Raymond L. Hackett

Prophylactic glutamine protects the intestinal mucosa from radiation injury.

Glutamine may be an essential dietary component, especially for the support of intestinal mucosal growth and function. This study evaluated the effects of a glutamine‐enriched elemental diet, administered before whole‐abdominal radiation on gut glutamine metabolism, mucosal morphometrics, and bacterial translocation. Rats were randomized to receive a nutritionally complete elemental diet that was glutamine‐enriched or glutamine‐free for 4 days. The animals were then subjected to a single dose of 1000 cGy x‐radiation to the abdomen. After irradiation, all animals received the glutamine‐free diet. Four days later the animals underwent laparotomy for sampling of arterial and portal venous blood, culture of mesenteric lymph nodes, and removal of the small intestine for microscopic examination. There was no difference in arterial glutamine or gut glutamine extraction between the two groups, but body weight loss was significantly diminished in the glutamine‐fed rats. Rats receiving the glutamine‐enriched elemental diet before radiation had a significant increase in jejunal villous number, villous height, and number of metaphase mitoses per crypt. Scanning electron microscopy confirmed the presence of an intact gut epithelium in eight of eight rats receiving prophylactic glutamine compared to one of eight animals in the glutamine‐free group. Three of eight rats fed glutamine had culture positive mesenteric lymph nodes compared with five of seven rats receiving the glutamine‐free diet. Glutamine exerts a protective effect on the small bowel mucosa by supporting crypt cell proliferation which may accelerate healing of the acutely radiated bowel.

Lipid Peroxidation in Ethylene Glycol Induced Hyperoxaluria and Calcium Oxalate Nephrolithiasis

PURPOSE To determine if lipid peroxidation plays a role in renal injury associated with experimental nephrolithiasis. MATERIALS AND METHODS Hyperoxaluria was produced in rats by ethylene glycol in drinking water. At 15, 30 and 60 days of treatment, urinary oxalate, lipid peroxide, calcium oxalate crystals, enzymes and tissue lipid peroxide were measured. RESULTS Urinary oxalate increased significantly at all time periods and was associated with crystalluria. Lipid peroxides in kidney tissue and urine increased at all time periods. Tissue calcium oxalate crystal deposits from 0 to 1+ were present on day 15, but present in all animals on days 30 and 60. Renal tubular cell damage was confirmed by an increase in urinary marker enzymes. CONCLUSIONS Renal cell damage is associated with lipid peroxide production indicating cell injury due to the production of free radicals. The damage appears due primarily to hyperoxaluria and is augmented by crystal deposition in the renal tubules.

Pathologic effects of ESWL on canine renal tissue

The introduction of extracorporeal shock wave lithotripsy (ESWL) has provided an avenue for dealing with many urinary stones noninvasively. The margin of safety for the kidney during shock wave administration is largely undefined. A pilot study was performed where six kidneys in five female mongrel dogs were shocked. Group A kidneys were given 1,776, 4,500, 6,000, or 8,000 shocks, respectively, at 18-24 kV. Group B kidneys received 1,600 and 8,000 shocks (18-24 kV). The number of shocks per electrode ranged from 500 to 4,538 and averaged 2,490. The dogs were sacrificed forty-eight to seventy-two hours (Group A) or twenty-eight to thirty-two days (Group B) post-treatment. Modest damage (hematoma and/or interstitial hemorrhage) was noted in all kidneys. Evidence of permanent change (fibrosis) was noted in both Group B kidneys. Complete necrosis of the kidney was not seen after administration of 8,000 shocks. These preliminary data indicate that lithotripsy can, in some circumstances, produce renal damage in the canine model.

Free radical scavengers, catalase and superoxide dismutase provide protection from oxalate-associated injury to LLC-PK1 and MDCK cells

PURPOSE Current studies have provided evidence that exposure of renal epithelial cells to oxalate and calcium oxalate crystals induces lipid peroxidation and injures the cells. Since oxidant/antioxidant balance is likely to play a critical role, we determined the effect of antioxidant scavengers on production of free radicals and injury to LLC-PK1 and MDCK cells from exposure to oxalate (Ox) or Ox + calcium oxalate monohydrate (COM) crystals. MATERIALS AND METHODS LLC-PK1 and MDCK cells were grown in monolayers and exposed to 1.0 mmol. Ox or 1.0 mmol. Ox + 500 microg. /ml. COM crystals for 120 or 240 minutes. We measured the release of lactate dehydrogenase (LDH) as a marker for cell injury and malondialdehyde (MDA) as a marker of lipid peroxidation. Superoxide and hydroxyl radicals were measured in the presence or absence of 400 U/ml. catalase, or superoxide dismutase (SOD). RESULTS Exposure of LLC-PK1 cells to Ox resulted in a significant increase in MDA and release of LDH, which was further elevated when COM crystals were added. MDCK cells responded similarly to both challenges, but showed significantly less impact when compared with LLC-PK1 cells. Both treatments were associated with significant increase in the generation of hydroxyl and superoxide radicals by both cell types. In both cell lines, the addition of catalase or SOD significantly reduced the increase of MDA and release of LDH. CONCLUSIONS Results of the present study indicate that both Ox and COM crystals are injurious to renal epithelial cells and the injury is associated with generation of free radicals. Cells of proximal tubular origin are more susceptible than those of distal tubules and collecting ducts. Free radical scavengers, catalase and SOD provide significant protection.

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.

Role of Organic Matrix in Urinary Stone Formation: An Ltrastructural Study of Crystal Matrix Interface of Calcium Oxalate Monohydrate Stones

Human calcium oxalate monohydrate (COM) urinary stones were decalcified by treatment with a mixture of ethylenediaminetetraacetic acid (EDTA) solution and Karnovskys fixative after embedding in bactoagar. Decalcified stones were examined by light microscopy, and also by scanning and transmission electron microscopy. Stones had distinct nuclei that were occupied by amorphous or apatitic calcium phosphate or aggregates of spherulitic COM crystals. EDTA insoluble matrix was ubiquitous in stones and consisted largely of finely matted fibrous material. It was organized in concentric laminations in the peripheral area of the stone but appeared highly disorganized in the stone center. Crystals were replaced by crystal ghosts. Organic matrix was present both inside the crystals and in the intercrystalline spaces. The study indicates a very close association between crystals and organic matrix. The relationship appears to begin early in crystal formation and persists throughout the formative and growth phases of the urinary stones.

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.

Urinary Enzymes and Calcium Oxalate Urolithiasis

Male Sprague-Dawley rats were challenged with various hyperoxaluric agents including ammonium oxalate, hydroxy-L-proline, and ethylene glycol. All treatments resulted in increased urinary oxalate. Associated with hyperoxaluria was an increase in urinary levels of renal enzymes, gamma-glutamyl transpeptidase, N-acetyl-beta-glucosaminidase, and alkaline phosphatase. Most of the rats did not demonstrate any significant change in urinary levels of beta-galactosidase. There was a highly significant positive correlation between urinary oxalate and N-acetyl-beta-glucosaminidase.

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.

Crystal retention by injured urothelium of the rat urinary bladder

Injuries caused by hydrochloric acid or Triton X 100 application to the rat bladder urothelium, and the effects on calcium oxalate crystal retention, were examined by light and scanning electron microscopy. Damage due to either compound resulted in desquamation of urothelial cells. The crystals appeared to be retained by a fibrillar material, some of which was identified as fibrin. Heparin treatment of injured urothelium was found to prevent crystal retention.