Proteomic analysis in peritoneal dialysis patients with different peritoneal transport characteristics

Peritoneal membranes can be categorized as high, high average, low average, and low transporters, based on the removal or transport rate of solutes. In this study, we used proteomic analysis to determine the differences in proteins removed by different types of peritoneal membranes. Peritoneal transport characteristics in patients who received peritoneal dialysis therapy were assessed by a peritoneal equilibration test. Two-dimensional differential gel electrophoresis technology followed by quantitative analysis was performed to study the variation in protein expression from peritoneal dialysis effluents (PDE) among different groups. Proteins were identified by MALDI-TOF-MS/MS analyses. Further validation in PDE or serum was performed utilizing ELISA analysis. Proteomics analysis revealed ten protein spots with significant differences in intensity levels among different groups, including vitamin D-binding protein, complement C3, apolipoprotein-A1, complement factor C4A, haptoglobin, alpha-1 antitrypsin, immunoglobulin kappa light chain, alpha-2-microglobulin, retinol-binding protein 4 and transthyretin. The levels of vitamin D-binding protein, complement C3, and apolipoprotein-A1 in PDE derived from different groups were greatly varied (P < 0.05). However, no significant difference was found in the serum levels of these proteins among different groups (P > 0.05 for all groups). This study provides a novel overview of the differences in PDE proteomes of four types of peritoneal membranes. Vitamin D-binding protein, complement C3, and apolipoprotein-A1 showed enhanced expression in PDE of patients with high transporter.     

Aquaporin-1 in the peritoneal membrane: Implications for water transport across capillaries and peritoneal dialysis

Peritoneal dialysis (PD) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced in the peritoneal cavity. The dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Computer simulations predicted that the membrane contains ultrasmall pores (radius < 3 A) responsible for the transport of solute-free water across the capillary endothelium during crystalloid osmosis. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water perfectly fit with the characteristics of the ultrasmall pore. Treatment with corticosteroids induces the expression of AQP1 in peritoneal capillaries and increases water permeability and ultrafiltration in rats, without affecting the osmotic gradient and the permeability for small solutes. Studies in knockout mice provided further evidence that osmotically-driven water transport across the peritoneal membrane is mediated by AQP1. AQP1 and endothelial NO synthase (eNOS) show a distinct regulation within the endothelium lining peritoneal capillaries. In acute peritonitis, the upregulation of eNOS and increased release of NO dissipate the osmotic gradient and result in ultrafiltration failure, despite the unchanged expression of AQP1. These data illustrate the potential of the peritoneal membrane to investigate the role and regulation of AQP1 in the endothelium. They also emphasize the critical role of AQP1 during peritoneal dialysis and suggest that manipulating AQP1 expression may be used to increase water permeability across the peritoneal membrane.     

Proteomics analysis of the peritoneal dialysate effluent reveals the presence of calcium-regulation proteins and acute inflammatory response

Background

Peritoneal dialysis (PD) is a form of renal replacement used for advanced chronic kidney disease. PD effluent holds a great potential for biomarker discovery for diagnosis and prognosis. In this study a novel approach to unravelling the proteome of PD effluent based-on dithiothreitol depletion followed by 2D-SDS-PAGE and protein identification using tandem mass spectrometry is proposed.

Results

A total of 49 spots were analysed revealing 25 proteins differentially expressed, among them many proteins involved in calcium regulation.

Conclusions

Remarkably, a group of proteins dealing with calcium metabolism and calcium regulation has been found to be lost through peritoneal dialysate effluent, giving thus a potential explanation to the calcification of soft tissues in patients subjected to peritoneal dialysis and kidney injury. Comparison of literature dealing with PD is difficult due to differences in sample treatment and analytical methodologies.     

An experimental, non-uremic rabbit model of peritoneal dialysis

Peritoneal dialysis (PD) is a well established method of depuration in uremic patients. Standard dialysis solutions currently in use are not biocompatible with the peritoneal membrane. Studying effects of dialysate on peritoneal membrane in humans is still a challenge. There is no consensus on the ideal experimental model so far. We, therefore, wanted to develop a new experimental non-uremic rabbit model of peritoneal dialysis, which would be practical, easy to conduct, not too costly, and convenient to investigate the long-term effect of dialysis fluids. The study was done on 17 healthy Chinchilla male and female rabbits, anesthetized with Thiopental in a dose of 0.5 mg/kg body mass. A catheter, specially made from Tro-soluset (Troge Medical GMBH, Hamburg, Germany) infusion system, was then surgically inserted and tunneled from animals' abdomen to their neck. The planned experimental procedure was 4 weeks of peritoneal dialysate instillation. The presented non-uremic rabbit model of peritoneal dialysis is relatively inexpensive, does not require sophisticated technology and was well tolerated by the animals. Complications such as peritonitis, dialysis fluid leakage, constipation and catheter obstruction were negligible. This model is reproducible and can be used to analyze the effects of different dialysis solutions on the rabbit peritoneal membrane.     

