Table 1.
Primers used in qPCR.
Fig 1.
Spectrin and ankyrin mRNA in mouse kidney.
Spectrin and ankyrin mRNA expression were measured by RT-PCR and qPCR with primers summarized in Table 1. (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.
Fig 2.
Spectrin and ankyrin abundance in kidney cortex and medulla.
(A) Western blot analysis of cortical and medullary tissue. Three kidneys were analyzed; samples from two are shown as paired lanes. Aquaporin1 (AQP1) and aquaporin2 (AQP2) were used as regional markers, AQP1 being evenly distributed while AQP2 marked medullary tissue. (B) Immunofluorescent micrographs of renal cortex and medulla stained for the dominant spectrins. IM, inner medulla; OM, outer medulla. While αΙΙ spectrin staining is uniform, the distributions of βΙΙ vs. βΙΙΙ spectrin tend to be complementary, especially in the inner medullary region.
Fig 3.
The beta spectrins favor different renal tubules.
(A) Double immunofluorescent micrographs of spectrin vs. α1-Na,K-ATPase. Spectrin βΙΙ stains both proximal (PT) and (more weakly) distal (DT) tubules, the latter being most prominently stained for α1-Na,K-ATPase. Spectrin βΙΙΙ staining is most prominent in the distal tubules and collecting duct (CD). All of the spectrins mark both the apical as well as basolateral membrane, with additional cytoplasmic staining. (Inserts) Enlarged view demonstrating the divergence of α1-Na,K-ATPsae staining from spectrin at the apical surface. (B) Immunofluorescent micrographs comparing the distribution of aquaporin’s 1 & 2 (AQP1 & 2) with βΙΙ and βΙΙΙ spectrin. βΙΙΙ spectrin is largely confined to the distal convoluted tubule and collecting duct, a pattern is shares with AQP2.
Fig 4.
Spectrins associate with internal organelles.
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.
Fig 5.
Distribution of alpha and beta spectrin.
(A,B)The distribution of spectrins βΙΙ and βΙΙΙ relate to NKCC2 or calbindin1. NKCC2 marks the thick ascending loop of Henle (TAL); calbindin1 marks the distal convoluted tubule (DCT). Note that βΙΙΙ spectrin spares the TAL, but marks the DCT. (C) Co-stains of αΙΙ spectrin (red) with βΙΙ or βΙΙΙ spectrin (green) show that αII spectrin, present throughout the kidney, is complemented by either βΙΙ or βΙΙΙ spectrin.
Fig 6.
Ankyrin B and ankyrin G expression in cortex and medulla.
(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.
Fig 7.
Ankyrin B clusters at the lateral microvillar in-folding of distal tubule cells. Ankyrin G is present at the apical and lateral membrane (LM) and on cytoplasmic vesicles (arrow heads) and near the brush border of proximal tubule cells (arrows). The boxed area of the left panel is shown at higher magnification in the right panel.
Fig 8.
Ankyrin B is expressed in the TAL and DCT.
(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).
Fig 9.
Spectrins and ankyrin distribution in the glomerulus.
ImmunoEM micrographs of spectrin and ankyrin in the glomerulus. Spectrin βΙΙ staining was largely confined to endothelial cells. Spectrin βΙΙΙ was present in both endothelial cells (arrow head) and podocytes (arrow). Ankyrin G largely spares the glomerulus except in the parietal layer of Bowman’s capsule. Ankyrin B was prominent in podocytes.
Fig 10.
Cartoon depicting the distribution of renal spectrin and ankyrin and their relationship to other membrane proteins.
The relative abundance of the proteins studied here is an estimate based on their localization and relative intensity of fluorescent staining. The distribution of the other proteins is derived from the published literature. While a variety of membrane and adapter proteins have been noted to interact with spectrin or ankyrin, no simple mapping of one protein to another is evident. The citations for the depicted distributions were as follows: AQP1,2 [28]; NKCC2 [32, 33]; calbindin1 [31]; protein 4.1 [2]; RhBG [61, 62]; NCX1 [63]; ENaC [64, 65]; IP3R [66, 67]; NHE3 [68]; NHE2 [68]; KCC3 [69]; KCC4 [69]; ClC5 [70]; ROMK [71]; NCC [64]; NCKX3 [72].