Understanding renal phosphate handling: unfinished business.

PURPOSE OF REVIEW: The purpose of this review is to highlight the publications from the prior 12-18 months that have contributed significant advances in the field of renal phosphate handling. RECENT FINDINGS: The discoveries include new mechanisms for the trafficking and expression of the sodium phosphate cotransporters; direct link between phosphate uptake and intracellular metabolic pathways; interdependence between proximal tubule transporters; and the persistent renal expression of phosphate transporters in chronic kidney disease. SUMMARY: Discovery of new mechanisms for trafficking and regulation of expression of phosphate transporters suggest new targets for the therapy of disorders of phosphate homeostasis. Demonstration of stimulation of glycolysis by phosphate transported into a proximal tubule cell expands the scope of function for the type IIa sodium phosphate transporter from merely a mechanism to reclaim filtered phosphate to a regulator of cell metabolism. This observation opens the door to new therapies for preserving kidney function through alteration in transport. The evidence for persistence of active renal phosphate transport even with chronic kidney disease upends our assumptions of how expression of these transporters is regulated, suggests the possibility of alternative functions for the transporters, and raises the possibility of new therapies for phosphate retention.

Analysis of sex difference in the tubular reabsorption of lithium in rats.

Lithium is used in the treatment of bipolar disorder. We previously demonstrated that two types of transporters mediate the tubular reabsorption of lithium in rats, and suggested that sodium-dependent phosphate transporters play a role in lithium reabsorption with high affinity. In the present study, we examined sex differences in lithium reabsorption in rats. When lithium chloride was infused at 60 µg/min, creatinine clearance and the renal clearance of lithium were lower, and the plasma concentration of lithium was higher in female rats. These values reflected the higher fractional reabsorption of lithium in female rats. In rats infused with lithium chloride at 6 µg/min, the pharmacokinetic parameters of lithium examined were all similar in both sexes. The fractional reabsorption of lithium was decreased by foscarnet, a representative inhibitor of sodium-dependent phosphate transporters, in male and female rats when lithium chloride was infused at the low rate. Among the candidate transporters mediating lithium reabsorption examined herein, the mRNA expression of only PiT2, a sodium-dependent phosphate transporter, exhibited sexual dimorphism. The present results demonstrated sex differences in the tubular reabsorption of lithium with low affinity in rats.

Novel mineral regulatory pathways in ovine pregnancy: I. phosphate, klotho signaling, and sodium-dependent phosphate transporters.

Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n = 3-14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P < 0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1-4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P < 0.05). Gestational day influenced endometrial expression of FGF7 (P < 0.001), and placental expression of FGF23 (P < 0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal-conceptus interface.

Modulation of Intestinal Phosphate Transport in Young Goats Fed a Low Phosphorus Diet.

The intestinal absorption of phosphate (Pi) takes place transcellularly through the active NaPi-cotransporters type IIb (NaPiIIb) and III (PiT1 and PiT2) and paracellularly by diffusion through tight junction (TJ) proteins. The localisation along the intestines and the regulation of Pi absorption differ between species and are not fully understood. It is known that 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) and phosphorus (P) depletion modulate intestinal Pi absorption in vertebrates in different ways. In addition to the apical uptake into the enterocytes, there are uncertainties regarding the basolateral excretion of Pi. Functional ex vivo experiments in Ussing chambers and molecular studies of small intestinal epithelia were carried out on P-deficient goats in order to elucidate the transepithelial Pi route in the intestine as well as the underlying mechanisms of its regulation and the proteins, which may be involved. The dietary P reduction had no effect on the duodenal and ileal Pi transport rate in growing goats. The ileal PiT1 and PiT2 mRNA expressions increased significantly, while the ileal PiT1 protein expression, the mid jejunal claudin-2 mRNA expression and the serum 1,25-(OH)2D3 levels were significantly reduced. These results advance the state of knowledge concerning the complex mechanisms of the Pi homeostasis in vertebrates.

Isolation and Characterization of Three Sodium-Phosphate Cotransporter Genes and Their Transcriptional Regulation in the Grass Carp Ctenopharyngodon idella.

