Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dent's disease, a X-linked hypercalciuric nephrolithiasis, is caused by mutations of the CLCN5 gene. The disease is characterised by low molecular weight proteinuria with variable presence of hypercalciuria, hyperphosphaturia, nephrocalcinosis, and kidney stones. CLCN5 encodes a chloride channel belonging to the voltage-gated chloride channel family, which is predominantly expressed in the endosomes of proximal tubular cells. By shunting the current of electrogenic H+-ATPase, ClC-5 is crucial for efficient acidification of renal endosomes. As shown in knock-out mouse models, the ClC-5 loss of function causes severe impairment of receptor-mediated endocytosis, as well as the endocytotic retrieval of plasma membrane proteins including megalin. In a minority of patients with classical Dent's disease, the analysis of CLCN5 coding sequences failed to identify causative mutations. It is conceivable that mutations in the 5' upstream regulatory regions could impair the correct processing and translation of CLCN5. The complexity of its promoter region seems to support this hypothesis. Molecular diagnosis of Dent's disease is now available; since the risk of developing renal insufficiency in adult life is elevated for this type of nephrolithiasis, the correct diagnosis could potentially modify the natural history of the disease by preventing the evolution towards uraemia.
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PMID:[Dent's disease: hereditary nephrolithiasis related to defective tubular endocytosis processes]. 1473 9

Diabetic kidney disease is initially associated with hypertension and increased urinary albumin excretion. The hypertension is mediated by enhanced volume expansion due to enhanced salt and water retention by the kidney. The increased urinary albumin is not only due to increased glomerular leak, but also to a decrease in albumin reabsorption by the proximal tubule. The precise molecular mechanisms underlying these two phenomena and whether there is any link between the increase in Na(+) retention and proteinuria remain unresolved. There is significant evidence to suggest that increased Na(+) retention by the proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3) can play a role in some forms of hypertension. Increased NHE3 activity in models of diabetes mellitus may explain, in part, the enhanced salt retention observed in patients with diabetic kidney disease. The NHE3 also plays a role in receptor-mediated albumin uptake in the proximal tubule. The uptake of albumin requires the assembly of a macromolecular complex that is thought to include the megalin/cubulin receptor, NHE3, the vacuolar type H(+)-ATPase (v-H(+)-ATPase), the Cl(-) channel ClC-5 and interactions with the actin cytoskeleton. The NHE3 seems to exist in two functionally distinct membrane domains, one involved with Na(+) reabsorption and the other involved in albumin uptake. The present review focuses on the evidence derived from in vivo studies, as well as complementary studies in cell culture models, for a dual role of NHE3 in both Na(+) retention and albumin uptake. We suggest a possible mechanism by which disruption of the proximal tubule albumin uptake mechanism in diabetes mellitus may lead to both increased Na(+) retention and proteinuria.
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PMID:Molecular changes in proximal tubule function in diabetes mellitus. 1519 16

The use of renal cortical slices in vitro and the data obtained in these studies have been subjects of controversy, largely due to uncertain viability, e.g., structural and functional integrity of the proximal and other tubules. However, detailed studies of tubule integrity have not been reported. To correlate functional and structural viability of the hand-cut rat renal cortical slices, incubated in optimally conditioned media for up to 25 h, we studied the time course of p-aminohippurate (PAH) uptake, the immunocytochemical distribution of several proteins that reside in the proximal tubule basolateral [Na/K-ATPase, organic anion transporters (OAT)1 and OAT3], or brush border [megalin, sodium-proton exchanger (NHE)3] membrane, as well as the general integrity of the tubule epithelium and its cytoskeleton (actin filaments, microtubules). PAH uptake in slices was proportional to time within 1 h of incubation and gradually declined thereafter. The immunostaining experiments indicated a fast, time-dependent loss of basolateral transporters, at a rate of OAT1 > Na/K-ATPase > OAT3. In the brush border membrane, the loss of megalin was faster than that of NHE3, and a partial redistribution of NHE3 into the basolateral domain indicated the loss of cell polarity. The loss of intracellular actin and tubulin cytoskeleton in the proximal tubule was already visible after 15 min of incubation and gradually increased with time, whereas a partial redistribution of actin to the basolateral domain indicated a compromised polarity of the cells. The data also revealed very early (after 15 min) necrotic events in the proximal tubule epithelium, with sloughing of brush border and cell debris into the tubule lumen, detachment of cells from the basal membrane, and opening and widening of the tubule lumen. We conclude that the loss of cellular structure, cytoskeleton, and cell membrane transporters in the nephron epithelium is a very early event in the incubated rat renal cortical slices.
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PMID:Immunocytochemical characterization of the incubated rat renal cortical slices. 1589 49