Aquaporin-1 in the peritoneal membrane: Implications for water transport across capillaries and peritoneal dialysis

Peritoneal dialysis (PD) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced in the peritoneal cavity. The dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Computer simulations predicted that the membrane contains ultrasmall pores (radius < 3 Å) responsible for the transport of solute-free water across the capillary endothelium during crystalloid osmosis. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water perfectly fit with the characteristics of the ultrasmall pore. Treatment with corticosteroids induces the expression of AQP1 in peritoneal capillaries and increases water permeability and ultrafiltration in rats, without affecting the osmotic gradient and the permeability for small solutes. Studies in knockout mice provided further evidence that osmotically-driven water transport across the peritoneal membrane is mediated by AQP1. AQP1 and endothelial NO synthase (eNOS) show a distinct regulation within the endothelium lining peritoneal capillaries. In acute peritonitis, the upregulation of eNOS and increased release of NO dissipate the osmotic gradient and result in ultrafiltration failure, despite the unchanged expression of AQP1. These data illustrate the potential of the peritoneal membrane to investigate the role and regulation of AQP1 in the endothelium. They also emphasize the critical role of AQP1 during peritoneal dialysis and suggest that manipulating AQP1 expression may be used to increase water permeability across the peritoneal membrane.     

Detection of Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, in dialysis fluid of patients with uremia

In order to estimate the exposure levels of mutagenic and carcinogenic heterocyclic amines in humans, we developed a high-performance liquid chromatography method to detect 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in dialysis fluid of patients with uremia. Using this methods, dialysis fluid of 12 patients who had received hemodialysis treatment or continuous ambulatory peritoneal dialysis was examined. Trp-P-1 was detected in dialysate of all uremic patients (727 +/- 282 pmoles, n = 12). In patients who had been treated with continuous ambulatory peritoneal dialysis, the average amount of Trp-P-1 found in whole dialysate (6 l) per day was 710 +/- 203 pmoles (mean +/- S.D., n = 8). Moreover, Trp-P-2 could be detected in 5 out of 12 patients (206 +/- 85 pmoles, n = 5). These results indicate that patients with uremia are actually exposed to carcinogenic tryptophan pyrolysis products. The average exposure level of Trp-P-1 in uremic patients apparently exceeded 710 pmoles (150 ng) per day.     

Aquaporin-1 in the peritoneal membrane: implications for peritoneal dialysis and endothelial cell function

PD (peritoneal dialysis) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced into the peritoneal cavity. The dialysis process involves diffusive and convective transports and osmosis through the PM (peritoneal membrane). Computer simulations predicted that the PM contains ultrasmall pores (radius <3 A, 1 A=10(-10) m), responsible for up to 50% of UF (ultrafiltration), i.e. the osmotically driven water movement during PD. Several lines of evidence suggest that AQP1 (aquaporin-1) is the ultrasmall pore responsible for transcellular water permeability during PD. Treatment with corticosteroids induces the expression of AQP1 in the PM and improves water permeability and UF in rats without affecting the osmotic gradient and permeability for small solutes. Studies in knockout mice provided further evidence that osmotically driven water transport across the PM is mediated by AQP1. AQP1 and eNOS (endothelial nitric oxide synthase) show a distinct regulation within the endothelium lining the peritoneal capillaries. In acute peritonitis, the up-regulation of eNOS and increased release of nitric oxide dissipate the osmotic gradient and prevent UF, whereas AQP1 expression is unchanged. These results illustrate the usefulness of the PM to investigate the role and regulation of AQP1 in the endothelium. The results also emphasize the critical role of AQP1 during PD and suggest that manipulation of AQP1 expression may be used to increase water permeability across the PM.     

Home peritoneal dialysis: 3 years' experience in Toronto.

