Novel Potential Application of Chitosan Oligosaccharide for Attenuation of Renal Cyst Growth in the Treatment of Polycystic Kidney Disease

Chitosan oligosaccharide (COS), a natural polymer derived from chitosan, exerts several biological activities including anti-inflammation, anti-tumor, anti-metabolic syndrome, and drug delivery enhancer. Since COS is vastly distributed to kidney and eliminated in urine, it may have a potential advantage as the therapeutics of kidney diseases. Polycystic kidney disease (PKD) is a common genetic disorder characterized by multiple fluid-filled cysts, replacing normal renal parenchyma and leading to impaired renal function and end-stage renal disease (ESRD). The effective treatment for PKD still needs to be further elucidated. Interestingly, AMP-activated protein kinase (AMPK) has been proposed as a drug target for PKD. This study aimed to investigate the effect of COS on renal cyst enlargement and its underlying mechanisms. We found that COS at the concentrations of 50 and 100 µg/mL decreased renal cyst growth without cytotoxicity, as measured by MTT assay. Immunoblotting analysis showed that COS at 100 µg/mL activated AMPK, and this effect was abolished by STO-609, a calcium/calmodulin-dependent protein kinase kinase beta (CaMKKβ) inhibitor. Moreover, COS elevated the level of intracellular calcium. These results suggest that COS inhibits cyst progression by activation of AMPK via CaMKKβ. Therefore, COS may hold the potential for pharmaceutical application in PKD

Effect of GlyH-101 on H₂O₂-induced osmotic tolerance of beta thalassemia/Hb E erythrocytes.

<p>The erythrocytes were incubated with PBS containing H₂O₂ (10 mM) with or without GlyH-101 (50 µM) at 37°C for 3 h in a shaking incubator. After centrifugation and removal of supernatant, cells were exposed to hypotonic solution (85% PBS) and centrifuged at 1,500 g at 4°C for 10 min. Absorbance of the supernatants was measured at 540 nm. Data were expressed as means ± S.E. *, p<0.05 compared with H₂O₂-treated group (n = 10).</p

Chemical structures of inhibitors of glutathione efflux transporters and CFTR expression in human erythrocytes.

<p>(A) Chemical structures of GlyH-101, a CFTR inhibitor, and MK571, a MRP1 inhibitors. (B) Expression of CFTR in erythrocytes of beta thalassemia/Hb E patients and normal healthy subjects.</p

Anti-oxidative properties of MK571 in erythrocytes of beta thalassemia/Hb E patients.

<p>(A) MK571 reduced H₂O₂-induced free radical production. Erythrocytes loaded with DCFH-DA were incubated for 1 h with DMSO (basal), DMSO plus MK571 (basal + MK571), H₂O₂ (10 mM), H₂O₂ plus MK571 (50 µM) or H₂O₂ plus MK571 and GlyH-101 (50 µM). Reactive oxygen species were measured by flow cytometry analysis at excitation wavelength of 490 nm and emission wavelength of 530 nm. Data were expressed as means ± S.E. *, p<0.05; **, p<0.001 compared with H₂O₂-treated group (n = 12). (B) Effect of MK571 on intracellular glutathione levels. Erythrocytes were treated for 1 h without (basal) or with H₂O₂ (10 mM) in the presence or absence of MK571 (50 µM) or MK571 plus GlyH-101 (50 µM) before measurements of GSH levels. Data were expressed as means ± S.E. NS, non-statistical significant difference from basal control; *, p<0.01 compared with basal control; #, p<0.05; ##, p<0.01 compared with H₂O₂-treated group (n = 8).</p

Effect of diclofenac on intracellular cAMP content in T84 cells.

<p>T84 cells were incubated for an hour with DMSO (vehicle), diclofenac (200 µM), forskolin (20 µM) or forskolin (20 µM) plus diclofenac (200 µM), followed by cell lysis and cAMP measurement using cAMP immunoassay kit. Data are expressed as means ± S.E.M. NS, non-statistical difference (n = 3).</p

Effect of diclofenac on CFTR Cl⁻ channel activity.

