Tae-Hwan Kwon

Rapid gating and anion permeability of an intracellular aquaporin

Aquaporin (AQP) water-channel proteins are freely permeated by water but not by ions or charged solutes. Although mammalian aquaporins were believed to be located in plasma membranes, rat AQP6 is restricted to intracellular vesicles in renal epithelia. Here we show that AQP6 is functionally distinct from other known aquaporins. When expressed in Xenopus laevis oocytes, AQP6 exhibits low basal water permeability; however, when treated with the known water channel inhibitor, Hg2+, the water permeability of AQP6 oocytes rapidly rises up to tenfold and is accompanied by ion conductance. AQP6 colocalizes with H+-ATPase in intracellular vesicles of acid-secreting α-intercalated cells in renal collecting duct. At pH less than 5.5, anion conductance is rapidly and reversibly activated in AQP6 oocytes. Site-directed mutation of lysine to glutamate at position 72 in the cytoplasmic mouth of the pore changes the cation/anion selectivity, but leaves low pH activation intact. Our results demonstrate unusual biophysical properties of an aquaporin, and indicate that anion-channel function may now be explored in a protein with known structure.

Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy

To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin‐digested using a gel‐assisted protocol, and quantified by label‐free LC‐MS/MS, using an MSE mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α‐1‐antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi‐quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level.

Reduced AQP1, -2, and -3 levels in kidneys of rats with CRF induced by surgical reduction in renal mass

Urinary concentration characteristically decreases in response to a reduction in renal mass in chronic renal failure (CRF). In the present study, we examined whether there are changes in the expression of aquaporins in rats where CRF was induced by 5/6 nephrectomy. Plasma creatinine levels were significantly elevated consistent with significant CRF: 135.7 ± 15.1 ( n = 17, CRF) vs. 33.9 ± 1.1 μmol/l ( n = 11, sham), P < 0.05. Two weeks after 5/6 nephrectomy, the remnant kidneys were hypertrophied, and total renal mass increased to 65 ± 3% of sham levels ( P < 0.05). Urine production increased markedly from 40 ± 2 to 111 ± 3 μl ⋅ min-1 ⋅ kg-1in CRF rats ( P < 0.05), whereas urine osmolality and solute-free water reabsorption decreased significantly. Quantitative immunoblotting of total kidney membrane fractions revealed a significant decrease in total kidney AQP2 expression in CRF rats to 43 ± 12% of sham levels ( P < 0.05). A similar reduction was observed for AQP1 and AQP3. Furthermore, the increased urine output and decreased urine osmolality persisted in CRF rats despite 7 days treatment with 1-desamino-[8-d-arginine]vasopressin (DDAVP, 0.1 μg/h sc) compared with untreated sham-operated controls. Also, there was no change in AQP2 expression (which remained at 38 ± 3% of sham levels, P < 0.05), urine output, or urine osmolality between CRF rats with or without DDAVP treatment. Immunocytochemistry confirmed the decreased AQP2 expression in collecting duct principal cells in CRF rats, with a predominant apical labeling. In conclusion, the results demonstrated that there was a significant vasopressin-resistant downregulation of AQP2 and AQP3 as well as downregulation of AQP1 associated with the polyuria in CRF rats.Urinary concentration characteristically decreases in response to a reduction in renal mass in chronic renal failure (CRF). In the present study, we examined whether there are changes in the expression of aquaporins in rats where CRF was induced by 5/6 nephrectomy. Plasma creatinine levels were significantly elevated consistent with significant CRF: 135.7 +/- 15.1 (n = 17, CRF) vs. 33. 9 +/- 1.1 micromol/l (n = 11, sham), P < 0.05. Two weeks after 5/6 nephrectomy, the remnant kidneys were hypertrophied, and total renal mass increased to 65 +/- 3% of sham levels (P < 0.05). Urine production increased markedly from 40 +/- 2 to 111 +/- 3 microliter. min-1. kg-1 in CRF rats (P < 0.05), whereas urine osmolality and solute-free water reabsorption decreased significantly. Quantitative immunoblotting of total kidney membrane fractions revealed a significant decrease in total kidney AQP2 expression in CRF rats to 43 +/- 12% of sham levels (P < 0.05). A similar reduction was observed for AQP1 and AQP3. Furthermore, the increased urine output and decreased urine osmolality persisted in CRF rats despite 7 days treatment with 1-desamino-[8-D-arginine]vasopressin (DDAVP, 0.1 microgram/h sc) compared with untreated sham-operated controls. Also, there was no change in AQP2 expression (which remained at 38 +/- 3% of sham levels, P < 0.05), urine output, or urine osmolality between CRF rats with or without DDAVP treatment. Immunocytochemistry confirmed the decreased AQP2 expression in collecting duct principal cells in CRF rats, with a predominant apical labeling. In conclusion, the results demonstrated that there was a significant vasopressin-resistant downregulation of AQP2 and AQP3 as well as downregulation of AQP1 associated with the polyuria in CRF rats.

