Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.

Effects of pathophysiological concentrations of albumin on NHE3 activity and cell proliferation in primary cultures of human proximal tubule cells

The progression of renal disease correlates strongly with hypertension and the degree of proteinuria, suggesting a link between excessive Na+ reabsorption and exposure of the proximal tubule to protein. The present study investigated the effects of albumin on cell growth and Na+ uptake in primary cultures of human proximal tubule cells (PTC). Albumin (1.0 mg/ml) increased cell proliferation to 134.1 +/- 11.8% (P < 0.001) of control levels with no change in levels of apoptosis. Exposure to 0.1 and 1.0 mg/ml albumin increased total Na-22(+) uptake to 119.1 &PLUSMN; 6.3% (P = 0.005) and 115.6 &PLUSMN; 5.3% (P < 0.006) of control levels, respectively, because of an increase in Na+/H+ exchanger isoform 3 (NHE3) activity. This was associated with an increase in NHE3 mRNA to 161.1 +/- 15.1% (P < 0.005) of control levels in response to 0.1 mg/ml albumin. Using confocal microscopy with a novel antibody raised against the predicted extracellular NH2 terminus of human NHE3, we observed in nonpermeabilized cells that exposure of PTC to albumin (0.1 and 1.0 mg/ml) increased NHE3 at the cell surface to 115.4 &PLUSMN; 2.7% (P < 0.0005) and 122.4 +/- 3.7% (P < 0.0001) of control levels, respectively. This effect was paralleled by significant increases in NHE3 in the subplasmalemmal region as measured in permeabilized cells. These albumin-induced increases in expression and activity of NHE3 in PTC suggest a possible mechanism for Na+ retention in response to proteinuria.

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio similar to50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S2O3)(2)](3-) resulted in formation of the adducts HSA + Au(S2O3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S2O3)(2)](3-) blocked the formation of gold adducts. (C) 2003 Elsevier Inc. All rights reserved.

Nedd4-2 functionally interacts with ClC-5 - Involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl- channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Pioglitazone increases renal tubular cell albumin uptake but limits proinflammatory and fibrotic responses

Background. Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. which are known to be critical factors in lipid metabolism, have also been reported to reduce proteinuria. The mechanism and its relevance to progressive nephropathy have not been determined. The aims of this study were to assess the direct effects of a PPARgamma agonist on tubular cell albumin uptake, proinflammatory and profibrotic markers of renal pathology, using an opossum kidney model of proximal tubular cells. Methods. Cells were exposed to pioglitazone (10 mumol/L) in the presence and absence of low-density lipoprotein (LDL) 100 mug/mL +/- exposure to albumin 1 mg/mL. Results were expressed relative to control (5 mmol/L glucose) conditions. Results. Pioglitazone caused a dose-dependent increase in tubular cell albumin uptake (P < 0.0001). Despite the increase in albumin reabsorption, no concurrent increase in inflammatory or profibrotic markers were observed. Exposure to LDL increased monocyte chemoattractant protein-1 (MCP-1) (P < 0.05) and transforming growth factor-beta1 (TGF-beta1) (P < 0.05) production. which were reversed in the presence of pioglitazone. LDL induced increases in MCP-1 and TGF-β1 were independent of nuclear factor-κB (NF-κB) transcriptional activity. In contrast. tubular exposure to albumin increased tubular protein uptake, in parallel with an increase in MCP-1 (P = 0.05): TGF-β1 (P < 0.02) and NF-kappaB transcriptional activity (P < 0.05). which were unaffected by concurrent exposure to pioglitazone. Conclusion. These findings suggest that dyslipidemia potentiates renal pathology through mechanisms that may be modified PPARγ activation independent of NF-κB transcriptional activitv. In contrast, tubular exposure to protein induces renal damage through NF-κB-dependent mechanisms that are Unaffected by PPARγ activation.