From November 1972 to November 1975, 52 males and 39 females aged 11 to 71 years were trained for home peritoneal dialysis. Dialysis was performed through a permanent catheter 4 nights a week. The first 11 patients used the manual system, exchanging 2 / of dialysate solution every 50 to 60 minutes. Subsequently 73 patients used the automatic cycler and commercially available dialysate and 7 patients used Tenckhoff''s reverse osmosis peritoneal dialysis machine. The average duration of training was 15, 11.6 and 15 dialysis days, respectively, for the three methods. For the 83 patients followed up, the average duration of home dialysis was 8.3 months (range, 0.5 to 33 months); the total number of dialyses at home was 10 571. Ten received a transplant, 20 were transferred to hospital peritoneal dialysis or hemodialysis, 8 died and 48 continued with home dialysis. Twenty-three patients had a total of 33 episodes of peritonitis, an incidence of 27.7% among the patients in the program for up to 3 years or 0.3% among all the dialyses. By November 1975, 46 patients had returned to their predialysis lifestyle, 18 were working part-time, 10 were able to work but were not doing so, and 9 were unable to work or care for themselves.     

Intraperitoneal insulin in uraemic diabetics undergoing continuous ambulatory peritoneal dialysis.

The kinetics of absorption of intraperitoneally administered insulin were studied in nine uraemic insulin-dependent diabetics undergoing continuous ambulatory peritoneal dialysis (CAPD). In each of three studies 20 U of regular insulin was directly injected as a bolus into the peritoneal cavity through an indwelling Tenckhoff catheter. In two procedures the insulin injection was followed by the instillation of either 2 litres of 1.5% dextrose dialysates or 2 litres of 4.5% dextrose dialysate. In the third 20 ml of saline was used to flush the tubing. Plasma free insulin values rose more rapidly and reached significantly higher concentrations (55.6 +/- 18.8 mU/l) when the insulin had been injected into an empty peritoneal cavity than when it was followed by dialysate. These differences were observed despite the fact that most of the insulin injected was retained by the patients. Since the plasma insulin values did not differ after instillations of dialysate containing 1.5% and 4.5% dextrose, the osmolality of the dialysate seemed not to affect insulin absorption, and the dilution of the insulin probably delayed its transfer through the peritoneum. These findings suggest that insulin given intraperitoneally to patients undergoing CAPD will be most effective if it is given into an empty peritoneal cavity at least 30 minutes before the dialysate is instilled.     

Effect of glucose concentration on peritoneal inflammatory cytokines in continuous ambulatory peritoneal dialysis patients

OBJECTIVE: It is known that glucose concentrations of peritoneal dialysis solutions are detrimental to the peritoneal membrane. In order to determine the effect of glucose concentration on cytokine levels of peritoneal fluid of continuous ambulatory peritoneal dialysis (CAPD) patients, a cross-sectional study was performed. METHODS: Nine non-diabetic CAPD patients participated in two 8-h dwell sessions of overnight exchanges in consecutive days, with 1.36% and 3.86% glucose containing peritoneal dialysis solutions (Baxter-Eczacibas). Peritoneal dialysis fluid tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 levels were measured. RESULTS: TNF-alpha levels after 1.36% and 3.86% glucose used dwells were 23+/-14 pg/ml and 28+/-4 pg/ml, respectively (p=0.78). The IL-6 levels were 106+/-57 pg/ml and 115+/-63 pg/ml (p=0.81), respectively. CONCLUSION: In our in vivo study we found that the glucose concentration of the conventional lactate-based CAPD solution has no effect on basal IL-6 and TNF-alpha levels of peritoneal fluid. Further in vivo studies with non-lactate-based CAPD solutions are needed in order to determine the effect of glucose concentration per se on cytokine release.     

MMP-7 affects peritoneal ultrafiltration associated with elevated aquaporin-1 expression via MAPK/ERK pathway in peritoneal mesothelial cells

Peritoneal membrane dysfunction and the resulting ultrafiltration failure are the major disadvantages of long-term peritoneal dialysis (PD). It becomes increasingly clear that mesothelial cells play a vital role in the pathophysiological changes of the peritoneal membrane. Matrix metalloproteinases (MMPs) function in the extracellular environment of cells and mediate extracellular matrix turnover during peritoneal membrane homeostasis. We showed here that dialysate MMP-7 levels markedly increased in the patients with PD, and the elevated MMP-7 level was negatively associated with peritoneal ultrafiltration volume. Interestingly, MMP-7 could regulate the cell osmotic pressure and volume of human peritoneal mesothelial cells. Moreover, we provided the evidence that MMP-7 activated mitogen-activated protein kinases (MAPKs)-extracellular signal-regulated kinase 1/2 (ERK) pathway and subsequently promoted the expression of aquaporin-1 (AQP-1) resulting in the change of cell osmotic pressure. Using a specific inhibitor of ERK pathway abrogated the MMP-7-mediating AQP-1 up-regulation and cellular homeostasis. In summary, all the findings indicate that MMP-7 could modulate the activity of peritoneal cavity during PD, and dialysate MMP-7 might be a non-invasive biomarker and an alternative therapeutic target for PD patients with ultrafiltration failure.     

