Marguerite Hatch

Enteric oxalate elimination is induced and oxalate is normalized in a mouse model of primary hyperoxaluria following intestinal colonization with Oxalobacter.

Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691-698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals.

The roles and mechanisms of intestinal oxalate transport in oxalate homeostasis.

The mammalian intestine has an important role in the dynamics of oxalate exchange and thereby is significant in the etiology of calcium oxalate nephrolithiasis. Here we review some of the phenomenologic observations that have led to the conclusion that anion exchangers (antiporters) are important mediators of secondarily active, net oxalate transport along the intestine (both absorptive and secretory). Understanding the mechanisms of transepithelial oxalate transport has been advanced radically in recent years by the identification of the solute-linked carrier (SLC)26 family of anion transporters, which has facilitated the identification of specific proteins mediating individual apical or basolateral oxalate transport pathways. Moreover, identification of specific exchangers has underscored their relative importance to oxalate homeostasis as revealed by using knockout mouse models and has facilitated studies of oxalate transport regulation in heterologous expression systems. Finally, the significance of oxalate degrading bacteria to oxalate homeostasis is considered from basic and applied perspectives.

Steatorrhea and Hyperoxaluria Occur after Gastric Bypass Surgery in Obese Rats Regardless of Dietary Fat or Oxalate

PURPOSE We determined the effect of dietary fat and oxalate on fecal fat excretion and urine parameters in a rat model of Roux-en-Y gastric bypass surgery. MATERIALS AND METHODS Diet induced obese Sprague-Dawley® rats underwent sham surgery as controls (16), or Roux-en-Y gastric bypass surgery (19). After recovery, rats had free access to a normal calcium, high fat (40%) diet with or without 1.5% potassium oxalate for 5 weeks and then a normal (10%) fat diet for 2 weeks. Stool and urine were collected after each period. Fecal fat was determined by gas chromatography and urine metabolites were evaluated by assay spectrophotometry. RESULTS Daily fecal fat excretion remained low in controls on either diet. However, Roux-en-Y gastric bypass rats ingested a food quantity similar to that of controls but had eightfold higher fecal fat excretion (p <0.001) and heavier stools (p = 0.02). Compared to controls, gastric bypass rats on the high fat diet with potassium oxalate had a fivefold increase in urine oxalate excretion (p <0.001), while gastric bypass rats without potassium oxalate had a twofold increase in urine calcium (p <0.01). Lowering dietary fat in gastric bypass rats with potassium oxalate led to a 50% decrease in oxalate excretion (p <0.01), a 30% decrease in urine calcium and a 0.3 U increase in urine pH (p <0.001). CONCLUSIONS In this Roux-en-Y gastric bypass model high fat feeding resulted in steatorrhea, hyperoxaluria and low urine pH, which were partially reversible by lowering the dietary fat and oxalate content. Roux-en-Y gastric bypass rats on normal fat and no oxalate diets excreted twice as much oxalate as age matched, sham operated controls. Although Roux-en-Y gastric bypass hyperoxaluria appears primarily mediated by gut and diet, secondary causes of oxalogenesis from liver or other mechanisms deserve further exploration.

Renal and Intestinal Handling of Oxalate following Oxalate Loading in Rats

Background: The enteric excretion of oxalate has been established in rats with chronic renal failure induced by 5/6 nephrectomy [Hatch et al.: Regulatory aspects of oxalate secretion in enteric oxalate elimination. JASN 1999;10:S324] and this response is mediated by angiotensin II receptor activation. However, the renal and intestinal handling of oxalate has not been evaluated for other common models of hyperoxaluria that simulate primary hyperoxaluria or oxalate stone disease. Methods: We assessed the renal clearances of creatinine, oxalate and calcium in three rat models: chronic hyperoxaluria (CH), chronic hyperoxaluria with hyperoxalemia (CHH) and acute hyperoxaluria (AH), and evaluated the transepithelial transport of oxalate and chloride in large intestinal segments of these models and their sensitivity to angiotensin II antagonism. Results: Hyperoxaluria alone (CH) was not associated with changes in colonic oxalate transport, whereas changes in net oxalate transport in distal colon from absorption to net secretion was observed in models with hyperoxalemia (CHH and AH). Angiotensin II receptor antagonism with losartan reduced net colonic oxalate secretion in AH but not CHH. Conclusions: Colonic secretion of oxalate is stimulated in rat models exhibiting hyperoxalemia suggesting a contribution of this extrarenal pathway to regulation of oxalate mass balance in clinical conditions manifesting hyperoxalemia. The transport avenues and regulatory mechanisms may not be identical to those observed during adaptive enteric oxalate secretion in chronic renal failure models.

