Loop diuretics have anxiolytic effects in rat models of conditioned anxiety

A number of antiepileptic medications that modulate GABA(A) mediated synaptic transmission are anxiolytic. The loop diuretics furosemide (Lasix) and bumetanide (Bumex) are thought to have antiepileptic properties. These drugs also modulate GABA(A) mediated signalling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signalling, we sought to investigate whether they also mediate anxiolytic effects. Here we report the first investigation of the anxiolytic effects of these drugs in rat models of anxiety. Furosemide and bumetanide were tested in adult rats for their anxiolytic effects using four standard anxiety models: 1) contextual fear conditioning; 2) fear-potentiated startle; 3) elevated plus maze, and 4) open-field test. Furosemide and bumetanide significantly reduced conditioned anxiety in the contextual fear-conditioning and fear-potentiated startle models. At the tested doses, neither compound had significant anxiolytic effects on unconditioned anxiety in the elevated plus maze and open-field test models. These observations suggest that loop diuretics elicit significant anxiolytic effects in rat models of conditioned anxiety. Since loop diuretics are antagonists of the NKCC1 and KCC2 cotransporters, these results implicate the cation-chloride cotransport system as possible molecular mechanism involved in anxiety, and as novel pharmacological target for the development of anxiolytics. In view of these findings, and since furosemide and bumetanide are safe and well tolerated drugs, the clinical potential of loop diuretics for treating some types of anxiety disorders deserves further investigation.     

Characterization of Rb uptake into Sf9 cells using cation chromatography: evidence for a K-Cl cotransporter

To assess cation-chloride cotransporter activity in Sf9 cells, cation chromatography was used to measure initial uptake rates of Rb. Rb exchanged with cellular K, with 30% of cellular K replaced after a 40 min exposure to Rb. Rb uptake into Sf9 cells was not inhibited by 50 μmol l(-1) ouabain. Rb uptake was approximately 65% inhibited by 250 μmol l(-1) bumetanide added to the assay solution, and was more than 95% inhibited when cells were pre-incubated for 20 min with bumetanide (100 and 1000 μmol l(-1)). Uptake of Rb and Cl followed simple Michaelis-Menten kinetics, with a K(m) for Rb of 17.1+/-2.2 mmol l(-1) and a K(m) for Cl of 93.7+/-5.6 mmol l(-1). Rb uptake was not dependent upon extracellular Na. Two min exposures to solutions with reduced [Na] or [Cl] produced small but significant changes in cellular Na content. We conclude that the primary Rb uptake pathway in Sf9 cells is a K-Cl cotransporter and that cation chromatography can be used to effectively study kinetic parameters of cotransporter function in tissue culture cells. Characterization of baseline cation-chloride cotransporter activity in Sf9 cells strengthens their utility as a tool for expression and characterization of exogenous proteins.     

Proteomics analysis identifies PARK7 as an important player for renal cell resistance and survival under oxidative stress

Renal fibrosis is a process that is characterized by declining excretory renal function. The molecular mechanisms of fibrosis are not fully understood. Oxidative stress pathways were reported to be involved in renal tissue deterioration and fibrosis progression. In order to identify new molecular targets associated with oxidative stress and renal fibrosis, differential proteomics analysis was performed with established renal cell lines (TK173 and HK-2). The cells were treated with oxidative stress triggering factor H(2)O(2) and the proteome alterations were investigated. Two dimensional protein maps were generated and differentially expressed proteins were processed and identified using mass spectrometry analysis combined with data base search. Interestingly the increase of ROS in the renal cell lines upon H(2)O(2) treatment was accompanied by alteration of a large number of proteins, which could be classified in three categories: the first category grouped the proteins that have been described to be involved in fibrogenesis (e.g. ACTA2, VIN, VIM, DES, KRT, COL1A1, COL4A1), the second category, which was more interesting involved proteins of the oxidative stress pathway (PRDX1, PRDX2, PRDX6, SOD, PARK7, HYOU1), which were highly up-regulated under oxidative stress, and the third category represented proteins, which are involved in different other metabolic pathways. Among the oxidative stress proteins the up-regulation of PARK7 was accompanied by a shift in the pI as a result of oxidation. Knockdown of PARK7 using siRNA led to significant reduction in renal cell viability under oxidative stress. Under H(2)O(2) treatment the PARK7 knockdown cells showed up to 80% decrease in cell viability and an increase in apoptosis compared to the controls. These results highlight for the first time the important role of PARK7 in oxidative stress resistance in renal cells.     

