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Calcium oxalate stone formation in genetic hypercalciuric stone-forming rats. Background Over 54 generations, we have successfully bred a strain of rats that maximizes urinary calcium excretion. The rats now consistently excrete 8 to 10 times as much calcium as controls, uniformly form poorly crystalline calcium phosphate kidney stones, and are termed genetic hypercalciuric stone-forming (GHS) rats. These rats were used to test the hypothesis that increasing urinary oxalate excretion would not only increase the supersaturation with respect to the calcium oxalate solid phase, but also would increase the ratio of calcium oxalate-to-calcium phosphate supersaturation and result in calcium oxalate stone formation. Methods To increase urine oxalate excretion an oxalate precursor, hydroxyproline, was added to the diet of male GHS rats. The GHS rats were fed a standard 1.2% calcium diet alone or with 1%, 3% or 5% trans-4-hydroxy-L-proline (hydroxyproline). Results The addition of 1% hydroxyproline to the diet of GHS rats led to an increase in urinary oxalate excretion, which did not increase further with the provision of additional hydroxyproline. The addition of 1% and 3% hydroxyproline did not alter calcium excretion while the provision of 5% hydroxyproline led to a decrease in urine calcium excretion. The addition of 1% hydroxyproline led to an increase in urinary calcium oxalate supersaturation, which did not further increase with additional hydroxyproline. The addition of 1% and 3% hydroxyproline did not alter urinary supersaturation with respect to calcium hydrogen phosphate while the addition of 5% hydroxyproline tended to lower this supersaturation. Compared to rats fed the control and the 3% hydroxyproline diet the addition of 5% hydroxyproline increased the ratio of calcium oxalate supersaturation to calcium phosphate supersaturation. Virtually all rats formed stones. In the control and 1% hydroxyproline group, all of the stones were composed of calcium and phosphate (apatite), in the 3% hydroxyproline group the stones were a mixture of apatite and calcium oxalate, while in the 5% hydroxyproline group all of the stones were calcium oxalate. Conclusions The provision of additional dietary hydroxyproline to GHS rats increases urinary oxalate excretion, calcium oxalate supersaturation and the ratio of calcium oxalate-to-calcium phosphate supersaturation, resulting in the formation of calcium oxalate kidney stones. Thus, with the addition of a common amino acid, the GHS rats now not only model the most common metabolic abnormality found in patients with nephrolithiasis, hypercalciuria, but form the most common type of kidney stone, calcium oxalate.

Shock waves generated during extracorporeal shock wave lithotripsy (ESWL†) therapy have produced both renal and extrarenal tissue injury.1–4 Renal injury, although not yet fully characterized, includes disruption of the vasculature, damage to the tubular epithelium, and apparent alterations in the renal interstitium. Whole animal studies are being used to document the types of tissue injury that occur during ESWL and are suitable for the investigation of how acute injury may lead to chronic alterations in kidney structure and function. However, in vivo studies may neither allow the adequate probing of the cellular basis of ESWL injury nor aid the understanding of the mechanism by which cells of the kidney are damaged. Such studies cannot precisely determine what role the various physical parameters of shock wave delivery play in producing cell injury. Therefore, investigators have employed cultured cells as model systems to investigate how shock waves injure cells and to determine the role of physical parameters in producing cell/tissue injury. A rationale for employing cultured cells as model systems in ESWL research is presented here, along with a discussion, from a tissue culture perspective, of the advantages and limitations of using isolated cells to better understand how shock waves injure cells of the intact kidney.

Substantial contributions to the field of RCD have been made over the past 15 years. Most intriguing is a growing awareness of acquired RCD and its complications. Data have been published and are reviewed here in support of a possibility that APKD and RCD acquired during hemodialysis are premalignant lesions. More data are needed before the possibility can be confirmed or denied. The collection of these data is an immediate need in the field of RCD.

Diuresis and natriuresis follow volume expansion by intravenous infusion of either blood or saline. However, the mechanisms underlying the renal responses seem different for the two types of expansion. It has been suggested that the response to saline expansion is due to changes in physical factors with alterations in the morphology of the lateral intercellular space (LIS). The present study examines the width of the LIS of the proximal tubule after a Ringer-Locke (1%, 2.3%, or 7% body weight) or whole blood (2.3% body weight) infusion and in nonexpanded control. After Ringer-Locke infusion, a significant widening of the LIS of the proximal tubule is seen at all levels of expansion. There is a linear relationship between the level of the Ringer-Locke infusion and the degree of expansion of the LIS. In contrast, after blood volume expansion, the width of the LIS is the same as nonexpanded controls. These studies are consistent with the view that the renal response to saline expansion is mediated at least in part by physical factors affecting the proximal tubule.

