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. 2019 Jan 1;316(1):G1-G14.
doi: 10.1152/ajpgi.00266.2018. Epub 2018 Oct 11.

Enhanced gastrointestinal passive paracellular permeability contributes to the obesity-associated hyperoxaluria

Mohamed Bashir  1 , Jon Meddings  2 , Altayeb Alshaikh  1 , Daniel Jung  1 , Kim Le  2 , Ruhul Amin  1 , Sireesha Ratakonda  1 , Sapna Sharma  1 , Ignacio Granja  3 , Mustafa Satti  1 , John Asplin  3 , Hatim Hassan  1
Affiliations

Enhanced gastrointestinal passive paracellular permeability contributes to the obesity-associated hyperoxaluria

Mohamed Bashir et al. Am J Physiol Gastrointest Liver Physiol. .

Abstract

Most kidney stones (KS) are composed of calcium oxalate and small increases in urine oxalate enhance the stone risk. Obesity is a risk factor for KS, and urinary oxalate excretion increases with increased body size. We previously established the obese ob/ob ( ob) mice as a model (3.3-fold higher urine oxalate) to define the pathogenesis of obesity-associated hyperoxaluria (OAH). The purpose of this study was to test the hypothesis that the obesity-associated enhanced small intestinal paracellular permeability contributes to OAH by increasing passive paracellular intestinal oxalate absorption. ob Mice have significantly higher jejunal (1.6-fold) and ileal (1.4-fold) paracellular oxalate absorption ex vivo and significantly higher (5-fold) urine [13C]oxalate following oral gavage with [13C]oxalate, indicating increased intestinal oxalate absorption in vivo. The observation of higher oxalate absorption in vivo compared with ex vivo suggests the possibility of increased paracellular permeability along the entire gut. Indeed, ob mice have significantly higher fractions of the administered sucrose (1.7-fold), lactulose (4.4-fold), and sucralose (3.1-fold) excreted in the urine, reflecting increased gastric, small intestinal, and colonic paracellular permeability, respectively. The ob mice have significantly reduced gastrointestinal occludin, zonula occludens-1, and claudins-1 and -3 mRNA and total protein expression. Proinflammatory cytokines and oxidative stress, which are elevated in obesity, significantly enhanced paracellular intestinal oxalate absorption in vitro and ex vivo. We conclude that obese mice have significantly higher intestinal oxalate absorption and enhanced gastrointestinal paracellular permeability in vivo, which would likely contribute to the pathogenesis of OAH, since there is a transepithelial oxalate concentration gradient to drive paracellular intestinal oxalate absorption. NEW & NOTEWORTHY This study shows that the obese ob/ob mice have significantly increased gastrointestinal paracellular oxalate absorption and remarkably enhanced paracellular permeability along the entire gut in vivo, which are likely mediated by the obesity-associated increased systemic and intestinal inflammation and oxidative stress. A transepithelial oxalate concentration gradient driving gastrointestinal paracellular oxalate absorption exists, and therefore, our novel findings likely contribute to the hyperoxaluria observed in the ob/ob mice and hence to the pathogenesis of obesity-associated hyperoxaluria.

Keywords: gastrointestinal paracellular permeability; hyperoxaluria; inflammation; intestinal oxalate absorption; obesity; oxidative stress; tight junction proteins.