<p>(A) Effect of diclofenac on CFTR Cl⁻ channel activity stimulated by forskolin (20 µM), CPT-cAMP (100 µM), or genistein (20 µM) in T84 cells. In this experiment, CFTR Cl⁻ channel activity was determined using apical Cl⁻ current analysis of T84 cell monolayers. Amphotericin B (250 µg/ml) was added into basolateral solutions to permeabilize basolateral membrane of T84 cells. Diclofenac at the indicated concentrations was added to both apical and basolateral sides. Representative current tracings of 4–7 experiments were shown. (B) Effect of diclofenac on CFTR Cl⁻ channel activity in human airway epithelial (Calu-3) cells. Diclofenac at the indicated concentrations was added into both apical and basolateral solutions after stimulation of CFTR-mediated apical Cl⁻ current by CPT-cAMP (100 µM). Apical Cl⁻ current analysis was performed with basolateral membrane of Calu-3 cells being permeabilized with amphotericin B (250 µg/ml). A representative current tracing of 5 separate experiments is shown. (C) Reversibility of inhibition of CFTR Cl⁻ channel activity by diclofenac (20 µM) in T84 cells. Apical Cl⁻ current analysis was performed with basolateral membrane permeabilization by amphotericin B. After stabilization of diclofenac -inhibited apical Cl⁻ current, solutions containing forskolin and diclofenac were removed, and hemichambers were gently washed three times and filled with fresh solutions containing only forskolin. At the end of experiment, CFTR_inh-172 (5 µM) was added into apical solutions. A representative current tracing of 4 separate experiments is shown.</p

Effects of diclofenac on cAMP-activated Cl⁻ secretion in T84 cells.

<p>(A) A model of Cl⁻ secretion by an enterocyte showing transport proteins involved in transepithelial Cl⁻ secretion. CFTR, cystic fibrosis transmembrane conductance regulator; IRC, inwardly rectifying Cl⁻ channel; CaCC, Ca²⁺-activated Cl⁻ channel; KCNQ1/KCNE3, cAMP-activated K⁺ channel; K_Ca3.1, Ca²⁺-activated K⁺ channel. (B) CFTR mediated cAMP-activated Cl⁻ secretion induced by forskolin. A representative short-circuit current tracing is shown (n = 5). (C) Effect of diclofenac on cAMP-activated Cl⁻ secretion. Diclofenac at the indicated concentrations was added into both apical and basolateral solutions (n = 4). (D) Polarity of inhibition by diclofenac on cAMP-activated Cl⁻ secretion. Diclofenac at the indicated concentrations was sequentially added into basolateral and apical solutions, respectively. A representative short-circuit current tracing is shown (n = 5). (E) The inhibition by diclofenac does not require metabolic activation. T84 cell monolayers were pretreated with 1-ABT, an inhibitor of CYP enzymes (1 mM). Diclofenac at the indicated concentrations was added into both apical and basolateral solutions. A representative short-circuit current tracing is shown (n = 5).</p

Effect of diclofenac on NKCC1 activity in T84 cells.

<p>(A) Diagrammatic protocol for Tl⁺ influx-based assay of NKCC1 activity. After loading with Tl⁺-sensitive dye, T84 cells were incubated for 15 min in the Cl⁻-free buffer containing Tl₂SO₄ and clotrimazole (to block K⁺ channels) with or without diclofenac (200 µM). Then, NaCl solution (final concentration of NaCl = 135 mM) was added to stimulate NKCC1-mediated Tl⁺ influx causing an increase in fluorescence from Tl⁺-sensitive dye. NKCC1 activity was analyzed from the slope of linear increases in fluorescent intensity within 15 s after NaCl addition. (B) Representatives of relative fluorescent signals from 5 separate experiments, without (control) and with diclofenac (200 µM). Bumetanide (100 µM), a known inhibitor of NKCC1, was used as a positive control.</p

Antidiarrheal application of diclofenac.