The present study details a new method for the exposure and viewing of individual microvessels located within the small intestine of rats. This procedure will selectively and consistently remove the outer muscle layers and underlying submucosa of the intestinal wall and thereby expose a variety of arterioles in their normal location within the tissue, with their normal relationship to each other undisturbed. The small intestine of the rat was initially fixed by vascular perfusion with 2.5% glutaraldehyde in 0.1 M cacodylate/HCL buffer, reinfused with heparinized whole blood, removed from the animal, and secured to a dissecting petri dish for further fixation. Subsequently, the external muscularis was dissected from the sample which exposed the submucosa. In order to remove the connective tissue elements from this layer and uncover the submucosal vasculature, the samples were first transferred to a solution of 30% potassium hydroxide for 2-5 minutes and then to a final digesting solution containing collagenase. Thereafter, the samples were routinely processed for light microscopy and for scanning (SEM) or transmission (TEM) electron microscopy. Examination of the samples revealed excellent preservation of the three-dimensional organization of the arteriolar wall with minimal membrane damage. This new technique now makes it possible to visualize the shape and position of individual smooth muscle cells along arterioles of differing size and branching order.

Unlike the case in rat, rabbit, and other species in which nephron formation continues into the newborn period, nephrogenesis in the guinea pig is completed well before the time of birth. Therefore, the marked increase in proximal tubule reabsorption that occurs during the postnatal period in that species can be attributed entirely to an increase in the absorptive capacity of existing nephron units. The purpose of the present morphometric studies was to correlate that change in proximal tubule function with changes in the apical and basolateral cell membrane surface areas. The apical and basolateral membrane surface densities were found to be approximately equal to each other and to remain constant throughout development. Because of increasing tubule volume, however, both membranes doubled in size between 1 and 3 wk of age and eventually increased by a factor of 3.5 in the adult. At the same time, there was little change in the length of tight function complexes measured in the plane of the luminal surface. Using previously published functional data and tubule length data, a good correlation was found between absolute absorption and total basolateral membrane surface area throughout the entire period of development in proximal tubules. Absorption per unit area of basolateral membrane was approximately 0.55, 0.41, 0.56, and 0.42 X 10(-6) nl.min-1.micron-2 in the 1st, 2nd, and 3rd wk, and in adult animals, respectively, and thus was similar to that reported for proximal tubule segments of adult rabbit and juvenile to adult rat.

A morphometric method to analyze scanning electron micrographs (SEM) of microvascular smooth muscle cells (SMC) is validated using intestinal arterioles. Features easily obtained from these microvasculature preparations are counted (number of tapers and number of complete wraps in the central 87% of the vessel, which is one-third of the vessel's circumference) or measured (vessel diameter and vessel segment length). These data allow the determination of wraps around the vessel per SMC, cell length, and cell width, which are not different from either the values as determined by circumferential examination and quantitation of the vascular segment or an alternative SEM quantitation method (Miller et al., 1986). The method described herein provides a relatively easy way to determine microvascular SMC parameters.

It is now well established that extracorporeal shock wave lithotripsy (ESWL†) produces significant acute renal side effects. There is strong evidence that chronic effects may also accompany ESWL therapy. The clinical and experimental data that document tissue injury from ESWL are compelling but do not adequately address the factors that are responsible for adverse acute side effects or the conditions that may lead to serious long-term health problems. In order to resolve these issues, it is necessary to determine the precise mechanism by which cells are damaged by ESWL. To accomplish this goal, animal experimentation is required so that the time course and severity of acute and chronic alterations can be followed. Animal models allow whole organ analysis of changes in both structural and functional features. These studies must be designed to give the best approximation of the treatment conditions that are applied to patients. To do this requires an animal model with a kidney that has similar structure and function to that of man. The kidneys of most small animals and many large animals, including the dog, do not mimic human renal anatomy and physiology. The laboratory pig appears to be the best large animal subject for ESWL research. The pig offers a model in which kidney size, renal/urinary anatomy, and characteristics of renal function closely resemble the human. Moreover, the methodology for correlative structure and function analysis in the pig is well established. Preliminary data on the mini-pig suggest that it is an excellent model to study the bioeffects of ESWL in that acute traumatic injury, similar to that reported for patients, is induced at therapeutic levels of shock wave delivery.