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Figures

Fig. 1.
Fig. 1.
Jejunal (A) and ileal (B) [14C]oxalate and [3H]mannitol absorptive fluxes in the ob mice and their lean controls. ~2-cm jejunal and ileal segments were obtained from the ob and control mice, and one end of each segment was sutured. The loop was filled with DMEM medium containing 1 mM mannitol, 0.1% FBS, 2 µM [14C]oxalate, and 0.02 µM [3H]mannitol, followed by closure of the other end. The loops were then transferred to wells with 2 ml of DMEM medium containing 1 mM mannitol, 0.1% FBS, 2 µM oxalate, and 0.02 µM mannitol and incubated at 37°C. Samples were taken at 30 min to determine the flux of [14C]oxalate and [3H]mannitol from the lumen to the medium outside bathing the loops. The ob mice have significantly higher jejunal (*P < 0.04 for ob compared with control, with regards to oxalate and mannitol, by unpaired t-test; n = 18–21) and ileal (*P < 0.02 and < 0.04 for ob compared with control, with regards to oxalate and mannitol, respectively, by unpaired t-test; n = 17–21).
Fig. 2.
Fig. 2.
Fraction of the administered [13C]oxalate excreted in the urine of the ob/ob (ob) mice and their lean controls. The ob mice and their controls were given a single dose of [13C]oxalate (5 µg/g) by oral gavage, followed by urine collection over the next 6-h period for [13C]oxalate and total oxalate measurements. The ob mice have significantly higher fractions of the administered [13C]oxalate excreted in the urine (*P < 0.02 for ob compared with controls, by unpaired t-test, n = 27 control mice and 32 ob mice).
Fig. 3.
Fig. 3.
Fractions of the administered sucrose (A), lactulose (B), mannitol (C), lactulose:mannitol ratio (D), sucralose (E), and creatinine (F) excreted in the urine of the ob/ob (ob) mice and their lean controls. Following food removal for 15 h, the ob mice and their controls received sucrose (100 mg per mouse; n = 10), lactulose (0.5 mg/g; n = 9–10), mannitol (0.32 mg/g; n = 9–10), sucralose (6 mg per mouse; n = 10), or creatinine (0.6 mg/g; n = 10) by oral gavage, and then the mice were placed in metabolic cages for 24 h for urine collection. The ob mice have significantly higher fractions of the administered sucrose, sucralose, and creatinine excreted in the urine (*P < 0.02, 0.03, and 0.0006, for ob compared with controls with regards to sucrose, sucralose, and creatinine, respectively, by unpaired t-test). The ob mice also have significantly higher fractions of the administered lactulose and mannitol excreted in the urine (*P < 0.002, 0.003, and 0.0001, for ob compared with controls with regards to lactulose, mannitol, and lactulose:mannitol ratio, respectively, by Mann-Whitney test).
Fig. 4.
Fig. 4.
Gastrointestinal occludin mRNA expression levels in the ob/ob (ob) mice and their lean controls. Total RNA was isolated from the stomach, jejunum, ileum, cecum, and distal colon (D. Colon) of the ob mice and their controls for real-time PCR analysis. Values are means ± SE of 5–6 independent experiments each of which was done in duplicate or triplicate. Relative occludin mRNA expression levels are expressed as a percentage of control normalized to GAPDH. The ob mice have significantly reduced gastric, jejunal, ileal, cecal, and distal colonic occludin mRNA expression levels (*P < 0.05, 0.03, 0.05, 0.05, and 0.05, for ob compared with controls with regards to stomach, jejunum, ileum, cecum, and distal colon, respectively, by one-sample Wilcoxon signed-rank test).
Fig. 5.
Fig. 5.
Gastrointestinal occludin total protein expression levels in the ob/ob (ob) mice and their lean controls. Representative Western blots analyses of occludin (Oc) total protein expression in protein samples (10–30 µg protein/lane) from the stomach (A), jejunum (B), ileum (C), cecum (D), and distal colon (E) of the ob mice and their controls (Con). The bottom of the same blots were probed with an anti-β-actin (Actin) antibody to normalize loading of protein in each lane. F: the densitometry of immunoblot results. Western blot band density was quantified using ImageJ software. Values are means ± SE for 4–5 independent experiments of relative occludin abundance to β-actin and are presented as a percentage of the control value. The ob mice have significantly reduced gastric, jejunal, ileal, cecal, and distal colonic occludin total protein expression levels (*P < 0.02, 0.03, 0.03, 0.008, and 0.008 for ob compared with controls with regards to stomach, jejunum, ileum, cecum, and distal colon, respectively, by Mann-Whitney test).
Fig. 6.
Fig. 6.
Gastrointestinal zonula occludens-1 (ZO-1) mRNA (A) and cecal (B) and distal colonic (C) ZO-1 total protein expression levels in the ob/ob (ob) mice and their lean controls. A: total RNA was isolated from the stomach, jejunum, ileum, cecum, and distal colon (D. Colon) of the ob mice and their controls for real-time PCR analysis. Values are means ± SE of 5–8 independent experiments, each of which was done in duplicate or triplicate. Relative ZO-1 mRNA expression levels are expressed as a percentage of control normalized to GAPDH. The ob mice have significantly reduced gastric, jejunal, ileal, cecal, and distal colonic ZO-1 mRNA expression levels (*P < 0.02, 0.02, 0.03, 0.05, and 0.05, for ob compared with controls with regards to stomach, jejunum, ileum, cecum, and distal colon, respectively, by one-sample Wilcoxon signed-rank test). B and C: representative Western blots analyses of ZO-1 total protein expression in protein samples (30 µg protein/lane) from the cecum (B) and distal colon (C) of the ob mice and their controls (Con). The bottom of the same blots were probed with an anti-GAPDH (GAP) antibody to normalize loading of protein in each lane (bottom). The corresponding densitometry of immunoblot results is shown on the right of each blot. Western blot band density was quantified using ImageJ software. Values are means ± SE for 4 independent experiments of relative ZO-1 abundance to GAPDH and are presented as a percentage of the control value. The ob mice have significantly reduced cecal and distal colonic ZO-1 total protein expression levels (*P < 0.03 and 0.03 for ob compared with controls with regards to cecum and distal colon, respectively, by Mann-Whitney test).