Kidney stone incidence and metabolic urinary changes after modern bariatric surgery: review of clinical studies, experimental models, and prevention strategies ☆

Bariatric surgery has been associated with increased metabolic kidney stone risk and post-operative stone formation. A MEDLINE search, performed for articles published between January 2005 and November 2013, identified 24 pertinent studies containing 683 bariatric patients with 24-hour urine profiles, 6,777 bariatric patients with kidney stone incidence, and 7,089 non-stone forming controls. Of all procedures reviewed, only Roux-en-Y gastric bypass (RYGB) was linked to post-operative kidney stone development, increasing stone incidence two-fold in non-stone formers (8.5%) and four-fold in patients with previous stone history (16.7%). High quality evidence from 7 studies (n=277 patients) before and after RYGB identified the following post-RYGB urinary lithogenic risk factors: 30% reduction in urine volume (the main driver of urinary crystal saturation), 40% reduction in urinary citrate (a potent stone inhibitor), and 50% increase in urinary oxalate (a stone promotor). Based on this, a summary of strategies to reduce calcium oxalate stone risk following RYGB is provided. Furthermore, recent experimental RYGB studies are assessed for insights into the pathophysiology of oxalate handling, and the literature in gut anion (oxalate) transport is reviewed. Finally, as a potential probiotic therapy for hyperoxaluria, primary data from our laboratory is presented, demonstrating a 70% reduction in urinary oxalate levels in four experimental RYGB animals after colonization with Oxalobacter formigines, a non-pathogenic gut commensal that uses oxalate as an energy source.Overall, urine profiles and kidney stone risk following bariatric surgery appear modifiable by dietary adjustments, appropriate supplementation, and lifestyle changes. For hyperoxaluria resistant to dietary oxalate restriction and calcium binding, well-designed human investigations are needed to identify additional means of lowering urinary oxalate, such as Oxalobacter colonization or empiric pyridoxine therapy. Further investigations are also needed to determine tolerability and compliance of stone prevention strategies, such as citrate supplementation and hydration, in this population.

Local upregulation of colonic angiotensin II receptors enhances potassium excretion in chronic renal failure

The role of angiotensin II (ANG II) in colonic secretion of K+ was examined in rats with chronic renal failure (CRF). The basal net secretory flux of86Rb+(as a tracer for K+) across the CRF distal colon (-0.20 ± 0.04 μeq ⋅ cm-2 ⋅ h-1) was reversed to an absorptive flux (0.35 ± 0.05 μeq ⋅ cm-2 ⋅ h-1) by injecting the rats with the AT1receptor antagonist, losartan. A similar result was observed when losartan was added to the CRF colonic tissue in vitro. In contrast, an AT2 receptor antagonist, PD-123319, did not reverse the CRF-induced alterations in Rb+ transport across the short-circuited colonic tissue. Plasma concentrations of ANG II, aldosterone, and K+, as well as the ANG II content of colonic tissues from CRF and normal rats, were similar. However, specific125I-labeled ANG II binding sites in rat distal colon increased twofold in CRF [maximal specific binding (Bmax) = 28.6 ± 1.6 fmol/mg protein] compared with normal (Bmax = 15.2 ± 0.4 fmol/mg protein). These studies suggest that CRF-induced secretion of K+ by the colon is mediated by an upregulation of AT1 receptors present in CRF.The role of angiotensin II (ANG II) in colonic secretion of K+ was examined in rats with chronic renal failure (CRF). The basal net secretory flux of 86Rb+ (as a tracer for K+) across the CRF distal colon (-0.20 +/- 0.04 mu eq.cm-2.h-1) was reversed to an absorptive flux (0.35 +/- 0.05 mu eq.cm-2.h-1) by injecting the rats with the AT1 receptor antagonist, losartan. A similar result was observed when losartan was added to the CRF colonic tissue in vitro. In contrast, an AT2 receptor antagonist, PD-123319, did not reverse the CRF-induced alterations in Rb+ transport across the short-circuited colonic tissue. Plasma concentrations of ANG II, aldosterone, and K+, as well as the ANG II content of colonic tissues from CRF and normal rats, were similar. However, specific 125I-labeled ANG II binding sites in rat distal colon increased twofold in CRF [maximal specific binding (Bmax) = 28.6 +/- 1.6 fmol/mg protein] compared with normal (Bmax = 15.2 +/- 0.4 fmol/mg protein). These studies suggest that CRF-induced secretion of K+ by the colon is mediated by an upregulation of AT1 receptors present in CRF.