Intact whole lungs from 14 d rat fetuses were explanted to organ culture and maintained fully submerged in medium or positioned at the gas-fluid interface. Striking differences were observed in the morphological development of submersion cultured and interface cultured explants after 7 d in vitro. Submersion cultured lung developed as an expanded hollow organ, whereas the tissue of interface cultures was glandular in appearance and attenuated against the culture substrate. This difference in the development of the cultured lung may be attributed to the influence of surface tension forces at the gas-fluid interface versus the fluid immersed environment. In submersion culture the fetal lung followed a unique pattern of morphogenetic development. Under these culture conditions the lung expanded in three dimensions and retained its discrete lobar structure. The primitive bronchial epithelial tree of the fetal lung at explantation exhibited substantial histoarchitectural changes in vitro. Progressive branching of the epithelium into the surrounding lung mesenchyme gave rise to a pulmonary parenchyma bearing characteristics of the presumptive alveolar region of the late gestation fetal lung. The epithelium consisted predominantly of large, glycogen rich cuboidal cells interspersed with numerous structurally differentiated type II cells, containing lamellar bodies. The pulmonary parenchyma of the submersion cultured explant occupied a narrow margin of the lung wall, surrounding a large central lumen. This unique form of the cultured lung made it possible to examine the pulmonary epithelium by scanning electron microscopy. Data from the present study suggest that submersion culture of the intact fetal lung may provide a useful model system for study of the maturation of epithelial cell surface features during pulmonary development.

Several examples of human renal cystic disease are associated with tubular epithelial hyperplasia. Micropapillary hyperplasia occurs in autosomal dominant polycystic kidney disease, in localized cystic disease, and in acquired cystic disease; neoplastic or severely dysplastic epithelial hyperplasia occurs in von Hippel-Lindau disease; a histopathologically distinctive epithelial hyperplasia occurs in tuberous sclerosis. In all of these conditions the epithelial hyperplasia appears to be responsible for cyst formation by causing tubular or ductal luminal obstruction, and in all of these conditions, save localized cystic disease (a rare condition with very few reported cases), epithelial hyperplasia imposes an increased risk of malignancy. The risk seems to be highest in patients under treatment with long-term hemodialysis for end-stage kidney disease. Some of these diseases may share common features, but it appears likely that the histopathological differences reflect different features converging on a common result.

To further evaluate the role of autonomic ganglia in the regulation of pelvic visceral activity, the neural elements in the major pelvic ganglion of the male rat have been studied with histochemical and electron microscopic techniques. The principal findings are that the ganglion is composed of cholinergic and adrenergic ganglion cells as well as small intensely fluorescent (SIF) cells. Polarity in the ganglion is indicated by clustering of small ganglion cells which stain intensely for acetylcholinesterase (AChE) along the pelvic nerve while larger cells, with weak to moderate AChE activity, collect near small branches of the hypogastric nerve. Some cholinergic ganglion cells are enclosed by a plexus of adrenergic terminals. SIF cells appear to be in contact with both cholinergic and adrenergic cells, although many of the fluorescent beads around adrenergic neurons may be short dendrites of ganglion cells, rather than processes of SIF cells. Two types of SIF cells may be distinguished on the basis of size and morphology of their granulated vesicles. Afferent synapses of the cholinergic type were common on SIF cells of the large granule and small granule type. Portions of SIF cells with large granules occur within the capsule of ganglion cells. Contacts seen here were interpreted as efferent synapses from SIF cells to the dendrites of ganglion cells.

Although diagnosis of polycystic kidney disease has been possible for many years, we still have only a limited understanding of the pathogenesis of the disease, partly because of the difficulty in obtaining human cystic kidneys in different stages of cyst formation. At present only a few human kidneys with early-onset polycystic changes have been evaluated for both structural and functional changes (Baert, 1978). Therefore, growing attention has been given to various animal models that possess lesions that mimic human polycystic kidney disease (Goodman et al., 1970; Carone et al., 1974; Gardner et al., 1976; Evan and Gardner, 1976, 1979; Evan et al., 1979). There are presently three types of animal models for experimental polycystic kidney disease: (1) chemically induced, (2) traumatically induced, and (3) genetically transmitted. The chemically induced models have received the most attention in that a large number of compounds have been reported to produce cysts in small laboratory animals. Furthermore, these models allow opportunity to follow the acquisition and sequential growth of cysts. Of the various renal cytogens, three compounds, all of which are antioxidants, have received the greatest attention. These substances are diphenylamine (DPA), nordihydro-guaiaretic acid (NDGA), and diphenylthiazole (DPT).