Fig. 7.
Fig. 7.
Jejunal and ileal claudin-1 mRNA (A) and total protein (B–D) expression levels in the ob/ob (ob) mice and their lean controls. A: total RNA was isolated from the jejunum and ileum of the ob mice and their controls for real-time PCR analysis. Values are means ± SE of 5–6 independent experiments each of which was done in duplicate or triplicate. Relative claudin-1 mRNA expression levels are expressed as a percentage of control normalized to GAPDH. The ob mice have significantly reduced jejunal and ileal claudin-1 mRNA expression levels (*P < 0.05 and 0.03 for ob compared with controls with regards to jejunum and ileum, respectively, by one-sample Wilcoxon signed-rank test). B and C: representative Western blots analyses of claudin-1 (Cld1) total protein expression in protein samples (10 µg protein/lane) from the jejunum (B) and ileum (C) of the ob mice and their controls (Con). The bottom of the same blots were probed with an anti-β-actin (Actin) antibody to normalize loading of protein in each lane (bottom). D: densitometry of immunoblot results. Western blot band density was quantified using ImageJ software. Values are means ± SE for 4–5 independent experiments of relative claudin-1 abundance to β-actin and are presented as a percentage of the control value. The ob mice have significantly reduced jejunal and ileal claudin-1 total protein expression levels (*P < 0.04 and 0.03 for ob compared with controls with regards to jejunum and ileum, respectively, by Mann-Whitney test).
Fig. 8.
Fig. 8.
Jejunal and ileal claudin-3 mRNA (A) and total protein (B–D) expression levels in the ob/ob (ob) mice and their lean controls. A: total RNA was isolated from the jejunum and ileum of the ob mice and their controls for real-time PCR analysis. Values are means ± SE of 6–7 independent experiments each of which was done in duplicate or triplicate. Relative claudin-3 mRNA expression levels are expressed as a percentage of control normalized to GAPDH. The ob mice have significantly reduced jejunal and ileal claudin-3 mRNA expression levels (*P < 0.02 and 0.03 for ob compared with controls with regards to jejunum and ileum, respectively, by one-sample Wilcoxon signed-rank test). B and C: representative Western blots analyses of claudin-3 (Cld3) total protein expression in protein samples (10 µg protein/lane) from the jejunum (B) and ileum (C) of the ob mice and their controls (Con). The bottom of the same blots were probed with an anti-β-actin (Actin) antibody to normalize loading of protein in each lane (bottom). D: densitometry of immunoblot results. Western blot band density was quantified using ImageJ software. Values are means ± SE for 4–6 independent experiments of relative claudin-3 abundance to β-actin and are presented as a percentage of the control value. The ob mice have significantly reduced jejunal and ileal claudin-3 total protein expression levels (*P < 0.05 and 0.03 for ob compared with controls with regards to jejunum and ileum, respectively, by Mann-Whitney test).
Fig. 9.
Fig. 9.
Effects of proinflammatory cytokines on [14C]oxalate and [3H]mannitol absorptive fluxes in vitro and ex vivo. A: effects of IFN-γ (IFN) and TNF-α (TNF) on [14C]oxalate and [3H]mannitol absorptive fluxes (apical to basolateral) in Caco2-BBE (Caco2) cells. Caco2 cells grown on snapwell inserts were pretreated basolaterally with IFN (10 ng/ml) for 24 h, followed by addition of TNF (10 ng/ml) for another 24 h (with continued presence of IFN) or vehicle (control), and then the cells were mounted in Ussing chambers. Papp, apparent permeability coefficient. Values are means ± SE of 17–18 monolayers. IFN + TNF significantly increased [14C]oxalate and [3H]mannitol fluxes absorptive fluxes (*P < 0.0008 and P < 0.0006 for IFN + TNF compared with control, with regards to oxalate and mannitol, respectively, by unpaired t-test). B: effects of TNF-α on mouse jejunal unidirectional [14C]oxalate and [3H]mannitol absorptive fluxes. BLAB/c mice were treated with TNF-α (5 µg ip) or vehicle (control), and then jejunal tissues (n = 24–35 tissues) were isolated and mounted in Ussing chambers 90 min later. TNF-α significantly enhanced [14C]oxalate and [3H]mannitol absorptive fluxes (*P < 0.008 and < 0.003 for TNF-α compared with control, with regards to oxalate and mannitol, respectively, by unpaired t-test).
Fig. 10.
Fig. 10.
Effects of H2O2 on [14C]oxalate and [3H]mannitol absorptive fluxes in Caco2-BBE (Caco2) cells (A) and BALB/c jejunal loops (B). A: Caco2 cells grown on snapwell inserts were mounted in Ussing chambers, and were then treated basolaterally with H2O2 (10 mM) or vehicle (control). [14C]oxalate and [3H]mannitol absorptive fluxes were performed as described in materials and methods. Papp, apparent permeability coefficient. Values are means ± SE of 7 monolayers. H2O2 significantly increased [14C]oxalate and [3H]mannitol absorptive fluxes (*P < 0.005 and P < 0.008 for H2O2 compared with control, with regards to oxalate and mannitol, by Mann-Whitney test). B: jejunal loops from BALB/c mice were developed as described in materials and methods and were then treated with H2O2 (1 mM inside and outside the loop) or vehicle (control). Samples were taken at 60 and 90 min to determine the 30-min flux of [14C]oxalate and [3H]mannitol from the lumen to the medium outside bathing the loops. H2O2 significantly increased jejunal [14C]oxalate and [3H]mannitol absorptive fluxes (*P < 0.009 and P < 0.05 for H2O2 compared with control, with regards to oxalate and mannitol, respectively, by Mann-Whitney test; n = 6).
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