Preliminary purification and characterization studies of a low molecular weight, high affinity cytosolic lead-binding protein in rat brain

Carrier-free 203Pb has been used to label high affinity lead-binding proteins in rat brain cytosol to allow their initial characterization. The low molecular weight 203Pb-protein complex collected from a Sephadex G-75 column eluate has been further purified by Sephadex DEAE chromatography and then partially characterized. The protein has a molecular weight of 23,000 daltons as determined by SDS polyacrylamide gel electrophoresis and significant levels of glutamic acid (9.3%), aspartic acid (10.8%) and cysteine (9.4%). Western blot studies conducted using the polyclonal antibody to the renal lead-binding proteins showed a lack of reactivity, indicating that the brain protein is immunologically distinct from that found in the kidney.     

Molecular Pathophysiology of Renal Tubular Acidosis

Renal tubular acidosis (RTA) is characterized by metabolic acidosis due to renal impaired acid excretion. Hyperchloremic acidosis with normal anion gap and normal or minimally affected glomerular filtration rate defines this disorder. RTA can also present with hypokalemia, medullary nephrocalcinosis and nephrolitiasis, as well as growth retardation and rickets in children, or short stature and osteomalacia in adults. In the past decade, remarkable progress has been made in our understanding of the molecular pathogenesis of RTA and the fundamental molecular physiology of renal tubular transport processes. This review summarizes hereditary diseases caused by mutations in genes encoding transporter or channel proteins operating along the renal tubule. Review of the molecular basis of hereditary tubulopathies reveals various loss-of-function or gain-of-function mutations in genes encoding cotransporter, exchanger, or channel proteins, which are located in the luminal, basolateral, or endosomal membranes of the tubular cell or in paracellular tight junctions. These gene mutations result in a variety of functional defects in transporter/channel proteins, including decreased activity, impaired gating, defective trafficking, impaired endocytosis and degradation, or defective assembly of channel subunits. Further molecular studies of inherited tubular transport disorders may shed more light on the molecular pathophysiology of these diseases and may significantly improve our understanding of the mechanisms underlying renal salt homeostasis, urinary mineral excretion, and blood pressure regulation in health and disease. The identification of the molecular defects in inherited tubulopathies may provide a basis for future design of targeted therapeutic interventions and, possibly, strategies for gene therapy of these complex disorders.Key Words: Renal tubular acidosis, acid-base homeostasis, molecular physiology, tubular transport, gene mutations.     

Functional characteristic and molecular topology of voltage independent sodium channels

The channel proteins so far known are transmembrane oligomers arranged in a manner that the polar residues are lining the central ion-conducting hydrophilic pore. In the last decade, electrophysiology and molecular biology studies revealed the principal similarity in the functional properties and membrane topology within a large family of sodium-conducting channels. Amiloride-sensitive channels are expressed in the apical membranes of renal epithelia. Moreover, in different mammalian cells non-voltage-gated sodium-selective channels have been recently found. According to molecular cloning of the respective DNAs and amino acid sequence analysis, epithelial channel subunits, degenerins and some other channel proteins display a significant homology in the regions forming two presumable transmembrane domains. This paper reviews some relevant data and current opinions of the superfamily of sodium-conducting cation channels.     

Urate Transport Across the Apical Membrane of Renal Proximal Tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or “urate transportsome,” that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.     

