Small intestine resection increases oxalate and citrate transporter expression and calcium oxalate crystal formation in rat hyperoxaluric kidneys

Abstract

Short bowel (SB) increases the risk of kidney stones. However, the underlying mechanism is unclear. Here, we examined how SB affected renal oxalate and citrate handlings for in vivo hyperoxaluric rats and in vitro tubular cells. SB was induced by small intestine resection in male Wistar rats. Sham-operated controls had no resection. After 7 days of recovery, the rats were divided into control, SB (both fed with distilled water), ethylene glycol (EG), and SB+EG (both fed with 0.75% EG for hyperoxaluric induction) groups for 28 days. We collected the plasma, 24 h of urine, kidney, and intestine tissues for analysis. Hypocitraturia was found and persisted up to 28 days for the SB group. Hypocalcemia and high plasma parathyroid hormone (PTH) levels were found in the 28-day SB rats. SB aggravated EG-mediated oxalate nephropathy by fostering hyperoxaluria and hypocitraturia, and increasing the degree of supersaturation and calcium oxalate (CaOx) crystal deposition. These effects were associated with renal up-regulations of the oxalate transporter solute carrier family 26 (Slc26)a6 and citrate transporter sodium-dependent dicarboxylate cotransporter-1 (NaDC-1) but not Slc26a2. The effects of PTH on the SB kidneys were then examined in NRK-52E tubular cells. Recombinant PTH attenuated oxalate-mediated cell injury and up-regulated NaDC-1 via protein kinase A (PKA) activation. PTH, however, showed no additive effects on oxalate-induced Slc26a6 and NaDC-1 up-regulation. Together, these results demonstrated that renal NaDC-1 upregulation-induced hypocitraturia weakened the defense against Slc26a6-mediated hyperoxaluria in SB kidneys for excess CaOx crystal formation. Increased tubular NaDC-1 expression caused by SB relied on PTH.

Keywords: calcium oxalate crystal; hyperoxaluria; hypocitraturia; parathyroid hormone; short bowel.

Figure 1. Anatomic diagram of the small intestine resection in SB rats

(A) The duodenum, small intestine, and cecum were exposed from the abdominal cavity under aseptic conditions after the abdomen was opened. (B) Four star signs (red) indicate the ligation of the vascular branches of the superior mesenteric artery, which were cut distal during the resection. Two scissor signs (blue) indicate the resection sites of the small intestine for further anastomosis.

Figure 2. Basic health parameters after 28 days of treatment

The data were obtained from a metabolic cage study collected over a 24-h period after 28 days of induction of body weight (BW) gain (A), daily food intake (B), feces amount (C), urine output (D), and kidney weight (KW) to BW ratio (E). A significant increase in the KW/BW ratio was found in the SB+EG group when compared with the EG or SB group. n=6 in each group. C, control; EG, ethylene glycol (hyperoxaluria); SB, small intestine resection; SB+EG, small intestine resection co-treated with ethylene glycol. ^#P<0.05, SB+EG vs. EG group; ^@P<0.05, SB+EG vs. SB group.

Figure 3. Changes in the plasma biochemistry and PTH levels

The blood was collected from the inferior vena cava after 28 days of treatment for determining the plasma levels of Ca²⁺ (A), oxalate (B), citrate (C), and PTH (D). The plasma levels of oxalate and citrate were similar among groups. Hypocalcemia and increases in the plasma PTH were found in both SB-treated groups. n=6 in each group. The group abbreviations are the same as in Figure 2. *P<0.05, SB vs. C group; ^#P<0.05, SB+EG vs. EG group.

Figure 4. Urinalysis and CaOx supersaturation

Urine was collected from metabolic cages over a 24-h period after 28 days of induction and analyzed for changes in the urinary pH (A), Ca²⁺ (B), oxalate (C), and citrate (D) excretion and Ccr, (E) and urinary LDH levels (F), and the degree of supersaturation as expressed as the AP(CaOx) index (G). Low urinary pH was found in both EG-treated rats. Urinary Ca²⁺ excretion and Ccr was similar among groups; these were associated with hyperoxaluria, hypocitraturia, enzymuria, and supersaturation in the SB or EG group with a more prominent change in the SB+EG group. U, units. n=6 in each group. The group abbreviations are the same as in Figure 2. *P<0.05, EG or SB vs. C group; ^#P<0.05, SB+EG vs. EG group; ^@P<0.05, SB+EG vs. SB group.

Figure 5. The effects of SB on renal CaOx crystal deposition

Representative micrographs of light microscopy after Pizzolato’s staining and counterstaining with Eosin Y in rat kidneys with 28 days of treatment showed CaOx crystal deposition (A). CaOx crystal deposition (indicated by open arrows) in the sections. CaOx crystals in the SB+EG kidney led to renal tubular obstruction and dilation (asterisk). A mild interstitial fibrosis (star signs) surrounding the dilated tubule and tubular atrophy (arrow head) were found in the SB+EG kidney. The amount of CaOx crystals in renal sections was counted under high-power field (HPF) (B). No crystals were found in the kidneys of the control and SB groups. The group abbreviations are the same as in Figure 2. *P<0.05, EG vs. C group; ^#P<0.05, SB+EG vs. EG group; ^@P<0.05, SB+EG vs. SB group.

Figure 6. Changes in oxalate and citrate transporter expression

The protein expressions of Slc26a6, Slc26a2, and NaDC-1 in whole kidneys (A–C) and intestinal (D–F) tissue extracts were examined by immunoblotting. Representative blots from three rats showed typical protein expression of Slc26a6, Slc26a2, and NaDC-1 (20 μg of total protein per lane) at appropriate molecular weights. The lower bar graph (n=6 in each group) showed the ratio of the band density of the interested protein to β-actin. Slc26a6, but not Slc26a2, was up-regulated in both EG-treated groups; this was associated with increases in the NaDC-1 expression in all experimental groups. The Slc26a6 expression was also increased in the intestines of SB group. The group abbreviations are the same as in Figure 2. *P<0.05, SB or EG vs. C group; ^#P<0.05, SB+EG vs. EG group; ^@P<0.05, SB+EG vs. SB group.

Figure 7. The effects of PTH on oxalate and citrate transporter expression

The responses to Oxa, PTH, and the PKA blockers, H89 and KT5720, were evaluated in NRK-52E cells. (A) LDH was released under the treatment of Oxa or PTH alone or in combination. (B) The cell viability was examined using the MTT assay. (C,D) Slc26a6 and NaDC-1 expression in cells treated with oxalate or PTH. (E,F) Slc26a6 and NaDC-1 expression in cells treated with PTH and PKA blockers, H89 or KT5720. The lower bar graphs (n=6 in each group, C–F) shows the ratio of the band density of the interested protein to β-actin. *P<0.05, Oxa or PTH vs. C group; ^#P<0.05, PTH+Oxa vs. Oxa group; ^@P<0.05, PTH+Oxa vs. PTH group.

Figure 8. Schematic diagram showing how SB induced hypocitraturia in hyperoxaluric kidneys

SB induced hypocalcemia and PTH release. PTH acted on renal tubules and increased citrate transporter NaDC-1 expression via the PKA pathway, which induced hypocitraturia and lowered the anticrystallization ability of SB kidneys. The oxalogenic treatment of EG increased the Slc26a6 expression in the kidney but not in the intestine of SB rats, aggravating hyperoxaluria for CaOx crystal formation.