Biomedical subjects
Publications and source records attributed to R L Hackett.
Sprague-Dawley rats were injected with known nephrotoxic doses of tobramycin and then subjected to varying amounts of warm renal ischemia. Analysis of serum creatinine at the time of sacrifice revealed no statistically significant difference among controls and the two doses of tobramycin at any given duration of ischemia. Light microscopy revealed no worsening of tubular necrosis with increasing doses of tobramycin.
Fragments of urinary stones resulting from extracorporeal shock wave lithotripsy were examined by light and scanning electron microscopy. Calcium oxalate monohydrate and uric acid stone fragments were homogeneous and regular whereas struvite stone fragments were irregular in shape. Examination of the fractured surfaces revealed that the process of stone fragmentation involved fracture and cleavage of the crystals at some places and their separation from each other at others. In stones whose crystals are organised in layers, for instance calcium oxalate monohydrate and uric acid, crystalline layers separated along the concentric laminations. In struvite stones, which are an agglomeration of struvite and calcium phosphate crystals, major fragmentation occurred along the crystalline interfaces.
A procedure based on scanning electron microscopic techniques is described for the identification of crystals in urinary sediments and stones. The crystals are identified by their morphology and elemental composition using scanning electron microscopy and x-ray microanalysis. The procedure has a number of advantages over conventional methods. It is easy to use. It is non-destructive so that both the exterior and interior of the same stone can be separately analyzed. It is the only technique in which information about spatial relationships between various crystals in a stone can be obtained easily. Scanning electron microscopic techniques can detect minor components, and analysis of a wide variety of materials ranging from amorphous substances to microcrystals to macroscopic stones is possible.
Organic material associated with the calcium oxalate crystals in urinary stones and experimentally induced nephrolithiasis was stained with colloidal iron and analysed by energy dispersive x-ray microanalysis using standard techniques. Iron was positively identified in the stained specimens indicating that some of the organic material is an acidic mucosubstance. The results also indicate that some of the organic material of urinary stones may originate in the kidneys.
Calcium oxalate bladder stones were induced in male rats by implanting plastic foreign bodies and by adding ethylene glycol to their drinking water. The foreign body surface was first coated with cellular debris and some amorphous material. Encrustation with crystals of calcium oxalate started on the third day of implantation. Within 2 weeks the entire surface of a foreign body was covered with crystals and some noncrystalline material. Calcium oxalate monohydrate crystals consisted of platelike crystallites arranged in hemispherulitic or spherulitic habit. Calcium oxalate dihydrate crystals wee basically dipyramidal, a majority of them showing interpenetrant twinning. The stone grew by confluent crystal growth and crystal aggregation. A transformation of calcium oxalate monohydrate crystals to calcium oxalate dihydrate also occurred. The matrix consisting of cellular debris and urinary macromolecules was universally distributed in the stone including the inside of crystal bodies.
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Calcium oxalate stone disease is the most common human urinary stone disease in the Western Hemisphere. To understand different aspects of the disease, calcium oxalate urolithiasis in the rat is used as a model. Spontaneous calcium oxalate urolithiasis is very rare in rats. Thus the disease is experimentally induced and the rats are generally made hyperoxaluric either by administration of excess oxalate, exposure to the toxin ethylene glycol, or various nutritional manipulations. All the experimental models show renal injury associated with crystal deposition. Calcium oxalate crystals are in most cases intraluminal in renal tubules and often attached to the basal lamina of the denuded epithelium. Rat renal papillary tips and fornices appear to be the preferential sites for the deposition of large calcium oxalate calculi. Where urinary supersaturation of calcium oxalate has been studied the crystal forming rat urines are shown to have higher urinary supersaturation of calcium oxalate than their controls. Oxalate metabolism in the rat is nearly identical to that in humans. Thus, in a number of respects, experimental calcium oxalate urolithiasis in the rat is similar to calcium oxalate stone disease in man.
Calcium oxalate crystalluria was induced in laboratory rats by subcutaneous implantation of potassium oxalate containing mini-osmotic pumps in their intercapsular region. Concentrations of major urinary ions were measured and urinary supersaturations of various urinary salts were calculated using a computer programme. The urines of experimental animals that received oxalate had calcium oxalate crystals and higher supersaturations for calcium oxalate compared to their controls. Oxalate levels of the urines of experimental animals were higher than their controls and this increase was proportional to the increase in urinary supersaturation of calcium oxalate. No significant difference was found in the calcium levels of urines from experimental and control animals.
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.
Human urinary stone fragments and sections were decalcified in ethylenediaminetetraacetic acid solution after embedding in 1% agar. These decalcified embedded stone specimens were then processed for scanning electron microscopy. After critical point drying the agar was removed and EDTA-insoluble stone residue was studied by scanning electron microscopy. This residue representing part of the stone matrix kept the original shape of the stone and was basically made of fibrous material. The fibers appeared to be arranged in matted sheets that were present as concentric laminations. In case of calcium oxalate dihydrate stones these fibrous sheets were also arranged as peripheral layers running parallel to the pyramidal faces of large calcium oxalate dihydrate crystals. In decalcified stones, crystals were represented by crystal ghosts. Often some vesicular material was present embedded in the fibrous sheets.
We have compiled a broad overview of the genesis of urinary stones. Central to the theses given are precise definitions of stone as opposed to stone disease. In terms of mechanisms, a kinetic point of view is essential to any formulation relating to stone formation. It is necessary to comprehend the kinetic approach, an understanding of nucleation, growth and aggregation, if one is to appreciate the final product--the stone. Certain fundamental concepts are reviewed which are critical in grasping these phenomena. Not to be forgotten is that the purely physical chemical aspects of stone formation are insufficient to explain all facets of the process. Therefore, interactions between crystals and tissue are explored in suggesting a unifying hypothesis for upper tract stone disease.