Urate transport across the apical membrane of renal proximal tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or "urate transportsome," that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.     

Hemoglobin binding sites on renal brush-border membranes

Prolonged exposure of renal tubules to hemoglobin markedly reduces kidney function and eventually leads to acute renal failure called pigment nephropathy. Intracellular hemoglobin toxicity is one of main pathomechanisms involved in the disease development. However, the process in which hemoglobin is taken up by renal tubular epithelium has not been characterized so far. Isolated renal brush-border membranes of the rat and radioiodinated rat and human hemoglobins were used. Binding properties were examined by the use of rapid filtration technique. Partial isolation of hemoglobin binding proteins was achieved by affinity chromatography. Our experiments showed that both human and rat hemoglobins can be specifically bound to renal brush-border membranes by one class of low affinity (Kd, 7.7 microM) and high capacity (Bmax, 0.18 nmol/mg protein) binding sites. The sites were relatively selective for hemoglobin. Albumin did not compete with hemoglobin. Cationic molecules cytochrome C and lysine exhibited some competition while strong competition of myoglobin was observed. The binding was affected by EGTA indicating a Ca2+ requirement for the interaction. There was a rise in binding in pH 5.4. Fall in binding activity after preincubation of the membranes with peptidases suggested the proteinaceous nature of the binding sites. Affinity chromatography of membrane proteins extract yielded heterogeneous preparation consisting of proteins with molecular masses of 110, 72, 38 and 27 kDa respectively. The existence of binding sites for hemoglobin in renal brush-border membranes strongly suggests that uptake of the protein by tubular epithelia occurs via adsorptive endocytosis. Increased binding of hemoglobin to the membranes under acidic conditions may explain exacerbation of hemoglobinuric acute renal failure in aciduric states.     

Kidney and adrenal mitochondria contain two forms of NADPH-adrenodoxin reductase-dependent iron-sulfur proteins. Isolation of the two porcine renal ferredoxins

Two NADPH-adrenodoxin reductase-dependent iron-sulfur proteins were detected in both porcine kidney and bovine adrenal mitochondria by using high resolution polyacrylamide electrophoresis. Adrenodoxin (Mr = 12,000) constituted the major ferredoxin activity in adrenal mitochondria and a similarly sized protein (Mr = 11,500) was isolated as the major renal ferredoxin activity. A second, higher molecular weight ferredoxin was observed in both adrenal (Mr = 13,300) and kidney (Mr = 13,000) mitochondria. The two renal ferredoxins were isolated by the use of ion exchange, gel exclusion, and preparative electrophoretic techniques. An absorption spectrum typical of [2Fe-2S] ferredoxins was obtained for each protein; however, the larger renal molecule had an unusually high 276 nm absorbance. Immunologic studies revealed a significant degree of antigenic commonality between the two renal proteins as well as specific cross-reactivity of adrenodoxin with antiserum raised against the renal proteins. A possible precursor-product relationship between the paired renal and adrenal ferredoxins is discussed.     

Renal Effects and Underlying Molecular Mechanisms of Long-Term Salt Content Diets in Spontaneously Hypertensive Rats

Several evidences have shown that salt excess is an important determinant of cardiovascular and renal derangement in hypertension. The present study aimed to investigate the renal effects of chronic high or low salt intake in the context of hypertension and to elucidate the molecular mechanisms underlying such effects. To this end, newly weaned male SHR were fed with diets only differing in NaCl content: normal salt (NS: 0.3%), low salt (LS: 0.03%), and high salt diet (HS: 3%) until 7 months of age. Analysis of renal function, morphology, and evaluation of the expression of the main molecular components involved in the renal handling of albumin, including podocyte slit-diaphragm proteins and proximal tubule endocytic receptors were performed. The relationship between diets and the balance of the renal angiotensin-converting enzyme (ACE) and ACE2 enzymes was also examined. HS produced glomerular hypertrophy and decreased ACE2 and nephrin expressions, loss of morphological integrity of the podocyte processes, and increased proteinuria, characterized by loss of albumin and high molecular weight proteins. Conversely, severe hypertension was attenuated and renal dysfunction was prevented by LS since proteinuria was much lower than in the NS SHRs. This was associated with a decrease in kidney ACE/ACE2 protein and activity ratio and increased cubilin renal expression. Taken together, these results suggest that LS attenuates hypertension progression in SHRs and preserves renal function. The mechanisms partially explaining these findings include modulation of the intrarenal ACE/ACE2 balance and the increased cubilin expression. Importantly, HS worsens hypertensive kidney injury and decreases the expression nephrin, a key component of the slit diaphragm.     