A human strain of Oxalobacter (HC-1) promotes enteric oxalate secretion in the small intestine of mice and reduces urinary oxalate excretion

Enteric oxalate secretion that correlated with reductions in urinary oxalate excretion was previously reported in a mouse model of primary hyperoxaluria, and in wild type (WT) mice colonized with a wild rat strain (OXWR) of Oxalobacter (Am J Physiol 300:G461–G469, 2010). Since a human strain of the bacterium is more likely to be clinically used as a probiotic therapeutic, we tested the effects of HC-1 in WT. Following artificial colonization of WT mice with HC-1, the bacteria were confirmed to be present in the large intestine and, unexpectedly, detected in the small intestine for varying periods of time. The main objective of the present study was to determine whether the presence of HC-1 promoted intestinal secretion in the more proximal segments of the gastrointestinal tract. In addition, we determined whether HC-1 colonization led to reductions in urinary oxalate excretion in these mice. The results show that the human Oxalobacter strain promotes a robust net secretion of oxalate in the distal ileum as well as in the caecum and distal colon and these changes in transport correlate with the beneficial effect of reducing renal excretion of oxalate. We conclude that OXWR effects on intestinal oxalate transport and oxalate homeostasis are not unique to the wild rat strain and that, mechanistically, HC-1 has significant potential for use as a probiotic treatment for hyperoxaluria especially if it is also targeted to the upper and lower gastrointestinal tract.

Transcellular oxalate and Cl− absorption in mouse intestine is mediated by the DRA anion exchanger Slc26a3, and DRA deletion decreases urinary oxalate

Active transcellular oxalate transport in the mammalian intestine contributes to the homeostasis of this important lithogenic anion. Several members of the Slc26a gene family of anion exchangers have a measurable oxalate affinity and are expressed along the gut, apically and basolaterally. Mouse Slc26a6 (PAT1) targets to the apical membrane of enterocytes in the small intestine, and its deletion results in net oxalate absorption and hyperoxaluria. Apical exchangers of the Slc26a family that mediate oxalate absorption have not been established, yet the Slc26a3 [downregulated in adenoma (DRA)] protein is a candidate mediator of oxalate uptake. We evaluated the role of DRA in intestinal oxalate and Cl(-) transport by comparing unidirectional and net ion fluxes across short-circuited segments of small (ileum) and large (cecum and distal colon) intestine from wild-type (WT) and DRA knockout (KO) mice. In WT mice, all segments demonstrated net oxalate and Cl(-) absorption to varying degrees. In KO mice, however, all segments exhibited net anion secretion, which was consistently, and solely, due to a significant reduction in the absorptive unidirectional fluxes. In KO mice, daily urinary oxalate excretion was reduced 66% compared with that in WT mice, while urinary creatinine excretion was unchanged. We conclude that DRA mediates a predominance of the apical uptake of oxalate and Cl(-) absorbed in the small and large intestine of mice under short-circuit conditions. The large reductions in urinary oxalate excretion underscore the importance of transcellular intestinal oxalate absorption, in general, and, more specifically, the importance of the DRA exchanger in oxalate homeostasis.