It is important to explore the regulatory mechanism of phosphorus homeostasis in fish, which help avoid the risk of P toxicity and prevent P pollution in aquatic environment. The present study obtained the full-length cDNA sequences and the promoters of three SLC20 members (slc20a1a, slc20a1b and slc20a2) from grass carp Ctenopharyngodon idella, and explored their responses to inorganic phosphorus (Pi). Grass carp SLC20s proteins possessed conservative domains and amino acid sites relevant with phosphorus transport. The mRNAs of three slc20s appeared in the nine tissues, but their expression levels were tissue-dependent. The binding sites of three transcription factors (SREBP1, NRF2 and VDR) were predicted on the slc20s promoters. The mutation and EMSA analysis indicated that: (1) SREBP1 binding site (-783/-771 bp) negatively but VDR (-260/-253 bp) binding site positively regulated the activities of slc20a1a promoter; (2) SREBP1 (-1187/-1178 bp), NRF2 (-572/-561 bp) and VDR(615/-609 bp) binding sites positively regulated the activities of slc20a1b promoter; (3) SREBP1 (-987/-977 bp), NRF2 (-1469/-1459 bp) and VDR (-1124/-1117 bp) binding sites positively regulated the activities of the slc20a2 promoter. Moreover, Pi incubation significantly reduced the activities of three slc20s promoters, and Pi-induced transcriptional inactivation of slc20s promoters abolished after the mutation of the VDR element but not SREBP1 and NRF2 elements. Pi incubation down-regulated the mRNA levels of three slc20s. For the first time, our study elucidated the transcriptional regulatory mechanisms of SLC20s and their responses to Pi, which offered new insights into the Pi homeostatic regulation and provided the basis for reducing phosphorus discharge into the waters.

Potential interaction of inflammatory hyperemia and hyperphosphatemia in tumorigenesis.

The present article proposes that the association of inflammation with cancer is potentially mediated by the interaction of inflammatory hyperemia and hyperphosphatemia. Hyperemia increases blood flow rate and blood volume, and hyperphosphatemia is caused by elevated serum levels of dysregulated inorganic phosphate. It is hypothesized that the interaction of inflammatory hyperemia and hyperphosphatemia circulates increased amounts of inorganic phosphate to the tumor microenvironment, where increased uptake of inorganic phosphate through sodium-phosphate cotransporters is sequestered in cells. Elevated levels of intracellular phosphorus increase biosynthesis of ribosomal RNA, leading to increased protein synthesis that supports tumor growth. The present article also proposes that the interaction of inflammatory hyperemia and hyperphosphatemia may help explain a chemopreventive mechanism associated with NSAIDs.

Tissue-Wide Gene Expression Analysis of Sodium/Phosphate Co-Transporters in Pigs.

Sodium/phosphate co-transporters are considered to be important mediators of phosphorus (P) homeostasis. The expression of specific sodium/phosphate co-transporters is routinely used as an immediate response to dietary interventions in different species. However, a general understanding of their tissue-specificity is required to elucidate their particular contribution to P homeostasis. In this study, the tissue-wide gene expression status of all currently annotated sodium/phosphate co-transporters were investigated in two pig trials focusing on a standard commercial diet (trial 1) or divergent P-containing diets (trial 2). A wide range of tissues including the gastrointestinal tract (stomach, duodenum, jejunum, ileum, caecum, and colon), kidney, liver, bone, muscle, lung, and aorta were analyzed. Both trials showed consistent patterns in the overall tissue-specific expression of P transporters. While SLC34A2 was considered as the most important intestinal P transporter in other species including humans, SLC34A3 appeared to be the most prominent intestinal P transporter in pigs. In addition, the P transporters of the SLC17 family showed basal expression in the pig intestine and might have a contribution to P homeostasis. The expression patterns observed in the distal colon provide evidence that the large intestine may also be relevant for intestinal P absorption. A low dietary P supply induced higher expressions of SLC20A1, SLC20A2, SLC34A1, and SLC34A3 in the kidney cortex. The results suggest that the expression of genes encoding transcellular P transporters is tissue-specific and responsive to dietary P supply, while underlying regulatory mechanisms require further analyses.

Pyrophosphate Stimulates the Phosphate-Sodium Symporter of Trypanosoma brucei Acidocalcisomes and Saccharomyces cerevisiae Vacuoles.

Inorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPi and other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Pi conductance in Xenopus oocytes expressing a Saccharomyces cerevisiae or Trypanosoma brucei Na+/Pi symporter. PPi is not taken up by Xenopus oocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPi generated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type or pho91Δ yeast strains expressing TbPHO91 but not from the pho91Δ strains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing Pi from S. cerevisiae vacuoles and T. brucei acidocalcisomes.IMPORTANCE Acidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities with S. cerevisiae vacuoles in their structure and composition. Both organelles share a Na+/Pi symporter involved in Pi release to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, at physiological cytosolic concentrations, stimulates the symporter expressed in either Xenopus oocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.

Renal phosphate handling and inherited disorders of phosphate reabsorption: an update.

Renal phosphate handling critically determines plasma phosphate and whole body phosphate levels. Filtered phosphate is mostly reabsorbed by Na+-dependent phosphate transporters located in the brush border membrane of the proximal tubule: NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit-2 (SLC20A2). Here we review new evidence for the role and relevance of these transporters in inherited disorders of renal phosphate handling. The importance of NaPi-IIa and NaPi-IIc for renal phosphate reabsorption and mineral homeostasis has been highlighted by the identification of mutations in these transporters in a subset of patients with infantile idiopathic hypercalcemia and patients with hereditary hypophosphatemic rickets with hypercalciuria. Both diseases are characterized by disturbed calcium homeostasis secondary to elevated 1,25-(OH)2 vitamin D3 as a consequence of hypophosphatemia. In vitro analysis of mutated NaPi-IIa or NaPi-IIc transporters suggests defective trafficking underlying disease in most cases. Monoallelic pathogenic mutations in both SLC34A1 and SLC34A3 appear to be very frequent in the general population and have been associated with kidney stones. Consistent with these findings, results from genome-wide association studies indicate that variants in SLC34A1 are associated with a higher risk to develop kidney stones and chronic kidney disease, but underlying mechanisms have not been addressed to date.

The role of sodium phosphate cotransporters in ectopic calcification.

Phosphate plays a critical role in many vital cellular processes. Deviations from normal serum phosphate levels, including alterations in the extracellular phosphate/pyrophosphate ratio, can cause severe consequences, such as ectopic calcification. Cellular phosphate levels are tightly controlled by sodium phosphate cotransporters, underscoring their importance in cellular physiology. The role of sodium phosphate cotransporters in ectopic calcification requires further elucidation, taking into account their important role in the control of intracellular phosphate levels and the synthesis of ATP, the main source of extracellular pyrophosphate (a potent endogenous inhibitor of calcification). In this review, we discuss the roles of phosphate and pyrophosphate homeostasis in ectopic calcification, with a specific focus on phosphate transporters. We concentrate on the five known sodium-dependent phosphate transporters and review their localisation and regulation by external factors, and the effects observed in knockout studies and in naturally occurring mutations.

5-Diphosphoinositol pentakisphosphate (5-IP7) regulates phosphate release from acidocalcisomes and yeast vacuoles.

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi-loaded giant vacuoles prepared from WT or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ yeast strains or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Nonlinear disposition of lithium in rats and saturation of its tubular reabsorption by the sodium-phosphate cotransporter as a cause.

We previously reported the contribution of sodium-phosphate cotransporter to the tubular reabsorption of lithium in rats. In the present study, the dose dependency of the renal handling of lithium was examined in rats. When lithium chloride at 1.25 mg/kg, 2.5 mg/kg and 25 mg/kg was intravenously injected as a bolus, the areas under the plasma concentration-time curve of lithium until 60 minutes were calculated to be 6.23 mEq·min/l, 8.77 mEq·min/l and 64.6 mEq·min/l, respectively. The renal clearance of lithium and its fractional excretion increased with increments in the dose administered. The renal clearance of lithium strongly correlated with the urinary excretion rate of phosphate in the 1.25 mg/kg group (r = 0.840) and 2.5 mg/kg group (r = 0.773), whereas this correlation was weak in the 25 mg/kg group (r = 0.306). The infusion of foscarnet, a typical inhibitor of sodium-phosphate cotransporter, decreased the fractional reabsorption of lithium in rats administered lithium chloride at 2.5 mg/kg, but did not affect it in rats administered 25 mg/kg. These results demonstrate the nonlinearity of the renal excretion of lithium in rats, with the saturation of lithium reabsorption by the sodium-phosphate cotransporter potentially being involved.

Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer.

Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.

Segmental diversity of phosphate transport along the intestinal axis in horses.

For horses, distinct differences in intestinal phosphate transport have been postulated to account for the unique features of hind gut fermentation compared to other monogastric animals and ruminants. So far published data on mechanisms and underlying transport proteins involved in intestinal phosphate transport in the horse are still missing. Therefore we investigated intestinal phosphate transport in horses at both functional and molecular levels. Segmental diversity of intestinal phosphate transport along the intestinal axis was documented using the Ussing chamber technique. A transcellular phosphate secretion in the jejunum was confirmed. Furthermore, 2 sodium-dependent phosphate cotransporters, NaPiIIb and PiT1, were first detected in the equine intestine at mRNA level with PiT1 being expressed in both the small and large intestine, and NaPiIIb being solely expressed in the large intestine. In the colon, unidirectional net flux rates of phosphate were significantly greater compared to flux rates in other segments ( < 0.005) suggesting the colon as a major site for phosphate absorption in horses. Phosphate transport in the colon was mainly transcellular and mediated by a sodium-gradient as documented by Ussing chamber experiments and uptake of phosphate into colonic brush border membrane vesicles. In summary, the present study demonstrated mechanisms and transporters of intestinal phosphate transport in equine intestinal tissues with distinct differences between intestinal segments providing a new basis for a better understanding of intestinal phosphate transport in horses.

The biochemical characterization of two phosphate transport systems in Phytomonas serpens.

Inorganic phosphate (Pi) is an essential nutrient for all organisms because it is required for a variety of biochemical processes, such as signal transduction and the synthesis of phosphate-containing biomolecules. Assays of 32Pi uptake performed in the absence or in the presence of Na+ indicated the existence of a Na+-dependent and a Na+-independent Pi transporter in Phytomonas serpens. Phylogenetic analysis of two hypothetical protein sequences of Phytomonas (EM1) showed similarities to the high-affinity Pi transporters of Saccharomyces cerevisiae: Pho84, a Na+-independent Pi transporter, and Pho89, a Na+-dependent Pi transporter. Plasma membrane depolarization by FCCP, an H+ ionophore, strongly decreased Pi uptake via both Na+-independent and Na+-dependent carriers, indicating that a membrane potential is essential for Pi influx. In addition, the furosemide-sensitive Na+-pump activity in the cells grown in low Pi conditions was found to be higher than the activity detected in the plasma membrane of cells cultivated at high Pi concentration, suggesting that the up-regulation of the Na+-ATPase pump could be related to the increase of Pi uptake by the Pho89p Na+:Pi symporter. Here we characterize for the first time two inorganic phosphate transporters powered by Na+ and H+ gradients and activated by low Pi availability in the phytopathogen P. serpens.

Absence of renal enlargement in fructose-fed proximal-tubule-select insulin receptor (IR), insulin-like-growth factor receptor (IGF1R) double knockout mice.

The major site of fructose metabolism in the kidney is the proximal tubule (PT). To test whether insulin and/or IGF1 signaling in the PT is involved in renal structural/functional responses to dietary fructose, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) in PT by Cre-lox recombination, using a γ-glutamyl transferase promoter. KO mice had slightly (~10%) reduced body and kidney weights, as well as, a reduction in mean protein-to-DNA ratio in kidney cortex suggesting smaller cell size. Under control diet, IR and IGF1R protein band densities were 30-50% (P < 0.05) lower than WT, and the relative difference was greater in male animals. Male, but not female KO, also had significantly reduced band densities for Akt (protein kinase B), phosphorylated AktT308 and IRY1162/1163 A high-fructose diet (1-month) led to a significant increase in kidney weight in WT males (12%), but not in KO males or in either genotype of female mice. Kidney enlargement in the WT males was accompanied by a small, insignificant fall in protein-to-DNA ratio, supporting hyperplasia rather than hypertrophy. Fructose feeding of male WT mice led to significantly higher sodium bicarbonate exchanger (NBCe1), sodium hydrogen exchanger (NHE3), sodium phosphate co-transporter (NaPi-2), and transforming growth factor-ß (TGF-ß) abundances, as compared to male KO, suggesting elevated transport capacity and an early feature of fibrosis may have accompanied the renal enlargement. Overall, IR and/or IGF1R appear to have a role in PT cell size and enlargement in response to high-fructose diet.

