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We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after ∼140 min) into the right jugular vein, and the fluorescence signal (at 650-670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30-40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 ± 0.26, mean ± SE, n = 10), indicating a "priming," or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 ± 0.07, n = 6; P < 0.01). Cy2-or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand β2-glycoprotein. Copyright © 2009 the American Physiological Society.

Evidence is accumulating that extracellular nucleotides act as autocrine/paracrine agents in most tissues, including the kidneys. Several families of surface-located enzymes, collectively known as ectonucleotidases, can degrade nucleotides. Using immunohistochemistry, we have examined the segmental distribution of five ectonucleotidases along the rat nephron. Perfusion-fixed kidneys were obtained from anesthetized male Sprague-Dawley rats. Cryostat sections of cortical and medullary regions were incubated with antibodies specific to the following enzymes: ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1, NTPDase2, NTPDase3, ectonucleotide pyrophosphatase phosphodiesterase 3 (NPP3), and ecto-5′-nucleotidase. Sections were then costained with Phaseolus vulgaris erythroagglutinin (for identification of proximal tubules) and antibodies against Tamm-Horsfall protein (for identification of thick ascending limb), calbindin-D28k(for identification of distal tubule), and aquaporin-2 (for identification of collecting duct). The tyramide signal amplification method was used when the ectonucleotidase and marker antibody were raised in the same species. The glomerulus expressed NTPDase1 and NPP3. The proximal tubule showed prominent expression of NPP3 and ecto-5′-nucleotidase in most, but not all, segments. NTPDase2 and NTPDase3, but not NPP3 or ecto-5′-nucleotidase, were expressed in the thick ascending limb and distal tubule. NTPDase3, with some low-level expression of ecto-5′-nucleotidase, was also found in cortical and outer medullary collecting ducts. Inner medullary collecting ducts displayed low-level staining for NTPDase1, NTPDase2, NTPDase3, and ecto-5′-nucleotidase. We conclude that these ectonucleotidases are differentially expressed along the nephron and may play a key role in activation of purinoceptors by nucleotides and nucleosides.

Proteomics is a promising new tool for functional genomics. In addition to two-dimensional gel electrophoresis, other methods that are based on liquid chromatography and mass spectrometry are now available to study proteins. In this brief article, we review the strengths and limitations of the proteomic approaches currently available to the researcher, and we provide examples of how proteomics has been, and can in the future be, used to study the kidney.

To test the proposal that the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) confers aldosterone specificity on mineralocorticoid receptors in the distal nephron by inactivating glucocorticoids, we performed a free-flow micropuncture study of distal tubular function in adrenalectomized rats infused with high-dose corticosterone. One-half of the rats were additionally given intravenous carbenoxolone (CBX; 6 mg/h) to inhibit renal 11β-HSD activity. Although this maneuver lowered fractional Na+excretion (1.1 ± 0.2 vs. 1.9 ± 0.2%, P < 0.01), Na+reabsorption within the accessible distal tubule was found to be similar in the two groups of animals. In contrast, distal tubular K+secretion was enhanced in CBX-treated rats: fractional K+deliveries to the early and late distal collection sites in the corticosterone-alone group were 13 ± 1 and 20 ± 3%, respectively (not significant), whereas corresponding data in the CBX-treated group were 9 ± 1 and 24 ± 2% ( P < 0.01). This stimulation of distal K+secretion provides the first direct in vivo evidence that 11β-HSD normally prevents corticosterone from exerting a mineralocorticoid-like effect in the distal tubule. The reduction in fractional Na+excretion during inhibition of 11β-HSD, in the absence of a change in end-distal Na+delivery, suggests enhanced Na+reabsorption in the collecting ducts.

P2 receptors have been identified in rat kidney by autoradiography, using the radioligand [3H]α,β-methylene ATP, and by immunohistochemistry, using a polyclonal antibody to the P2X1 purinoceptor. They have been localized to the vascular smooth muscle of intrarenal arteries, including arcuate and interlobular arteries, and afferent arterioles, but not glomeruli, postglomerular efferent arterioles, or renal tubules. We conclude that at least some of the P2 receptors present on vascular smooth muscle are of the P2X1 subtype. The functional significance of these findings in the vascular control of the kidney is discussed.

Micropuncture techniques were used to investigate the nephron site(s) responsible for the vasopressin-induced reductions in lithium clearance and fractional lithium excretion (FELi) in anesthetized Brattleboro rats lacking endogenous vasopressin. In rats treated intravenously with the vasopressin analogue 1-desamino-8-D-arginine vasopressin (DDAVP; 40 pg/min), FELi was significantly lower than in untreated animals (0.23 +/- 0.01 vs. 0.28 +/- 0.02, P < 0.05). Free-flow micropuncture showed that fractional lithium delivery (FDLi) to late proximal convolutions was identical in the two groups, whereas at the early distal tubule both FDLi (0.28 +/- 0.02 vs. 0.33 +/- 0.01, P < 0.05) and the tubular fluid-to-plasma lithium concentration ratio (1.18 +/- 0.04 vs. 1.36 +/- 0.06, P < 0.05) were lower in the DDAVP-treated group. No differences between the groups with respect to lithium handling beyond the early distal tubule were observed. In rats subjected to in vivo microperfusion of loops of Henle, intravenous infusion of DDAVP (40 pg/min) increased fractional lithium reabsorption in the loop from 0.56 +/- 0.03 to 0.66 +/- 0.04 (P < 0.05) and from 0.39 +/- 0.02 to 0.45 +/- 0.02 (P < 0.05) at perfusion rates of 10 and 15 nl/min, respectively. We conclude that DDAVP stimulates lithium reabsorption in the loop of Henle and suggest that this results from an increased transepithelial potential difference in the thick ascending limb.

The contribution of the loop segments to lithium reabsorption in sodium-replete, anesthetized Sprague-Dawley rats was examined by perfusing superficial loops of Henle between late proximal convolutions and early distal tubules. Preliminary experiments in which lithium was initially present only inside or outside the perfused loop confirmed substantial permeability of one or more of the loop segments to lithium. In subsequent experiments, in which lithium was infused intravenously and included in the perfusate so that the perfusate-to-plasma lithium concentration ratio was close to that found in late proximal tubules during lithium clearance studies, lithium reabsorption was inversely related to the perfusion rate: values for fractional lithium reabsorption (FRLi) at perfusion rates of 10, 20, and 30 nl/min were 58 +/- 3, 17 +/- 2, and 2 +/- 2%, respectively. Bumetanide (10(-6) M) markedly inhibited FRLi but also reduced water reabsorption, suggesting an effect on the pars recta at this dose; 10(-7) M bumetanide, which was without effect on water reabsorption, had only a small effect on FRLi at a perfusion rate of 10 nl/min but reduced FRLi by approximately 70% at 20 nl/min. We argue that the remarkable flow dependency of lithium reabsorption, together with its bumetanide sensitivity, provides evidence for significant voltage-driven lithium reabsorption in the thick ascending limb of Henle.