Increased colonic sodium absorption in rats with chronic renal failure is partially mediated by AT1 receptor agonism

To test the hypothesis that colonic Na(+) transport is altered in the 5/6 nephrectomized rat model of chronic renal failure (CRF), we measured Na(+) fluxes across distal colon from control (CON), CRF, and CRF rats treated with the angiotensin II (ANG II) receptor antagonist losartan (+LOS). We also evaluated overall fluid and Na(+) balance and compared colonic protein and mRNA expression profiles for electroneutral [sodium-hydrogen exchanger (NHE)] and electrogenic Na(+) transport [epithelial sodium channel (ENaC)] in these groups. Consistent with a 60% enhancement in colonic Na(+) absorption in CRF, urinary Na(+) excretion increased by about 50% while serum Na(+) homeostasis was maintained. These CRF-induced changes in Na(+) handling were normalized by treatment with LOS. Net Na(+) absorption was also stimulated in in vitro tissues from CON rats following acute serosal addition of ANG II (10(-7) M), and this increase was blocked by AT(1) antagonism but not by an AT(2) antagonist. In CRF, colonic protein and mRNA expression variably increased for apical NHE2, NHE3, and ENaC alpha-, beta-, gamma-subunits, whereas expression of basolateral NHE1 and Na(+)-K(+)-ATPase (alpha-isoform) remained unaltered. Upregulation of the ENaC subunit mRNA was attenuated somewhat by LOS treatment. Previously, we showed that colonic AT(1) receptor protein is upregulated twofold in CRF, and here we find that AT(1) and AT(2) mRNA and AT(2) protein abundance is unchanged in CRF. We conclude that Na(+) absorption in CRF rat distal colon is increased due to elevated expression of proteins mediating electroneutral and electrogenic uptake and that it is partially mediated by AT(1) receptors.

Sulfate secretion and chloride absorption are mediated by the anion exchanger DRA (Slc26a3) in the mouse cecum

Inorganic sulfate (SO₄²⁻) is essential for a multitude of physiological processes. The specific molecular pathway has been identified for uptake from the small intestine but is virtually unknown for the large bowel, although there is evidence for absorption involving Na⁺-independent anion exchange. A leading candidate is the apical chloride/bicarbonate (Cl⁻/HCO₃⁻) exchanger DRA (down-regulated in adenoma; Slc26a3), primarily linked to the Cl⁻ transporting defect in congenital chloride diarrhea. The present study set out to characterize transepithelial ³⁵SO₄²⁻ and ³⁶Cl⁻ fluxes across the isolated, short-circuited cecum from wild-type (WT) and knockout (KO) mice and subsequently to define the contribution of DRA. The cecum demonstrated simultaneous net SO₄²⁻ secretion (-8.39 ± 0.88 nmol·cm⁻²·h⁻¹) and Cl⁻ absorption (10.85 ± 1.41 μmol·cm⁻²·h⁻¹). In DRA-KO mice, SO₄²⁻ secretion was reversed to net absorption via a 60% reduction in serosal to mucosal SO₄²⁻ flux. Similarly, net Cl⁻ absorption was abolished and replaced by secretion, indicating that DRA represents a major pathway for transcellular SO₄²⁻ secretion and Cl⁻ absorption. Further experiments including the application of DIDS (500 μM), bumetanide (100 μM), and substitutions of extracellular Cl⁻ or HCO₃⁻/CO₂ helped to identify specific ion dependencies and driving forces and suggested that additional anion exchangers were operating at both apical and basolateral membranes supporting SO₄²⁻ transport. In conclusion, DRA contributes to SO₄²⁻ secretion via DIDS-sensitive HCO₃⁻/SO₄²⁻ exchange, in addition to being the principal DIDS-resistant Cl⁻/HCO₃⁻ exchanger. With DRA linked to the pathogenesis of other gastrointestinal diseases extending its functional characterization offers a more complete picture of its role in the intestine.