The Role of Sodium-Dependent Phosphate Transporter in Phosphate Homeostasis.

Inorganic phosphate (Pi) is an essential compound for several biologic functions. Pi levels outside the normal range, however, contribute to several pathological processes. Hypophosphatemia leads to bone abnormalities, such as rickets/osteomalacia. Hyperphosphatemia contributes to vascular calcification in patients with chronic kidney disease and hemodialysis patients and is independently associated with cardiac mortality.Pi homeostasis is regulated by the coordinated function of renal and intestinal sodium-dependent phosphate (NaPi) transporters with dietary Pi, parathyroid hormone, 1,25-dihydroxyvitamin D3, and fibroblast growth factor 23. The type II NaPi transporter/SLC34 family, with three members identified to date, is mainly responsible for Pi homeostasis in the body. SLC34A1 and SCL34A3 are predominantly expressed in the kidney, whereas SLC34A2 is expressed in the small intestine. The role of each SLC34 in the body was recently established by studies of gene-targeted mice. Mutation of SLC34A1 causes Fanconi syndrome and mutation of SLC34A3 causes autosomal recessive hereditary hypophosphatemic rickets with hypercalciuria. SLC34A2 is thought to be a major intestinal NaPi transporter and mutation of SLC34A2 causes pulmonary alveolar microlithiasis. A detailed understanding of Pi regulation in the body is important toward maintaining health.

NEDD4-2 and salt-sensitive hypertension.

PURPOSE OF REVIEW: NEDD4-2 is an ubiquitin-protein ligase that was originally identified as an interactor of the epithelial Na+ channel (ENaC); this interaction is defective in Liddle's syndrome, causing elevated ENaC activity and salt-sensitive hypertension. In this review we aim to highlight progress achieved in recent years demonstrating that NEDD4-2 is involved in the control of Na+ transporters that are different from ENaC, but which also play a role in salt-sensitive hypertension. RECENT FINDINGS: It has been shown that NEDD4-2 interacts with ubiquitylates and negatively regulates the thiazide-sensitive NCC (Na+,Cl- -cotransporter), both in vitro and in vivo in inducible, nephron-specific Nedd4-2 knockout mice. Moreover, evidence has been provided that NEDD4-2 is also involved in the regulation of human NHE3 (Na+,H+-exchanger 3) and NKCC2 (Na+,K+,2Cl- -cotransporter 2). SUMMARY: The emerging role of NEDD4-2 in the regulation of different Na+ transporters along the nephron and the identification of human polymorphisms in the NEDD4-2 gene (Nedd4L) related to salt-sensitive hypertension makes this ubiquitin-protein ligase an interesting target for the development of antihypertensive drugs.

iTRAQ quantitative clinical proteomics revealed role of Na(+)K(+)-ATPase and its correlation with deamidation in vascular dementia.

Dementia is a major public health burden characterized by impaired cognition and loss of function. There are limited treatment options due to inadequate understanding of its pathophysiology and underlying causative mechanisms. Discovery-driven iTRAQ-based quantitative proteomics techniques were applied on frozen brain samples to profile the proteome from vascular dementia (VaD) and age-matched nondementia controls to elucidate the perturbed pathways contributing to pathophysiology of VaD. The iTRAQ quantitative data revealed significant up-regulation of protein-l-isoaspartate O-methyltransferase and sodium-potassium transporting ATPase, while post-translational modification analysis suggested deamidation of catalytic and regulatory subunits of sodium-potassium transporting ATPase. Spontaneous protein deamidation of labile asparagines, generating abnormal l-isoaspartyl residues, is associated with cell aging and dementia due to Alzheimer's disease and may be a cause of neurodegeneration. As ion channel proteins play important roles in cellular signaling processes, alterations in their function by deamidation may lead to perturbations in membrane excitability and neuronal function. Structural modeling of sodium-potassium transporting ATPase revealed the close proximity of these deamidated residues to the catalytic site during E2P confirmation. The deamidated residues may disrupt electrostatic interaction during E1 phosphorylation, which may affect ion transport and signal transduction. Our findings suggest impaired regulation and compromised activity of ion channel proteins contribute to the pathophysiology of VaD.