ClC-5 is a chloride (Cl(-)) channel expressed in renal tubules and is critical for normal tubular function. Loss of function nonsense or missense mutations in ClC-5 are associated with Dent's disease, a condition in which patients present with low molecular weight (LMW) proteinuria (including albuminuria), hypercalciuria and nephrolithiasis. Several key studies in ClC-5 knockout mice have shown that the proteinuria results from defective tubular reabsorption of proteins. ClC-5 is typically regarded as an intracellular Cl(-) channel and thus the defect in this receptor-mediated uptake pathway was initially attributed to the failure of the early endosomes to acidify correctly. ClC-5 was postulated to play a key role in transporting the Cl(-) ions required to compensate for the movement of H(+) during endosomal acidification. However, more recent studies suggest additional roles for ClC-5 in the endocytosis of albumin. ClC-5 is now known to be expressed at low levels at the cell surface and appears to be a key component in the assembly of the macromolecular complex involved in protein endocytosis. Furthermore, mutations in ClC-5 affect the trafficking of v-H(+)-ATPase and result in decreased expression of the albumin receptor megalin/cubulin. Thus, the expression of ClC-5 at the cell surface as well as its presence in endosomes appears to be essential for normal protein uptake by the renal proximal tubule.
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PMID:ClC-5: a chloride channel with multiple roles in renal tubular albumin uptake. 1622 13

The origin of albuminuria remains controversial owing to difficulties in quantifying the actual amount of albumin filtered by the kidney. Here we use fluorescently labeled albumin, together with the powerful technique of intravital 2-photon microscopy to show that renal albumin filtration in non-proteinuric rats is approximately 50 times greater than previously measured and is followed by rapid endocytosis into proximal tubule cells (PTCs). The endocytosed albumin appears to undergo transcytosis in large vesicles (500 nm in diameter), identified by immunogold staining of endogenous albumin by electron microscopy, to the basolateral membrane where the albumin is disgorged back to the peritubular blood supply. In nephrotic rats, the rate of uptake of albumin by the proximal tubule (PT) is decreased. This is consistent with reduced expression of clathrin, megalin, and vacuolar H(+)-ATPase A subunit, proteins that are critical components of the PT endocytotic machinery. These findings strongly support the paradigm-shifting concept that the glomerular filter normally leaks albumin at nephrotic levels. Albuminuria does not occur as this filtered albumin load is avidly bound and retrieved by PTCs. Dysfunction of this retrieval pathway leads to albuminuria. Thus, restoration of the defective endocytotic and processing function of PT epithelial cells might represent an effective strategy to limit urinary albumin loss, at least in some types of nephrotic syndrome.
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PMID:The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states. 1800 14