Crystalluria was induced in male Sprague-Dawley rats by implanting potassium-oxalate-containing mini-osmotic pumps. Urine of all experimental animals had abundant crystals of calcium oxalate, hydroxypatite, struvite, and calcium phosphate. These crystals were similar in morphology to the crystals found in human crystalluria. Histological examination of kidneys and tissue samples from other organs revealed no abnormality. One group of animals was injected with sodium oxalate in addition to implantation of potassium-oxalate-containing mini-osmotic pumps. Bladder urine from these animals contained calcium oxalate microstones and their kidneys had deposits of calcium oxalate crystals.
A procedure for studying urinary stones by various microscopic techniques is described. The stones are sectioned into approximately 0.2 to 1.0 mm. thick pieces using a low-speed saw. The sections are then embedded in agar and decalcified using 0.25 M ethylenediaminetetracetic acid at pH 7.2. The decalcified residue is then processed for light microscopy and scanning and transmission electron microscopy as with any other biological tissue. The results indicate that the ethylenediaminetetracetic acid-insoluble stone matrix keeps its architectural integrity and can be studied like other biological materials.
All urinary concretions are composed of a crystalline or mineral phase and a non-crystalline phase. Both phases vary markedly in their chemical nature. There are a number of theories about the role and morphogenesis of the organic phase or matrix. In our opinion, at least a part of the matrix is formed by adsorption of urinary proteins onto crystal surfaces. It has already been shown that naturally occurring polymers have an affinity for calcium oxalate crystal surfaces and that spaces filled with amorphous substances exist between calcium oxalate monohydrate crystals of whewellite renal stones. We wanted to visualize these crystal surfaces with adsorbed organic material. We studied calcium oxalate monohydrate crystals from urinary stones, and crystal made in a crystallizer and incubated in gamma globulin or bovine serum albumin. We also studied calcium oxalate monohydrate crystals experimentally induced in rat renal tubules. They were studied using light, scanning and transmission electron microscopy with, as well as without, EDTA digestion. All crystals were surrounded by an amorphous coat which may have originated by adsorption of proteins on crystal surfaces.
Calcium oxalate nephrolithiasis in rats, induced by single intraperitoneal injection of sodium oxalate, is associated with pathologic changes in the renal papillary tip. Calcium oxalate crystals appear in the tubular lumens, in the intercellular spaces between epithelial cells, and attached to the tubular epithelial basal lamina. Unusual paracrystalline structures also develop in the distal tubule associated with the basal lamina. Speculations are made about the role of these structures. The epithelial changes are primarily necrotic and are similar to those described in experimental papillary necrosis. Complete morphologic recovery occurs in 1-2 weeks.
The formation of urinary oxamide stones was induced in rats by feeding them oxamide mixed with powdered rat chow. The structure of these stones and the changes in the rat renal papillary structure following oxamide administration were studied using bright field and polarizing light microscopy as well as scanning electron microscopy. Oxamide appeared in the papillary collecting ducts, pelvis, ureters and urinary bladder of the rats in the form of yellow spherulitic units composed of dendritic crystals. Oxamide stones in turn were aggregates of these spherulites. Our results indicate the renal stone formation started with the crystallization of oxamide in the tubular lumina of the collecting ducts of the papillae. Crystal formation in the tubules was associated with epithelial necrosis. Some of the crystals became attached to injured epithelium, thus impeding urinary flow. The attachment of the crystals resulted in their retention in the renal tubules. These oxamide deposits then grew or aggregated to form stones. The formation of oxamide deposits in the ducts of Bellini resulted in dilatation, compression of the epithelium and destruction of the papillary urothelium. These factors resulted in the deformation of the papillary tip of the kidney.
The formation of calcium oxalate crystals in rat kidneys was induced by intraperitoneal challenge with sodium oxalate. The structure of these crystals and the effect of their formation on the architecture of the tubular epithelium was studied by SEM. From the results we conclude that the crystals were formed intraluminally in the renal tubules. During the growth of the microliths they were closely adherent to the luminal surface of the tubular epithelial cells. Two types of the crystals were identified. Calcium oxalate monohydrate crystals consisted of plate-like crystallites and were more common. Dihydrate crystals on the other hand were tetragonal dipyramids and were less common. Elemental analysis of the crystal surface revealed them to be closely similar to the crystals present in calcium oxalate stones. The renal tubules were morphologically altered. Their epithelial cells underwent focal necrosis. There was a focal loss of the microvillous brush border of the proximal tubules. The epithelial cells formed large blebs which protruded into the tubular lumen. Sometimes the tubular lumen was completely occluded by the crystals. Crystals were often covered with cellular debris. It is suggested that the cellular debris derived from from necrotic cells become incorporated into the calculi. A similar process may be involved in the formation of mucoproteinaceus matrix of the human renal stones.
A model system is described for the induction of renal calcium oxalate crystals with intraperitineal injections of sodium oxalate in rats. Early crystals are formed predominantly in cortical areas. Massive amounts of calcium are associated with this process, as demonstrated by potassium pyroantimonate staining. Actual crystal formation appears to be an involved process associated with calcium, oxalate, and cellular membranes. Although overt stone formation was not observed, we feel that the intimate involvement of membranes during crystal formation may be similar to that found in renal stones.