studied mice with selective deletion of prostanoid receptors and generated conditionally immortalized podocyte lines from mice with either COX-2 deletion or overexpression. Podocytes that overexpressed COX-2 were virtually indistinguishable from wild-type podocytes but were significantly more sensitive to PAN-induced injury, produced more prostaglandin E2 and thromboxane B2, and had greater expression of prostaglandin E2 receptor subtype 4 (EP4) and thromboxane receptor (TP). Treatment of COX-2-overexpressing podocytes with a TP antagonist reduced apoptosis, but treatment with an EP4 antagonist did not. In contrast, podocytes from COX-2-knockout mice exhibited increased apoptosis, markedly decreased cell adhesion, and prominent stress fibers. In vivo, selective deletion of podocyte EP4 did not alter the increased sensitivity to adriamycin-induced injury observed in mice overexpressing podocyte COX-2. In contrast, genetic deletion of TP in these mice prevented adriamycin-induced injury, with attenuated albuminuria and foot process effacement. These results suggest that basal COX-2 may be important for podocyte survival, but overexpression of podocyte COX-2 increases susceptibility to podocyte injury, which is mediated, in part, by activation of the thromboxane receptor

Ankyrin B and ankyrin G expression in cortex and medulla.

<p>(A&B) Immunofluorescent micrographs of ankyrin and α1-Na,K-ATPase in renal cortex and medulla. Both ankyrin B and G are found on both apical and basolateral membrane of tubule cells. Ankyrin G is widely distributed, but tapers in the medulla and DCT. Ankyrin B is enriched in the medullar kidney, especially in the distal tubule and collecting duct.</p

Ankyrin B is expressed in the TAL and DCT.

<p>(A) Distribution of ankyrin B overlaps that of calbindin1, a marker of the distal convoluted tubule (DCT). (B) Distribution of ankyrin B overlaps that of NKCC2, a marker of the thick ascending loop of Henle (TAL).</p

Spectrin and ankyrin mRNA in mouse kidney.

<p>Spectrin and ankyrin mRNA expression were measured by RT-PCR and qPCR with primers summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.t001" target="_blank">Table 1</a>. (A) Amplimers were detected for spectrins βΙ, βΙΙ, βΙΙΙ, and αΙΙ and for ankyrins R (Ank1), G (Ank2), and B (Ank3). NC is no-RNA control. (B) The levels of the various transcripts varied widely, as measured by qPCR. Relative abundance is presented. Results shown for three separate determinations. Error bars ±1SD from mean. (Inset) RT-PCR detected two alternative transcripts of βΙΙ spectrin (βΙΙΣ1 & βΙΙΣ2), but only one of two potential transcripts of βΙ spectrin.</p

Spectrins associate with internal organelles.

<p>ImmunoEM micrographs highlight a pool of αΙΙ/βΙΙ spectrin in association with a variety of organelles including canaliculi (arrow heads) and coated vesicles (arrows) in the cytoplasm and along the lateral and apical membranes of proximal and distal tubule cells and the collecting duct. The boxed areas are enlarged in the right column. PC, principal cell; IC, intercalated cell.</p