Randomized trial of 8 Gy in 1 versus 20 Gy in 5 fractions of radiotherapy for neuropathic pain due to bone metastases (Trans-Tasman Radiation Oncology Group, TROG 96.05)

Background and purpose: Despite numerous randomized trials investigating radiotherapy (RT) fractionation schedules for painful bone metastases, there are very few data on RT for bone metastases causing pain with a neuropathic component. The Trans-Tasman Radiation Oncology Group undertook a randomized trial comparing the efficacy of a single 8 Gy (8/1) with 20 Gy in 5 fractions (20/5) for this type of pain. Materials and methods: Eligible patients had radiological evidence of bone metastases from a known malignancy with no change in systemic therapy within 6 weeks before or anticipated within 4 weeks after RT, no other metastases along the distribution of the neuropathic pain and no clinical or radiological evidence of cord/cauda equina compression. All patients gave written informed consent. Primary endpoints were pain response within 2 months of commencement of RT and time to treatment failure (TTF). The hypothesis was that 8/1 is at least as effective as 20/5 and the planned sample size was 270 patients. Results: Between February 1996 and December 2002, 272 patients were randomized (8/1:20/5 = 137:135) from 15 centres (Australia 11, New Zealand 3, UK 1). The commonest primary cancers were lung (31%), prostate (29%) and breast (8%); index sites were spine (89%), rib (9%), other (2%); 72% of patients were males and the median age was 67 (range 2989). The median overall survival (95% CI) for all randomized patients was 4.8 mo (4.2-5.7 mo). The intention-to-treat overall response rates (95% Cl) for 8/1 vs 20/5 were 53% (45-62%) vs 61% (53-70%), P = 0.18. Corresponding figures for complete response were 26% (18-34%) vs 27% (19-35%), P = 0.89. The estimated median TTFs (95% CI) were 2.4 mo (2.0-3.3 mo) vs 3.7 mo (3.1-5.9 mo) respectively. The hazard ratio (95% Cl) for the comparison of TTF curves was 1.35 (0.99-1.85), log-rank P = 0.056. There were no statistically significant differences in the rates of re-treatment, cord compression or pathological fracture by arm. Conclusions: 8/1 was not shown to be as effective as 20/5, nor was it statistically significantly worse. Outcomes were generally poorer for 8/1, although the quantitative differences were relatively small. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Urinary albumin levels in the normal range determine arterial wall thickness in adults with Type 2 diabetes: a FIELD substudy

Aim Cardiovascular disease (CVD) rates are substantially higher among patients with Type 2 diabetes than in the general population. The objective of this study was to identify the determinants of carotid intima media thickness (IMT) in patients with Type 2 diabetes. Methods We measured the thickness of the intima media layer of the carotid artery, a strong predictor of the risk of future vascular events, in 397 Type 2 diabetic patients drawn from the Fenofibrate Intervention and Event Lowering in Diabetes study, prior to treatment allocation. Results The mean IMT was 0.78 mm [interquartile range (IQR) 0.23 mm], and the maximum IMT was 1.17 mm (IQR 0.36 mm). By multivariate analysis, age, sex, duration of diabetes, triglycerides, and total cholesterol were independently correlated with IMT, as was urine albumin-creatinine ratio (ACR) (P < 0.001). The effect of ACR on IMT was further examined by tertile. Clinically significant differences in IMT were associated with ACR > 0.65 mg/mmol, approximately one-fifth the standard clinical threshold for microalbuminuria (P < 0.01). Long-term diabetes, independent of other parameters, was associated with a 50% increase in age-related thickening. Conclusions IMT in people with Type 2 diabetes is independently and continuously related to urine albumin levels and to the duration of diabetes. These results support previous data linking urine albumin measurements within the normal range with increased ischaemic cardiac mortality in the setting of Type 2 diabetes, and strongly suggest that urine albumin levels within this range should trigger a formal evaluation for CVD.