Reduced abundance of aquaporins in rats with bilateral ischemia-induced acute renal failure : prevention by α-MSH

We examined the effect of temporary renal ischemia (30 min or 60 min) and reperfusion (1 day or 5 days) on the expression of renal aquaporins (AQPs) and urinary concentration in rats with bilateral ischemia-induced acute renal failure (ARF). Next, we tested whether reducing ischemia/reperfusion (I/R) injury by treatment with alpha-melanocyte stimulating hormone (alpha-MSH) affects the expression of AQPs and urine output. Rats with ARF showed significant renal insufficiency, and urinary concentration was markedly impaired. In rats with mild ischemic injury (30 min), urine output increased significantly to a maximum at 48 h, and then nearly normalized within 5 days. Consistent with this, semiquantitative immunoblotting revealed that kidney AQP1 and AQP2 abundance was significantly decreased after 24 h to 30 +/- 5% and 40 +/- 11% (n = 8) of controls (n = 9), respectively (P < 0.05). Five days after ischemia, AQP2 abundance was not significantly decreased and urine output was normalized. In contrast, severe ischemic injury (60 min) resulted in a marked reduction in urine output at 24 h, despite a significant decrease in urine osmolality and solute-free water reabsorption, T(c)H(2)O. AQP1 and AQP2 abundance was markedly decreased to 51 +/- 5% and 31 +/- 9% (n = 10) of controls (n = 8) at 24 h (P < 0.05). After 5 days, the rats developed gradually severe polyuria and had very low AQP2 and AQP1 levels [11 +/- 4% and 6 +/- 2% (n = 5) of controls (n = 8), respectively; P < 0.05]. A similar reduction was observed for AQP3. The reduction in AQP expression in the proximal tubule and inner medullary collecting duct was confirmed by immunocytochemistry. Next, we found that intravenous alpha-MSH treatment of rats with ARF significantly reduced the ischemia-induced downregulation of renal AQPs and reduced the polyuria. In conclusion, the I/R injury is associated with markedly reduced expression of the collecting duct and proximal tubule AQPs, in association with an impairment of urinary concentration. Moreover, alpha-MSH treatment significantly prevented the reduction in expression of AQPs and renal functional defects. Thus decreased AQP expression is likely to contribute to the impairment in urinary concentration in the postischemic period.We examined the effect of temporary renal ischemia (30 min or 60 min) and reperfusion (1 day or 5 days) on the expression of renal aquaporins (AQPs) and urinary concentration in rats with bilateral ischemia-induced acute renal failure (ARF). Next, we tested whether reducing ischemia/reperfusion (I/R) injury by treatment with α-melanocyte stimulating hormone (α-MSH) affects the expression of AQPs and urine output. Rats with ARF showed significant renal insufficiency, and urinary concentration was markedly impaired. In rats with mild ischemic injury (30 min), urine output increased significantly to a maximum at 48 h, and then nearly normalized within 5 days. Consistent with this, semiquantitative immunoblotting revealed that kidney AQP1 and AQP2 abundance was significantly decreased after 24 h to 30 ± 5% and 40 ± 11% ( n = 8) of controls ( n = 9), respectively ( P < 0.05). Five days after ischemia, AQP2 abundance was not significantly decreased and urine output was normalized. In contrast, severe ischemic injury (60 min) resulted in a marked reduction in urine output at 24 h, despite a significant decrease in urine osmolality and solute-free water reabsorption, TcH2O. AQP1 and AQP2 abundance was markedly decreased to 51 ± 5% and 31 ± 9% ( n = 10) of controls ( n = 8) at 24 h ( P < 0.05). After 5 days, the rats developed gradually severe polyuria and had very low AQP2 and AQP1 levels [11 ± 4% and 6 ± 2% ( n = 5) of controls ( n = 8), respectively; P < 0.05]. A similar reduction was observed for AQP3. The reduction in AQP expression in the proximal tubule and inner medullary collecting duct was confirmed by immunocytochemistry. Next, we found that intravenous α-MSH treatment of rats with ARF significantly reduced the ischemia-induced downregulation of renal AQPs and reduced the polyuria. In conclusion, the I/R injury is associated with markedly reduced expression of the collecting duct and proximal tubule AQPs, in association with an impairment of urinary concentration. Moreover, α-MSH treatment significantly prevented the reduction in expression of AQPs and renal functional defects. Thus decreased AQP expression is likely to contribute to the impairment in urinary concentration in the postischemic period.

Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with H+-ATPase.

To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H+-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and H+-ATPase and analysis by confocal images showed that ClC-5 and H+-ATPase colocalized in these ClC-5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the H+ secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney.

Ser-256 phosphorylation dynamics of Aquaporin 2 during maturation from the ER to the vesicular compartment in renal cells

Aquaporin 2 (AQP2) phosphorylation at Ser‐256 by protein kinase A (PKA) is a key signal for vasopressin‐stimulated AQP2 insertion into the plasma membrane in renal cells. This study underscores the possible role of phosphorylation at Ser‐256 in regulating AQP2 maturation. AQP2‐transfected renal CD8 cells were incubated with brefeldin A (BFA) to accumulate newly synthesized AQP2 in the endoplasmic reticulum (ER), and AQP2 flow from ER to the vesicular compartment was analyzed after BFA washout. We found that a) in the ER, AQP2 is weakly phosphorylated; b) the amount of phosphorylated AQP2 (p‐AQP2) at Ser‐256 increased significantly during transit in the Golgi, even in the presence of the PKA inhibitor H89; and c) AQP2 transport from the Golgi to the vasopressin‐regulated vesicular compartment occurred with a concomitant decrease in p‐AQP2 at Ser‐256. These results support the hypothesis that AQP2 transition in the Golgi apparatus is associated with a PKA‐independent increase in AQP2 phosphorylation at Ser‐256. Conversely, impaired constitutive phosphorylation in a Golgiassociated compartment occurring in cells expressing mutated S256A‐AQP2 or E258K‐AQP2 causes phosphorylation‐defective AQP2 routing to lysosomes. This result might explain the molecular basis of the dominant form of nephrogenic diabetes insipidus caused by the mutation E258K‐AQP2, in which the phenotype is caused by an impaired routing of AQP2.

Beta ig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with α3β1 integrin

Betaig-h3 (βig-h3) is a secretory protein composed of fasciclin I-like repeats containing sequences that allows binding of integrins and glycosaminoglycans in vivo. Expression of βig-h3 is responsive to TGF-β and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating βig-h3 as an ECM adhesive protein of developmental processes. We previously observed predominant expression of βig-h3 expression in the basement membrane of proximal tubules of kidney. In this study, the physiological relevance of such localized expression of βig-h3 was examined in the renal proximal tubular epithelial cells (RPTEC). RPTEC constitutively expressed βig-h3 and the expression was dramatically induced by exogenous TGF-β1 treatment. βig-h3 and its second and fourth FAS1 domain were able to mediate RPTEC adhesion, spreading and migration. Two known α3β1 integrin-interaction motifs including aspartatic acid and isoleucine residues, NKDIL and EPDIM in βig-h3 were responsible to mediate RPTEC adhesion, spreading, and migration. By using specific antibodies against integrins, we confirmed that α3β1 integrin mediates the adhesion and migration of RPTECs on βig-h3. In addition, it also enhanced proliferation of RPTECs through NKDIL and EPDIM. These results indicate that βig-h3 mediates adhesion, spreading, migration and proliferation of RPTECs through the interaction with α3β1 integrin and is intimately involved in the maintenance and the regeneration of renal proximal tubular epithelium.

