Paula N. Shevock

Acute Hyperoxaluria, Renal Injury and Calcium Oxalate Urolithiasis

Single intraperitoneal injections of three, seven, or 10 mg. of sodium oxalate per 100 gm. of rat body weight were administered to male Sprague-Dawley rats. At various times after the injection, urine samples were analyzed for oxalate, and urinary enzymes, alkaline phosphatase, leucine aminopeptidase, gamma-glutamyl transpeptidase, and N-acetyl-beta-glucosaminidase. The kidneys were processed for light microscopy and renal calcium and oxalate determination. Oxalate administration resulted in an increase in urinary oxalate and formation of calcium oxalate crystals in the kidneys. The amount and duration of urinary excretion of excess oxalate and retention of crystals in the kidneys correlated with the dose of sodium oxalate administered. At a low oxalate dose of three mg./100 gm., crystals moved rapidly down the nephron and cleared the kidneys. At higher doses crystals were retained in kidneys and at a dose of 10 mg./100 gm. were still there seven days post-injection. Crystal retention was associated with enhanced excretion of urinary enzymes indicating renal tubular epithelial injury.

Madin-Darby canine kidney cells are injured by exposure to oxalate and to calcium oxalate crystals.

The reaction of Madin-Darby canine kidney cells (MDCK) to potassium oxalate (KOx), calcium oxalate monohydrate (COM) crystals, or a combination of the two was studied. The most noticeable effect of exposure of the cells to either KOx or COM crystals was loss of cells from the monolayer ranging from 20% to 30%, depending upon the particular treatment. Cellular enzyme values in the media were elevated significantly by 12h of exposure, although in specific instances, elevated levels occurred at earlier time periods. As regards the monolayer, trypan blue exclusion was decreased significantly, although amounting to only a 4–5% reduction. Specific tritiated release occurred at 4 and 12 h after exposure to KOx and at 12 h after exposure to crystals. Structurally, COM-cell interactions were complex and extensive endocytosis was noted. Cells were released from culture either as cellcrystal complexes or from the intercellular spaces after exocytosis. When treatment were combined the effects were only slightly additive, but the two treatments potentiated each other: all media enzyme levels (with one exception) were elevated at 2 h, tritiated adenine release was present at 4 h, and there was more extensive cell loss from the culture monolayer. These data suggest that both KOx and COM crystals damage MDCK cells when applied alone, and in concert they act synergistically.

Cell Injury Associated Calcium Oxalate Crystalluria

Renal tubular cell damage, resulting in membranuria, was induced by the administration of subcutaneous gentamicin to male Sprague-Dawley rats. One group of rats received gentamicin only, while a second group was given gentamicin plus ethylene glycol in drinking water at a concentration which increased urine oxalate but alone did not cause calcium oxalate crystalluria. Crystalluria occurred early in the combined treatment groups and persisted for the duration of the experiment. Crystalluria was not present in animals receiving gentamicin or ethylene glycol only. These results suggest that cellular fragments can serve as heterogeneous foci for the nucleation of calcium oxalate crystals.

MAGNESIUM OXIDE ADMINISTRATION AND PREVENTION OF CALCIUM OXALATE NEPHROLITHIASIS

We studied the effect of oral administration of magnesium oxide (MgO) on calcium oxalate (CaOx) nephrolithiasis in rats. Nephrolithiasis was induced by administration of 1.0% ethylene glycol (EG) in drinking water. Magnesium oxide was given mixed with food at 500 mg./100 g. rat chow. Dispensation of MgO resulted in a significant increase of urinary pH and a modest increase in urinary excretion of citrate. Urinary excretion of oxalate started to decline by day 14 and was significantly reduced on days 21 and 28. All rats receiving EG displayed crystalluria. From the group receiving EG only, 3 of 4 rats sacrificed on day 15 and 2 of 4 rats sacrificed on day 29 had CaOx crystal deposits in their kidneys. None of the 8 rats who received both EG and MgO had CaOx nephrolithiasis. Thus our findings indicate that dispensation of magnesium as MgO can be beneficial against calcium oxalate nephrolithiasis.

Effect of magnesium on calcium oxalate urolithiasis.