We recently reported that megalin is subjected to regulated intramembrane proteolysis (RIP) and includes 1) protein kinase C (PKC)-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-bound megalin COOH-terminal fragment (MCTF) and 2) gamma-secretase-mediated cleavage of the MCTF producing a soluble megalin intracellular domain (MICD). Based on studies of RIP of other receptors, the MICD is predicted to target to the nucleus and regulate gene expression. To determine whether RIP of megalin regulates proximal tubule gene expression, we stably expressed the transfected MCTF (tMCTF) or transfected MICD (tMICD) in opossum kidney proximal tubule (OKP) cells and examined the resulting phenotype. Immunoblotting and immunocytochemical analysis of tMCTF cells showed the tMCTF was expressed and constitutively processed by gamma-secretase. Analysis of specific protein expression in tMCTF- and tMICD-transfected cells using Western blot showed endogenous megalin and Na(+)/H(+) exchanger 3 (NHE3) protein expression to be dramatically lower than that of control cells. Expression of other proteins including myosin VI, beta-adaptin, and the Na-K-ATPase appeared unchanged. Analysis of specific mRNA expression using quantitative real-time PCR showed megalin and NHE3 mRNA levels were significantly lower in tMCTF- and tMICD-transfected cells compared with controls. Inhibition of gamma-secretase activity in tMCTF cells resulted in an 8- to 10-fold recovery of megalin mRNA within 4 h. These data show that the COOH-terminal domain of megalin regulates expression of specific proteins in OKP cells and provides the first evidence that RIP of megalin may be part of a signaling pathway linking protein absorption and gene expression in proximal tubule.
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PMID:The COOH terminus of megalin regulates gene expression in opossum kidney proximal tubule cells. 1849 14

Angiotensin II (ANG II) stimulates proximal tubule (PT) sodium and water reabsorption. We showed that treating rats acutely with the angiotensin-converting enzyme inhibitor captopril decreases PT salt and water reabsorption and provokes rapid redistribution of the Na(+)/H(+) exchanger isoform 3 (NHE3), Na(+)/Pi cotransporter 2 (NaPi2), and associated proteins out of the microvilli. The aim of the present study was to determine whether acute ANG II infusion increases the abundance of PT NHE3, NaPi2, and associated proteins in the microvilli available for reabsorbing NaCl. Male Sprague-Dawley rats were infused with a dose of captopril (12 microg/min for 20 min) that increased PT flow rate approximately 20% with no change in blood pressure (BP) or glomerular filtration rate (GFR). When ANG II (20 ng x kg(-1) x min(-1) for 20 min) was added to the captopril infusate, PT volume flow rate returned to baseline without changing BP or GFR. After captopril, NHE3 was localized to the base of the microvilli and NaPi2 to subapical cytoplasmic vesicles; after 20 min ANG II, both NHE3 and NaPi2 redistributed into the microvilli, assayed by confocal microscopy and density gradient fractionation. Additional PT proteins that redistributed into low-density microvilli-enriched membranes in response to ANG II included myosin VI, DPPIV, NHERF-1, ezrin, megalin, vacuolar H(+)-ATPase, aminopeptidase N, and clathrin. In summary, in response to 20 min ANG II in the absence of a change in BP or GFR, multiple proteins traffic into the PT brush-border microvilli where they likely contribute to the rapid increase in PT salt and water reabsorption.
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PMID:Angiotensin II stimulates trafficking of NHE3, NaPi2, and associated proteins into the proximal tubule microvilli. 1986 1

Renal Fanconi syndrome (FS) is a generalized dysfunction of proximal tubular epithelial cells leading to the urinary leak of essential metabolites like phosphate, uric acid, glucose, amino acids and low molecular weight proteins. From inherited forms involving mutations on apparently unrelated genes to acquired forms induced by drugs, heavy metals or monoclonal immunoglobulin (Ig) light chains (LC), heterogeneous causalities of FS have complicated the understanding of this pathology for a long time. Experimental models of FS have allowed researchers to face the challenge and have helped unravel the main mechanisms disturbing proximal tubule reabsorption. Administration of cadmium to animals first demonstrated an inhibition of Na/K/ATPase activity, highlighting how a single toxic component could induce the general sodium-linked transport defect observed in FS. Today, genetically modified mice allow the development of reliable and reproducible experimental models for inherited or acquired forms of FS. One of the most exciting advances offered by these models is the unexpected major role of endocytosis in the function of the proximal tubule revealed by megalin and ClC-5 knockout mice. Using gene-targeted insertion, a transgenic mouse for LC-associated FS, the most frequent adult form of FS, has also been recently developed and represents a major step in the development of models of this pathology. Beyond deciphering molecular and cellular events at the origin of FS, these models also represent essential tools for the development of therapeutic strategies.
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PMID:Toward understanding renal Fanconi syndrome: step by step advances through experimental models. 2125 24