Alloxan is known to induce diabetic renal changes as well as causing nephrotoxic alterations. However, no ultrastructural study has been performed to differentiate diabetic verses toxic affects of alloxan to the tubule and/or glomerulus. Therefore the present study used the "protected" kidney model to prevent one kidney from being exposed to the alloxan while allowing the other to receive the drug immediately. In all experimental animals the right renal hilum was gently occluded for 5 minutes and then released. This was performed prior to the injection of alloxan. Subsequently, the left renal hilum was occluded at the time of, and for 5 minutes after, alloxan administration (40 mg/kg i.v.). The experimental rats were divided into three groups: untreated diabetics, diabetics treated with protamine-zinc-insulin, and alloxan-treated rats that failed to become diabetic. Three groups of controls were included: one group received an equal volume of saline diluent as the experimental rats but no clamping of either renal hilum; another group received the saline and had the left renal hilum occluded for 5 minutes; and a third group had both the right and left renal hila occluded. All animals were followed and sacrificed after 9 weeks. Endogenous creatinine clearance did not change among groups. Alloxan-treated nondiabetic rats displayed marked interstitial nephritis in unprotected kidneys, while protected kidneys were normal. The diabetic state resulted in mesangial proliferation and focal glomerular basement membrane thickening as well as glomerular capillary endothelial abnormalities and visceral epithelial foot-process fusion. The endothelial changes consisted of focal areas showing a reduction in the size of endothelial fenestrae. All glomerular changes were ameliorated by insulin treatment. We conclude: 1) alloxan per se is distinctly nephrotoxic; and 2) the glomerular endothelium and epithelium are involved early in the course of experimental diabetes.

To elucidate the time course of glomerular and arterial endothelial injury resulting from pulsatile perfusion preservation of human kidneys, we examined two kidneys, one at 16 and the other at 42 hr, for which no suitable recipient could be found. The scanning electron microscope revealed subtle changes at 16 hr in the filtration barrier. These included mild endothelial swelling with an increase in the appearance of bulbous processes, and elongated fenestrae. The visceral epithelial surface was normal as was the arterial endothelial surface. By 42 hr the glomerular endothelial surface displayed very prominent cytoplasmic ridges and clearly distorted fenestrae. The arterial endothelium exhibited a tendency to separate from the vessel wall. The proximal tubular epithelium revealed scattered loss of microvilli. These changes are similar in kind to, albeit less severe than, those described after 60 hr of perfusion. They may represent cell swelling following ischemia, or be the result of altered cell permeability engendered by low temperature. The possibility remains that such changes could be minimized by modifying the perfusate. Scanning electron microscopy provides a versatile tool in the study of vascular and other surfaces of tissues stored with perfusion preservation.

Accelerated renal cyst development in deconditioned germ-free rats. The influence of environment on renal cyst growth was assessed in experiments utilizing 56 germ-free and 32 conventional male Sprague–Dawley rats fed 2% nordihydroguaiaretic acid (NDGA). Among three groups of six germ-free rats allowed ad lib intake of NDGA for six weeks and deconditioned (placed into the ambient laboratory environment) at the start, at the midpoint, or not at all, cyst formation and interstitial changes (both severe) were noted only among animals deconditioned at the midpoint. Among 14 germ–free rats that were offered 0.4g NDGA daily, deconditioned after two weeks, and sacrificed 0 to 15 days thereafter, nephron dilation and thymidine uptake by renal tubular epithelium appeared within one to three days and correlated in magnitude with each other and with the duration of the contamination interval. Deconditioning, a prior period of four or more days of exposure to NDGA in the germ-free state, the duration of the contamination period, and the presence of cecal flora were identified as prerequisites to accelerated cyst formation in this model, while the dose of ingested NDGA, the presence of bacteria within the kidney, an action of bacteria on NDGA within the colon, and the type(s) of organisms colonizing the host were not. These findings provide experimental evidence that environmental circumstance can modulate the expression of renal cystic disease.