TRPM6 and TRPM7--Gatekeepers of human magnesium metabolism

Human magnesium homeostasis primarily depends on the balance between intestinal absorption and renal excretion. Magnesium transport processes in both organ systems - next to passive paracellular magnesium flux - involve active transcellular magnesium transport consisting of an apical uptake into the epithelial cell and a basolateral extrusion into the interstitium. Whereas the mechanism of basolateral magnesium extrusion remains unknown, recent molecular genetic studies in patients with hereditary hypomagnesemia helped gain insight into the molecular nature of apical magnesium entry into intestinal brush border and renal tubular epithelial cells. Patients with Hypomagnesemia with Secondary Hypocalcemia (HSH), a primary defect in intestinal magnesium absorption, were found to carry mutations in TRPM6, a member of the melastatin-related subfamily of transient receptor potential (TRP) ion channels. Before, a close homologue of TRPM6, TRPM7, had been characterized as a magnesium and calcium permeable ion channel vital for cellular magnesium homeostasis. Both proteins share the unique feature of an ion channel fused to a kinase domain with homology to the family of atypical alpha kinases. The aim of this review is to summarize the data emerging from clinical and molecular genetic studies as well as from electrophysiologic and biochemical studies on these fascinating two new proteins and their role in human magnesium metabolism.     

TRPM6 and TRPM7—Gatekeepers of human magnesium metabolism

Human magnesium homeostasis primarily depends on the balance between intestinal absorption and renal excretion. Magnesium transport processes in both organ systems – next to passive paracellular magnesium flux – involve active transcellular magnesium transport consisting of an apical uptake into the epithelial cell and a basolateral extrusion into the interstitium. Whereas the mechanism of basolateral magnesium extrusion remains unknown, recent molecular genetic studies in patients with hereditary hypomagnesemia helped gain insight into the molecular nature of apical magnesium entry into intestinal brush border and renal tubular epithelial cells. Patients with Hypomagnesemia with Secondary Hypocalcemia (HSH), a primary defect in intestinal magnesium absorption, were found to carry mutations in TRPM6, a member of the melastatin-related subfamily of transient receptor potential (TRP) ion channels. Before, a close homologue of TRPM6, TRPM7, had been characterized as a magnesium and calcium permeable ion channel vital for cellular magnesium homeostasis. Both proteins share the unique feature of an ion channel fused to a kinase domain with homology to the family of atypical alpha kinases. The aim of this review is to summarize the data emerging from clinical and molecular genetic studies as well as from electrophysiologic and biochemical studies on these fascinating two new proteins and their role in human magnesium metabolism.     

Critical role of mitochondrial dysfunction and impaired mitophagy in diabetic nephropathy