Proteomics-based analysis of potential therapeutic targets in patients with peritoneal dialysis-associated peritonitis

BackgroundPeritoneal dialysis-associated peritonitis (PDAP) is the most common complication in peritoneal dialysis patients. We propose screening for characteristic expressed proteins in the dialysate of PDAP patients to provide clues for the diagnosis of PDAP and its therapeutic targets.MethodsDialysate samples were collected from patients with a first diagnosis of PDAP (n = 15) and from patients who had not experienced peritonitis (Control, n = 15). Data-independent acquisition (DIA) proteomic analysis was used to screen for differentially expressed proteins (DEPs). Co-expression networks were constructed via weighted gene co-expression network analysis (WGCNA) for detection of gene modules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for functional annotation of DEPs and gene modules. Hub proteins were validated using the parallel reaction monitoring (PRM) method.ResultsA total of 142 DEPs in the dialysate of PDAP patients were identified. 70 proteins were upregulated and 72 proteins were downregulated. GO and KEGG analysis showed that DEPs were mainly enriched in cell metabolism, glycolysis/glycogenesis and hypoxia-inducible factor-1 signaling pathway. Subsequently, a co-expression network was constructed and four gene modules were detected. Myeloperoxidase (MPO) and myeloperoxidase (HP) were the key proteins of the blue and turquoise modules, respectively. Additionally, PRM analysis showed that the expression of MPO and HP was significantly upregulated in the PDAP group compared to the non-peritonitis group, which was consistent with our proteomics data.ConclusionMPO and HP were differentially expressed in the dialysate of PDAP patients and may be potential diagnostic and therapeutic targets for PDAP.     

Large-scale phosphoproteomic analysis of membrane proteins in renal proximal and distal tubule

Recent advances in mass spectrometry (MS) have provided means for large-scale phosphoproteomic profiling of specific tissues. Here, we report results from large-scale tandem MS [liquid chromatography (LC)-MS/MS]-based phosphoproteomic profiling of biochemically isolated membranes from the renal cortex, with focus on transporters and regulatory proteins. Data sets were filtered (by target-decoy analysis) to limit false-positive identifications to <2%. A total of 7,125 unique nonphosphorylated and 743 unique phosphorylated peptides were identified. Among the phosphopeptides identified were sites on transporter proteins, i.e., solute carrier (Slc, n = 63), ATP-binding cassette (Abc, n = 4), and aquaporin (Aqp, n = 3) family proteins. Database searches reveal that a majority of the phosphorylation sites identified in transporter proteins were previously unreported. Most of the Slc family proteins are apical or basolateral transporters expressed in proximal tubule cells, including proteins known to mediate transport of glucose, amino acids, organic ions, and inorganic ions. In addition, we identified potentially important phosphorylation sites for transport proteins from distal nephron segments, including the bumetanide-sensitive Na-K-2Cl cotransporter (Slc12a1 or NKCC2) at Ser(87), Thr(101), and Ser(126) and the thiazide-sensitive Na-Cl cotransporter (Slc12a3 or NCC) at Ser(71) and Ser(124). A subset of phosphorylation sites in regulatory proteins coincided with known functional motifs, suggesting specific regulatory roles. An online database from this study (http://dir.nhlbi.nih.gov/papers/lkem/rcmpd/) provides a resource for future studies of transporter regulation.     

Liposome supported peritoneal dialysis in rat amitriptyline exposure with and without intravenous lipid emulsion

Liposome supported peritoneal dialysis is a recently described technique which may eventually be applicable in the clinical scenario of the intoxicated patient. We evaluated the hypothesis that intravenous injection of lipid emulsion (ILE) would augment acidic pH gradient liposome supported peritoneal dialysis (LSPD). Orogastrically amitriptyline dosed rats were treated with either Sodium bicarbonate (NaHCO3) intravenously and standard intraperitoneal dialysate (Group A); NaHCO3 intravenously and LSPD (Group B); or ILE and LSPD (Group C). The primary endpoint was dialysate amitriptyline concentration after a 60?min dwell. Secondary analysis included an estimate of extraction ratio for peritoneal blood flow (ERs). There were significantly higher intraperitoneal concentrations of amitriptyline and ERs in the two groups treated with LSPD (Group B, p?=?0.02, Group C, p?<?0.01 vs. Group A). There was no observed effect for ILE on intraperitoneal amitriptyline concentration or ERs (p?>?0.20). LSPD increased the amitriptyline concentration in peritoneal dialysate. No further increase was demonstrated with ILE. This may be either because such an effect is absent, or type II error. Exploratory analysis suggests LSPD may be driven by total rather than free drug concentrations.     