During organogenesis of the kidney, small S-shaped metanephric vesicles form from the undifferentiated metanephric mesoderm. The cephalic part of each primitive renal tubule invaginates to form a cleft within which mesenchymal cells, destined to become the mesangium, accumulate. The formation of a continuous basement membrane serves to separate the intracleftal mesenchymal cells from the tubular cells. As the mesenchymal cells increase in number, vascular tissue enters the cleft to form the capillaries of the glomerular tuft. Eventually, the spherical glomerular capsule is formed and encloses the capillaries, leaving the afferent and efferent arterioles to maintain continuity with the vascular system.

Kidney slices of 7–17-week-old human fetuses were incubated for 90 min or less in Krebs-Ringer solution containing aminoisobutyric acid (AIB). Cell to medium ratios of AIB decrease with increasing fetal age. Uptake rate is slower than for mature human tissue. Both water content and extracellular space is high in embryonic tissue as compared to adult.

Morphometric comparison of rabbit cortical connecting tubules and collecting ducts. Connecting tubule (CNT) segments of the rabbit distal nephron were examined by scanning electron microscopy and computer-assisted morphometric analysis of transmission electron micrographs. CNT were very similar to the cortical collecting ducts (CCD) described previously. The epithelium of both segments contains two cell types, both of which can be modeled as simple cuboidal cells, and two distinct systems of extracellular channels. The lateral intercellular channels are comparable to the spaces between simple cuboidal cells but are modified by short projecting microvilli which produce a modest increase in lateral cell surface area. The basal infolded channels are best developed in the connecting tubule cells of CNT and contribute 63% of all channel-associated membranes in CNT. Total membrane areas are similar in CNT and CCD. The two segments differ only in the degree of extracellular channel dilation and the distribution of infolded membrane relative to cell height in the connecting tubule and principal cells. The relatively minor morphometric differences between CNT and CCD do not correlate well with the marked difference in transtubular volume flow induced in the two segments by ADH and an osmotic gradient. Comparaison morphometrique des tubules connecteurs et des tubes collecteurs corticaux de lapin. Des segments de tubules connecteurs (CNT) de nephron distal de lapin ont ete examines par microscopie electronique a balayage, et par analyse morphometrique avec ordinateur de micrographies electroniques en transmission. Les CNT etaient tres semblables aux tubes collecteurs corticaux (CCD) decrits anterieurement. L'epithelium des deux segments contient deux types cellulaires, qui peuvent etre modelises comme des cellules cuboides simples et deux systemes distincts de canaux extracellulaires. Les canaux intercellulaires lateraux sont comparables aux espaces entre les cellules cuboides simples, mais sont modifies par les projections de courtes microvillosites qui produisent une augmentation moderee de la surface laterale cellulaire. Les canaux basaux contournes sont les plus developpes dans les cellules de tubules de connection des CNT et forment 63% de l'ensemble des membranes associees aux canaux des CNT. Les surfaces membranaires totales sont identiques dans les CNT et les CCD. Les deux segments different seulement par le degre de dilatation des canaux extracellulaires et par la distribution des membranes invaginees par rapport a la hauteur cellulaire dans le tubule de connection et les cellules principales. Les differences morphometriques relativement minimes entre CNT et CCD ne sont pas bien correlees avec les differences marquees dans le flux volumique transtubulaire induit dans ces deux segments par l'ADH et un gradient osmotique.

This study investigates the development of the vascular system of the puppy kidney (1-21 days after birth) after preparing casts of the renal vessels. At two days, the intrarenal vascular system distal to the afferent arteriole is strikingly different than that of the adult. The glomeruli of the outer cortex consist of a single dilated vessel while those of the mid and inner cortex posses an increasingly larger number of capillary loops. The efferent arterioles vary greatly in appearance from outer to inner cortex. Those in the nephrogenic zone are characteristically short and narrow and join a larger venous vessel termined a sinusoidal capillary. An efferent system somewhat similar to that of the adult is seen in the mid and inner cortex. One of the most obvious differences noted between the puppy and adult kidney is the relative lack of peritubular capillary networks throughout the cortex of the puppy kidney. The puppy possesses large, irregular vessels termed sinusoidal capillaries. The most rudimentary sinusoids are found in the outer cortex with more mature vessels in the inner cortex. The vascular arrangement of the efferent arteriole and sinusoidal capillary appears as a post-glomerular shunt. Functionally, the shunt would direct blood flow away from the proximal tubule and thus could result in a low extraction ratio and Tm for secreted solutes.