Mitochondrial dynamics play a critical role in deciding the fate of a cell under normal and diseased condition. Recent surge of studies indicate their regulatory role in meeting energy demands in renal cells making them critical entities in the progression of diabetic nephropathy. Diabetes is remarkably associated with abnormal fuel metabolism, a basis for free radical generation, which if left unchecked may devastate the mitochondria structurally and functionally. Impaired mitochondrial function and their aberrant accumulation have been known to be involved in the manifestation of diabetic nephropathy, indicating perturbed balance of mitochondrial dynamics, and mitochondrial turnover. Mitochondrial dynamics emphasize the critical role of mitochondrial fission proteins such as mitochondrial fission 1, dynamin-related protein 1 and mitochondrial fission factor and fusion proteins including mitofusin-1, mitofusin-2 and optic atrophy 1. Clearance of dysfunctional mitochondria is aided by translocation of autophagy machinery to the impaired mitochondria and subsequent activation of mitophagy regulating proteins PTEN-induced putative kinase 1 and Parkin, for which mitochondrial fission is a prior event. In this review, we discuss recent progression in our understanding of the molecular mechanisms targeting reactive oxygen species mediated alterations in mitochondrial energetics, mitophagy related disorders, impaired glucose transport, tubular atrophy, and renal cell death. The molecular cross talks linking autophagy and renoprotection through an intervention of 5′-AMP-activated protein kinase, mammalian target of rapamycin, and SIRT1 factors are also highlighted here, as in-depth exploration of these pathways may help in deriving therapeutic strategies for managing diabetes provoked end-stage renal disease.     

Role of matrix metalloproteinases in development of diabetic nephropathy

This review considers molecular mechanisms that underlie disorders in the structure and metabolism of renal extracellular matrix in diabetic nephropathy. The contribution of the increased synthesis of renal extracellular matrix proteins in the accumulation of renal mesangial matrix is considered, and the important role of the degradation system of the extracellular matrix proteins in the development of fibrosis is also shown. Data on changes in mRNA expression for the matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in various forms of diabetic nephropathy are presented. A correlation is established between changes in the balance of MMP proteolytic activity and TIMP activity and the accumulation of extracellular matrix.     

Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.     

Identification of P-glycoprotein in renal brush border membranes

A monoclonal antibody (C219) that recognizes the P-glycoprotein (Mr = 170,000) in plasma membranes of multidrug-resistant Chinese hamster ovary (CHO) cell lines was used to assay renal brush border membrane (BBM) and basolateral membrane (BLM) fractions for the presence of a cross-reactive polypeptide. The C219 antibody bound to a 155,000 dalton protein in immunoblots of rat BBM but not BLM proteins resolved by sodium dodecyl sulfate gel electrophoresis. The corresponding human kidney BBM and dog kidney BBM proteins had molecular weights of 170,000 and 160,000 respectively. The glycoprotein nature of the renal protein was shown by its sensitivity to N-glycanase treatment which reduced the apparent molecular weight of the dog protein to 120,000. In addition, dog P-glycoprotein could be bound to and eluted from immobilized wheat germ agglutinin. The molecular weight, antibody crossreactivity, glycosidase sensitivity and lectin binding show that this protein is a normal kidney analogue of the P-glycoprotein induced in multidrug resistant cell lines.     

Quantitative Global Proteome and Lysine Succinylome Analyses Reveal the Effects of Energy Metabolism in Renal Cell Carcinoma

In light of the increasing incidence of renal cell carcinoma (RCC), its molecular mechanisms have been comprehensively explored in numerous recent studies. However, few studies focus on the influence of multi‐factor interactions during the occurrence and development of RCC. This study aims to investigate the quantitative global proteome and the changes in lysine succinylation in related proteins, seeking to facilitate a better understanding of the molecular mechanisms underlying RCC. LC‐MS/MS combined with bioinformatics analysis are used to quantitatively detect the perspectives at the global protein level. IP and WB analysis were conducted to further verify the alternations of related proteins and lysine succinylation. A total of 3,217 proteins and 1,238 lysine succinylation sites are quantified in RCC tissues, and 668 differentially expressed proteins and 161 differentially expressed lysine succinylation sites are identified. Besides, expressions of PGK1 and PKM2 at protein and lysine, succinylation levels are significantly altered in RCC tissues. Bioinformatics analysis indicates that the glycolysis pathway is a potential mechanism of RCC progression and lysine succinylation may plays a potential role in energy metabolism. These results can provide a new direction for exploring the molecular mechanism of RCC tumorigenesis.