Dissipation of acetochlor and its distribution in surface and sub-surface soil fractions during laboratory incubations

Pesticides in soil are subject to a number of processes that result in transformation and biodegradation, sorption to and desorption from soil components, and diffusion and leaching. Pesticides leaching through a soil profile will be exposed to changing environmental conditions as different horizons with distinct physical, chemical and biological properties are encountered. The many ways in which soil properties influence pesticide retention and degradation need to be addressed to allow accurate predictions of environmental fate and the potential for groundwater pollution. Degradation and sorption processes were investigated in a long-term (100 days) study of the chloroacetanilide herbicide, acetochlor. Soil cores were collected from a clay soil profile and samples taken from 0-30cm (surface), 1.0-1.3m (mid) and 2.7-3.0m (deep) and treated with acetochlor (2.5, 1.25, 0.67 mu g acetochlor g(-1) dry wt soil, respectively). In sterile and non-sterile conditions, acetochlor concentration in the aqueous phase declined rapidly from the surface and subsoil layers, predominantly through nonextractable residue (NER) formation on soil surfaces, but also through biodegradation and biotic transformation. Abiotic transformation was also evident in the sterile soils. Several metabolites were produced, including acetochlor-ethane sulphonic acid and acetochlor-oxanilic acid. Transformation was principally microbial in origin, as shown by the differences between non-sterile and sterile soils. NER formation increased rapidly over the first 21 days in all soils and was mainly associated with the macroaggregate (> 2000 mu m diameter) size fractions. It is likely that acetochlor is incorporated into the macroaggregates through oxidative coupling, as humification of particulate organic matter progresses. The dissipation (ie total loss of acetochlor) half-life values were 9.3 (surface), 12.3 (mid) and 12.6 days (deep) in the non-sterile soils, compared with 20.9 [surface], 23.5 [mid], and 24 days [deep] in the sterile soils, demonstrating the importance of microbially driven processes in the rapid dissipation of acetochlor in soil.

PKC-alpha-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKC-α in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker bisindolylmaleimide-1 and the isoform-specific PKC blockers Go-6976 and 2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether, indicating a role for PKC-α. Overexpression of a kinase deficient PKC-α(K368R) but not wild-type PKC-α significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKC-α-green fluorescent protein with the membrane fraction within 10-20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or overexpressing kinase-deficient PKC-α(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKC-α in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.

Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds - Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney ( OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.

CIC-5: A chloride channel with multiple roles in renal tubular albumin uptake

CIC-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 CIC-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 CIC-5 knockout mice have shown that the proteinuria results from defective tubular reabsorption of proteins. CIC-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. CIC-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 CIC-5 in the endocytosis of albumin. CIC-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 CIC-5 affect the trafficking of v-H+-ATPase and result in decreased expression of the albumin receptor megalin/cubulin. Thus, the expression of CIC-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. (c) 2005 Elsevier Ltd. All rights reserved.

Experimental Calcification of HEMA-Based Hydrogels in the Presence of Albumin and a Comparison to the in Vivo Calcification

The precipitation patterns and characteristics of calcium phosphate (CaP) phases deposited on HEMA-based hydrogels upon incubation in simulated body fluid (SBF-2) containing a protein (human serum albumin) have been investigated in relation to the calcification in an organic-free medium (SBF-1) and to that occurring after subcutaneous implantation in rats. In SBF-2, the deposits occurred exclusively as a peripheral layer on the surface of the hydrogels and consisted mainly of precipitated hydroxyapatite, a species deficient in calcium and hydroxyl ions, similarly to the deposits formed on the implanted hydrogels, where the deposited layer was thicker. In SBF-1, the deposits were mainly of brushite type. There was no evidence that albumin penetrated the interstices of hydrogels. As the X-ray diffraction patterns of the CaP deposits generated in SBF-2 showed a similar nature with those formed on the implanted hydrogel, it was concluded that the calcification in SBF-2 can mimic to a reliable extent the calcification process taking place in a biological environment.