Previous studies have shown that hypomagnesuria induced by magnesium deficient diet causes calcium oxalate crystal deposition in renal tubules of hyperoxaluric rats and administration of magnesium to these rats results in prevention of calcium oxalate crystallization in their kidneys. Based on these studies magnesium was claimed to be beneficial for calcium oxalate stone patients. However, hypomagnesuria is not a common phenomenon. To better understand the role of magnesium as an inhibitor of calcium oxalate crystallization in urine, we studied the effect of magnesium on calcium oxalate urolithiasis in rats on a regular diet and a hyperoxaluric protocol. Excess magnesium was administered to male rats on regular diet and a lithogenic protocol. Magnesium administration to hyperoxaluric rats did not result in significant changes in urinary excretion of calcium or oxalate or in calcium oxalate relative supersaturation. Urinary excretion of citrate was also not significantly altered. Some animals from both groups, those on magnesium therapy and those not on magnesium therapy had crystals deposited in their renal tubules. We conclude that excess magnesium has no significant effect on calcium oxalate urolithiasis in normomagnesuric conditions.

In Vitro Precipitation of Calcium Oxalate in the Presence of Whole Matrix or Lipid Components of the Urinary Stones

Organic matrix of human calcium oxalate urinary stones was obtained by demineralizing with EDTA. Lipids were extracted from the EDTA-insoluble matrix by chloroform methanol treatment. The whole matrix and its total lipid extract were then incubated in a metastable solution of calcium oxalate and depletion of calcium and oxalate ions from the calcifying solution was determined. Results of our studies described here show that urinary calcium oxalate stone matrix and its total lipid contents were capable of binding calcium and oxalate ions and of catalysing calcium oxalate crystal formation from a metastable calcium oxalate solution.

Presence of lipids in urinary stones: Results of preliminary studies

SummaryThe presence of lipids in urinary stones was determined by histochemical and biochemical methods. When crystals of calcium oxalate, made by mixing calcium chloride and potassium oxalate solutions and sections of human calcium oxalate urinary stones, were exposed to osmium vapors, there was no staining of the pure crystals whereas the stone sections were stained. De-paraffinized sections of demineralized calcium oxalate stones showed positive sudanophilia on staining with Sudan black B. Both these experiments indicate the presence of lipids in calcium oxalate stones. Lipids were extracted from uric acid, struvite, and calcium oxalate stones using standard techniques. Phospholipids were separated by one-dimensional thin layer chromatography. All the stones studied contained lipids. In calcium oxalate stones they accounted for 10.15% of the matrix. Calcium oxalate and struvite stones contained more phospholipids than uric acid stones. Cardiolipin, sphingomyelin, phosphatidyl choline, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl glycerol were identified in lipid extracts. Demineralization by ethylenediaminetetraacetate (EDTA) treatment increased lipid output from calcium oxalate stones by 15.5%.

Membrane-associated crystallization of calcium oxalatein vitro

SummaryIncubation of proximal tubular brush border membrane in a metastable calcium oxalate solution of low supersaturation resulted in the equimolar depletion of calcium and oxalate and the formation of monoclinic calcium oxalate crystals. We propose that membrane fragments from sloughed epithelial cells of the nephron can similarly induce crystallization in urine that is metastable for calcium oxalate.

Urinary chemistry of the normal Sprague-Dawley rat.

SummaryOn the basis of metabolic in vivo studies using the adult male Sprague-Dawley rat, we present a comprehensive summary of urine species measurements conducted in our laboratory over the last several years, as well as those present in the literature.

Inhibition of Calcium Oxalate Monohydrate Seed Crystal Growth is Decreased in Renal Injury

We have demonstrated previously that sub-lithogenic doses of ethylene glycol (EG), when combined with administration of the proximal tubule nephrotoxin, gentamicin sulfate (GS), cause calcium oxalate (CaOx) crystalluria in association with cellular degradation products1. In this model, consistent elevation of CaOx relative supersaturation (RSS) occurs as calculated by the ion speciation program EQUIL2. Because EQUIL calculations do not consider large molecules, we examined the growth inhibitory activity of compounds > or < 10,000 mol. wt. (10K MW) using a multi-well crystallizer technique.