<p>(A) Effect of diclofenac on cholera toxin (CT)-induced Cl⁻ secretion in T84 cells. Short-circuit current measurements were performed in T84 cells. After stimulation of Cl⁻ secretion by CT (1 µg/ml), diclofenac was added into both apical and basolateral solutions (n = 5). (B) Effect of diclofenac on cAMP-induced Cl⁻ secretion in mouse intestinal sheets. Mouse intestinal sheets were mounted in Ussing chambers and Cl⁻ secretion was stimulated by forskolin (20 µM). Diclofenac was added into both apical and basolateral solutions (n = 4). (C) Effect of diclofenac on CT-induced intestinal fluid secretion in mice. Ileal loops were instilled with PBS or PBS containing CT (1 µg/loop) with or without concomitant intraperitoneal administration of diclofenac (30 mg/kg). Four h later, ileal loops were removed for loop weight/length ratio measurements; (left) representative photographs of ileal loops, (right) summary of data. Data are expressed as means of loop weight/length ratio ± S.E.M. *, p<0.05 compared with CT-treated control (n = 8). (D) Effect of diclofenac on <i>V. cholerae</i>-induced intestinal fluid secretion in mice. Ileal loops were inoculated with PBS or PBS containing <i>V. cholerae</i> (10⁷ CFU/loop) with or without concomitant intraperitoneal administration of diclofenac (30 mg/kg). Twelve hours post-inoculation, ileal loops were removed for measurements of loop weight/length ratio; (left) representative photographs of ileal loops, (right) summary of data. Data are expressed as means of loop weight/length ratio ± S.E.M. **, p<0.01 compared with <i>V. cholerae</i>-inoculated control (n = 6) (E) Effect of diclofenac on intestinal fluid absorption. Ileal loops were instilled with PBS with or without intraperitoneal administration of diclofenac (30 mg/kg). Twenty or forty min later, ileal loops were removed for loop weight/length ratio measurements; (left) representative photographs of ileal loops, (right) summary of data. Ileal loops at 1 min after PBS instillation and ileal loops without PBS instillation (-PBS) were shown for comparisons. Data are expressed as means of loop weight/length ratio ± S.E.M. (n = 6–8). NS, non-statistical difference compared with control at the same time point.</p

Effects of diclofenac on CaCC, IRC and Ca²⁺-activated basolateral K⁺ channel.

<p>(A) Inhibition of CaCC-mediated Cl⁻ transport by diclofenac. (left) Representative tracing of CaCC-mediated apical Cl⁻ current with basolateral membrane permeabilization. CFTR_inh-172 (5 µM) and ATP (100 µM) were added into apical solutions before addition of diclofenac into both apical and basolateral solutions (n = 5). (right) Diclofenac had no effect on ATP-induced CaMKII phosphorylation. T84 cells were incubated for 20 min with vehicle (control), ATP (100 µM), or ATP (100 µM) plus diclofenac (20 µM). CaMKII phosphorylation was investigated using immunoblot analysis of phosphorylated CaMKII. Results of band intensity analysis are expressed as relative band intensity. NS, non-statistical difference; *, p<0.05 compared with control (n = 3). (B) Inhibition of IRC-mediated Cl⁻ transport by diclofenac. In this experiment, apical Cl⁻ current analysis was performed. CFTR_inh-172 (5 µM) was added into apical solution before IRC activation by forskolin (20 µM). Diclofanac was added into both apical and basolateral solutions (n = 5). (C) Inhibition of Ca²⁺-activated basolateral K⁺ channel (K_Ca3.1) by diclofenac. In this experiment, basolateral K⁺ current measurements were performed in the presence of BaCl₂ (5 mM) in the apical solution. DMSO (control) or diclofenac was added into both apical and basolateral solutions before activation of K_Ca3.1 by ATP (100 µM) (n = 4–6).</p