Experimental study of chronic ambulatory peritoneal dialysis. I. Inhibition of protein and amino acid losses by single amino acids

A successful dog model of the continuous ambulatory peritoneal dialysis patient was developed. These preparations were employed in initial studies of the effects of single amino acid-containing dialysis solutions on the losses of protein and amino acids into the dialysate. A decrease of about 40% in the loss of total amino acids into the dialysate was observed when DL-serine-containing dialysis solutions were employed. The addition of DL-serine, L-lysine, or DL-alanine to the dialysis solutions diminished protein loss into the dialysate by 27-55%. DL-Glutamic acid and DL-aspartic acid were ineffective in this regard.     

Calcitriol decreases TGF-β1 and angiotensin II production and protects against chlorhexide digluconate-induced liver peritoneal fibrosis in rats

Peritoneal fibrosis is a major complication of peritoneal dialysis that can lead to ultrafiltration failure. This study investigates the protective effects of calcitriol on chlorhexidine digluconate-induced peritoneal fibrosis in rats. Peritoneal fibrosis was induced in Sprague-Dawley rats by daily administration of 0.5 mL 0.1% chlorhexidine digluconate in normal saline via peritoneal dialysis for 1 week. Rats received daily intravenous injections of calcitriol (low-dose, 10 ng/kg; or high-dose, 100 ng/kg) for 1 week. After 7 days, conventional 4.25% Dianeal (30 mL) was administered via peritoneal dialysis over 4 h. Peritoneal solute transport was calculated from the dialysate concentration relative to its concentration in the initial infused dialysis solution (D₄/D_{0 glucose}) for glucose, and the dialysate-to-plasma concentration ratio (D₄/P_{4 urea}) at 4 h for urea. Rats were then sacrificed and the liver peritoneum was harvested for immunohistochemical analysis via microscopy. After dialysis, the D₄/P_{4 Urea} level was reduced; increases were observed in the D₄/D_{0 glucose} level and the levels of active transforming growth factor-β1 and angiotensin II in serum and dialysate; the liver peritoneum and muscle peritoneum was markedly thickened, and the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and phosphorylated Smad2/3 (P-Smad2/3)-positive cells in the liver peritoneum was elevated in the peritoneal fibrosis group compared with the vehicle group. Calcitriol decreased the serum and dialysate active transforming growth factor-β1 and angiotensin II level, decreased the thickness of the liver peritoneum and muscle peritoneum, and decreased the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and P-Smad2/3-positive cells in liver peritoneum cells. High-dose calcitriol exhibited better protective effects against peritoneal fibrosis than did the lower dose. Calcitriol protected against chlorhexidine digluconate-induced peritoneal fibrosis in rats by decreasing transforming growth factor-β1 and angiotensin II production.     

Expression and activity of cGMP-dependent phosphodiesterases is up-regulated by lipopolysaccharide (LPS) in rat peritoneal macrophages

It has been shown that cyclic GMP (cGMP) modulates the inflammatory responses of macrophages, but the underlying molecular mechanisms are still poorly understood. Looking for proteins potentially regulated by cGMP in rat peritoneal macrophages (PMs), in this study we analyzed expression and activity of cGMP-hydrolyzing and cGMP-regulated phosphodiesterases (PDEs). It was found that freshly isolated peritoneal exudate macrophages (PEMs) express enzymes belonging to families PDE1-3, PDE5, PDE10, and PDE11. Analysis of substrate specificity, sensitivity to inhibitors, and subcellular localization showed that PDE2 and PDE3 are the main cGMP-regulated PDE isoforms in PEMs. The profile of PDE expression was altered by maintaining PEMs in culture and treatment with bacterial endotoxin (LPS). After 24 h culture, PDE5 was not present and the levels of PDE2, PDE3, and PDE11 were markedly decreased. However, their expression and activity was recovered after treatment of cultured cells with LPS. A similar pattern of changes was observed for the expression of TNFalpha, but not for guanylyl cyclase A (GC-A). LPS up-regulated PDE expression also in resident peritoneal macrophages (RPMs), although not all PDEs present in PEMs were detected in RPMs. Taken together, our results show that in rat PMs expression of cGMP-dependent PDEs positively correlates with the activation state of cells. Moreover, the fact that most of these PDEs hydrolyze also cAMP indicates that cGMP can play a role of potent regulator of cAMP signaling in macrophages.     

Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct

In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane "raft" proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n = 22) or immunoblotting (n = 6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.