A disintegrin and metalloprotease 10 (ADAM10) is a zinc protease that mediates ectodomain shedding of numerous receptors including Notch and members of the amyloid precursor protein family (APP, APLP1, and APLP2). Ectodomain shedding frequently activates a process called regulated intramembrane proteolysis (RIP) that links cellular events with gene regulation. To characterize ADAM10 in kidney and in opossum kidney proximal tubule (OKP) cells, we performed indirect immunofluorescence microscopy and immunoblotting of renal membrane fractions using specific antibodies. These studies show that ADAM10 and APLP2 are coexpressed in the proximal tubule and in OKP cells. To study the role of ADAM10 activity in the proximal tubule, we stably overexpressed wild-type ADAM10 or an inactive mutant ADAM10 in OKP cells. We found a direct correlation between the amount of active ADAM10 expressed and 1) the amount of APLP2 ectodomain shed into the culture supernatant and 2) the amount of Na(+)/H(+) exchanger 3 (NHE3) and megalin mRNA and protein expressed compared with control proteins. To establish a link between ADAM10-mediated shedding of APLP2 and the effect on NHE3 and megalin mRNA expression we performed RNA interference experiments using APLP2-specific short hairpin RNA (shRNA) in OKP cells. Cells expressing the APLP2 shRNA showed >80% knock down of APLP2 protein and mRNA as well as 60-70% reduction in NHE3 protein and mRNA. Levels of megalin and Na-K-ATPase protein and mRNA were not changed. These studies show 1) ADAM10 and APLP2 are expressed in proximal tubule cells and, 2) ADAM10 activity has a pronounced effect on expression of specific brush-border proteins. We postulate that ADAM10 and APLP2 may represent elements of a here-to-fore unknown signaling pathway in proximal tubule that link events at the brush border with control of gene expression.
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PMID:A disintegrin and metalloprotease 10 activity sheds the ectodomain of the amyloid precursor-like protein 2 and regulates protein expression in proximal tubule cells. 2132 36

Besides its location at the plasma membrane, CFTR is present in intracellular vesicles along both the exocytic and the endocytic pathways. Immunostaining and subcellular fractionation studies of mouse kidney demonstrate that CFTR is located in endosomes of the cells lining the terminal part of the proximal tubule (PT). The PT cells efficiently reabsorb the ultrafiltered low molecular weight (LMW) proteins by apical endocytosis involving the multiligand receptors megalin and cubilin. The progression from early endosomes to lysosomes depends on the integrity of the cytoskeleton, as well as on vesicular acidification. The latter is mediated by the vacuolar H+-ATPase (V-ATPase) and requires an anionic conductance to dissipate the transmembrane potential gradient. CFTR might ensure such chloride conductance, thereby participating to endosomal acidification and protein uptake by PT cells. Immunostaining with well-characterized antibodies shows that CFTR is located in the terminal segment of PT, where it co-distributes with megalin and cubilin. Subcellular fractionation of total mouse kidneys through Percoll gradients demonstrates the co-localization of CFTR with the V-ATPase and early endosome markers including the Cl-/H+ exchanger, ClC-5, and the small GTPase, Rab5a. Deglycosylation studies and immunoblotting show a distinct glycosylation pattern for CFTR in mouse kidney and lung. The segmental and subcellular distribution of CFTR in mouse kidney supports a role for CFTR in PT receptor-mediated endocytosis of ultrafiltered LMW proteins.
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PMID:Segmental and subcellular distribution of CFTR in the kidney. 2159 92


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