Targeting Bladder Tumor Cells In vivo and in the Urine with a Peptide Identified by Phage Display

Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXRX/RC showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer. (Mol Cancer Res 2007;5(1):11–19)

Altered expression of Na transporters NHE-3, NaPi-II, Na-K-ATPase, BSC-1, and TSC in CRF rat kidneys.

In chronic renal failure (CRF), reduction in renal mass leads to an increase in the filtration rates of the remaining nephrons and increased excretion of sodium per nephron. To address the mechanisms involved in the increased sodium excretion, we determined the total kidney levels and the densities per nephron of the major renal NaCl transporters in rats with experimental CRF. Two weeks after 5/6 nephrectomy (reducing the total number of nephrons to ∼24 ± 8%), the rats were azotemic and displayed increased Na excretion. Semiquantitative immunoblotting revealed significant reduction in the total kidney levels of the proximal tubule Na transporters NHE-3 (48% of control), NaPi-II (13%), and Na-K-ATPase (30%). However, the densities per nephron of NHE-3, NaPi-II, and Na-K-ATPase were not significantly altered in remnant kidneys, despite the extensive hypertrophy of remaining nephrons. Immunocytochemistry confirmed the reduction in NHE-3 and Na-K-ATPase labeling densities in the proximal tubule. In contrast, there was no significant reduction in the total kidney levels of the thick ascending limb and distal convoluted tubule NaCl transporters BSC-1 and TSC, respectively. This corresponded to a 3.6 and 2.5-fold increase in densities per nephron, respectively (confirmed by immunocytochemistry). In conclusion, in this rat CRF model: 1) increased fractional sodium excretion is associated with altered expression of proximal tubule Na transporter expression (NHE-3, NaPi-II, and Na-K-ATPase), consistent with glomerulotubular imbalance in the face of increased single-nephron glomerular filtration rate; and 2) compensatory increases in BSC-1 and TSC expression per nephron occur in distal segments.

Aquaporin-5: A Marker Protein for Proliferation and Migration of Human Breast Cancer Cells

Aquaporin (AQP) is a family of transmembrane proteins for water transport. Recent studies revealed that AQPs are likely to play a role in tumor progression and invasion. We aimed to examine the potential role of AQP5 in the progression of human breast cancer cells. Expression of AQP5 mRNA and protein was seen in human breast cancer cell line (both MCF7 and MDA-MB-231) by RT-PCR and immunoblotting analysis. Immunoperoxidase labeling of AQP5 was observed at ductal epithelial cells of human breast tissues. In benign tumor, AQP5 labeling was mainly seen at the apical domains of ductal epithelial cells. In contrast, in invasive ductal carcinoma, prominent AQP5 labeling was associated with cancer cells, whereas some ducts were unlabeled and apical polarity of AQP5 in ducts was lost. Cell proliferation (BrdU incorporation assay) and migration of MCF7 cells were significantly attenuated by lentivirus-mediated AQP5-shRNA transduction. Hyperosmotic stress induced by sorbitol treatment (100 mM, 24 h) reduced AQP5 expression in MCF7 cells, which was also associated with a significant reduction in cell proliferation and migration. Taken together, prominent AQP5 expression in breast cancer cells with the loss of polarity of ductal epithelial cells was seen during the progression of breast carcinoma. shRNA- or hyperosmotic stress-induced reduction in AQP5 expression of MCF7 cells was associated with significantly reduced cell proliferation and migration. In conclusion, AQP5 overexpression is likely to play a role in cell growth and metastasis of human breast cancer and could be a novel target for anti-breast cancer treatment.