Calcium oxalate crystal deposition in kidneys of hypercalciuric mice with disrupted type IIa sodium-phosphate cotransporter

Abstract

The most common theories about the pathogenesis of idiopathic kidney stones consider precipitation of calcium phosphate (CaP) within the kidneys critical for the development of the disease. We decided to test the hypothesis that a CaP substrate can promote the deposition of calcium oxalate (CaOx) in the kidneys. Experimental hyperoxaluria was induced by feeding glyoxylate to male mice with knockout (KO) of NaP(i) IIa (Npt2a), a sodium-phosphate cotransporter. Npt2a KO mice are hypercalciuric and produce CaP deposits in their renal tubules. Experimental hyperoxaluria led to CaOx crystalluria in both the hypercalciuric KO mice and the normocalciuric control B6 mice. Only the KO mice produced CaOx crystal deposits in their kidneys, but the CaOx crystals deposited separately from the CaP deposits. Perhaps CaP deposits were not available for a CaOx overgrowth. These results also validate earlier animal model observations that showed that CaP substrate is not required for renal deposition of CaOx and that other factors, such as local supersaturation, may be involved. The absence of CaOx deposition in the B6 mice despite extreme hyperoxaluria also signifies the importance of both calcium and oxalate in the development of CaOx nephrolithiasis.

Fig. 1

Nephrolithiasis in mice with disrupted type IIa sodium-phosphate cotransporter (Npt2a). A: von Kossa-stained paraffin section of a kidney from a 5-day-old male knockout (KO) mouse. The von Kossa method specifically detects calcium deposits. Section shows von Kossa-positive calcium phosphate (CaP) deposits throughout the kidney. Most deposits are seen in the renal cortex and outer medulla. B: von Kossa-stained paraffin section of a kidney from a 4-mo-old male KO mouse showing the presence of a very few crystal deposits. Original magnification ×1.2.

Fig. 2

A: hematoxylin and eosin (H & E)-stained paraffin section of a kidney from a 5-day-old KO mouse showing CaP deposits in outer medulla. Crystals are mostly intraluminal. B: renal papilla of the same kidney showing patent collecting ducts (L) and CaP deposits in the renal interstitium. C: same deposit after von Kossa staining. Original magnification ×20.

Fig. 3

A: von Kossa-stained paraffin section of a kidney from a 2-mo-old KO mouse showing interstitial deposits in the renal cortex. Renal tubules are completely devoid of any crystals. Original magnification ×20. B: higher magnification showing ringlike substructure in the crystal deposit. Original magnification ×40. C: scanning electronic microscopic (SEM) image of a similar CaP crystal deposit showing internal concentric laminations.

Fig. 4

A: von Kossa-stained paraffin section of a kidney from a 2-mo-old KO mouse showing a large CaP deposit in the inner medullary portion of the renal papilla. Original magnification ×20. B: SEM image of a similar deposit. The size and shape of crystal deposits indicates that they started in the renal tubules, grew beyond the tubular dimension, and secondarily came to lie in the renal interstitium.

Fig. 5

SEM analyses of a renal CaP crystal deposit. A: surface of a fractured CaP deposit. B: high magnification of the surface showing its rough appearance. C: high magnification of the central part of the CaP deposit showing amorphous nature. D: energy-dispersive X-ray microanalysis of the peripheral area of the deposit showing major peaks for calcium (Ca) and phosphorus (P). Ca peak is slightly higher than P peak. E: energy-dispersive X-ray microanalysis of the central area of the deposit showing major peaks for Ca and P. Here the Ca peak is slightly lower than the P peak.

Fig. 6

Weight change during 28 days of the experiment. Control (Norm) B6 and KO mice maintained their weight, while both types of experimental (Tx) mice who received glyoxylate (Gox) in food lost their weight. There was no significant difference in weight between the normal B6 and KO mice at any time during the experimental period. However, there was a gradual reduction in weight of the experimental KO mice compared with the control KO mice, and experimental mice had significantly reduced weight compared with their controls on days (D) 14 (P < 0.001), 21 (P < 0.001), and 28 (P < 0.001).

Fig. 7

Urinary pH. There was no significant difference in urinary pH between control (Norm) B6 and control KO mice except on day 21, when KO mice showed significant increase in urinary pH (P < 0.003). Experimental (Tx) mice, B6 as well as KO, generally had lower urinary pH than their respective controls. This difference reached significant levels on day 21 (P < 0.001) and slight significance (P < 0.059) on day 28.

Fig. 8

Urinary calcium. There was no significant difference in urinary calcium excretion between control (Norm) and experimental (Tx) B6 mice for the first week. However, urinary excretion of calcium by the experimental B6 mice became significantly lower on days 21 (P < 0.037) and 28 (P < 0.001). Urinary calcium excretion by the experimental (Tx) KO mice, however, did not differ significantly from the control KO mice.

Fig. 9

Urinary oxalate. By day 7 of Gox treatment, urinary excretion of oxalate by the experimental (Tx) B6 as well as KO mice increased significantly compared with their respective controls. Oxalate remained significantly high in urine of the experimental B6 mice compared with their B6 controls and on days 14, 21, and 28 compared with the experimental KO mice. On the other hand, oxalate excretion started to go down in the urine of the KO mice by day 14, and by day 21 it was significantly lower than the oxalate excretion by KO mice on day 7.

Fig. 10

SEM images of urinary crystals of mice treated with Gox. A and B: calcium oxalate (CaOx) monohydrate (COM) crystals, which appeared as interpenetrant twins of platelike crystals. CaOx dehydrate crystals (not shown) appeared as tetragonal bipyramids. Bar, 6 μm.

Fig. 11

H & E-stained paraffin section of a kidney of an experimental KO mouse on day 14 of Gox treatment showing both birefringent CaOx crystal deposits and dark CaP crystal deposits. Birefringent CaOx crystals are mostly intraluminal. Epithelial cell of a renal tubule also contains an intracellular CaOx crystal (arrow). CaP deposit appears to be surrounded by a ring of inflammatory cells (dashed arrows). Original magnification ×20.

Fig. 12

SEM analyses of renal CaOx crystal deposit. A: deposit shows highly compact aggregate of platelike COM crystals. B: energy-dispersive X-ray microanalysis of the deposit shows major peak for calcium.