Renal cystic disease induced by diphenylthiazole. Intranephron hydrostatic pressures were monitored while microperfusing proximal nephrons in diphenylthiazole (DPT)-exposed kidneys, a model in which increased compliance of tubular basement membrane is thought to predispose to cyst formation. Structural studies subsequently were done on these and additional kidneys. Results were compared to those obtained from the study of kidneys from normal rats. Intranephron hydrostatic pressures were shown to rise with microperfusion and did so at lower rates of perfusion among DPT exposed nephrons. For example, at four perfusion rates between 17 and 36 nl/min (60 to 130% of the SNGFR in DPT-fed rats), intranephron hydrostatic pressures were 6 to 14cm H2O higher (P < 0.05) in DPT-exposed kidneys. Subsequent light and electron microscopic examination of DPT-exposed kidneys showed micropolyps partially occluding inner medullary and intrapapillary collecting ducts. DPT-induced renal cystic disease resembles other forms of chemically induced renal cystic disease in its functional and structural parameters save that micropolyp formation appears to occur nearer the papillary tip. We conclude that conditions in the DPT-exposed rat kidney resemble more closely those predicted by the partial obstruction rather than by the increased compliance hypothesis of renal cyst formation.Maladie kystique renale induite par le diphenylthiazole. Les pressions hydrostatiques intra-néphroniques ont été contrôlées pendant la micro-perfusion de néphrons proximaux de reins exposés au diphénylthiazole (DPT), un modèle dans lequel la compliance augmentée de la membrane basale tubulaire pourrait prédisposer à la formation de kystes. Des études de structure ont ensuite été faites sur ces reins, et sur d'autres reins. Les résultats ont été comparés avec ceux obtenus dans les reins de rats normaux. Les pressions hydrostatiques intra-néphroniques augmentaient avec la micro-perfusion, mais dans les néphrons exposés au DPT, elles le faisaient pour des vitesses de perfusions plus fabiles. Par exemple, pour quatre vitesses de perfusions, entre 17 et 36 nl/mn (60 à 130% de la SNGFR chez les rats nourris au DPT), les pressions hydrostatiques intra-néphroniques étaient de 6 à 14cm H2O plus élevées (P < 0.05) dans les reins exposés au DPT. Ultérieurement l'examen au microscope optique et électronique des reins exposés au DPT a révélé des micropolypes, obstruant partiellement les canaux collecteurs médulaires internes et intra-papillaires. La maladie des kystes rénaux induite par le DPT ressemble aux autres formes de maladies kystiques rénales induites chimiquement dans ses caractéristiques fonctionnelles et structurelles à la réserve près que la formation de micropolypes semble se produire plus près de la pointe de la papille. Nous concluons que les conditions existant dans le rein de rat exposé au DPT ressemblent plus à celles attendues lors d'une obstruction partielle qu'à celles attendues dans l'hypothèse d'une augmentation de la compliance à l'origine de la formation des kystes rénaux.

Male rats were studied between 20 and 60 days of age. Standard renal clearance techniques were used. The efficiency of the diuretic response, the sodium response, and the potassium response were calculated as defined previously. Results indicate that both diuretic and sodium efficiency is lower between the ages of 20 and 30 days as compared to 40 and 60 days of age, whereas potassium efficiency shows no age deoendence. In morphological studies, it has been observed that the proximal tubule cells of 22-day-old rats show changes in lateral intercellular spaces with comparable features to those of mature animals. The spaces widened during volume expansion. The change is quantitatively the same as that found in mature rats. Both blood pressure and hematocrit show continuous increase with age. The response of Ringer infusion in both immature and mature animals may be a mixed response involving changes in physical factors and renal responses to volume expansion per se.

The study of diabetic nephropathy in experimental animals generally relies on the chemical induction of the diabetic state. Streptozotocin is commonly employed to that end; however, streptozotocin has an inherent nephrotoxic potential. We studied the effects of both streptozotocin, and diabetes on the kidneys of rats given streptozotocin 60 mg/kg, which was sufficient to induce severe diabetes. Our studies, which utilized both transmission and scanning electron microscopy, considered the pharmacokinetics of streptozotocin in renal tissue, as well as the effect of insulin treatment. Renal tissue kinetics were altered by occluding the renal hilum of one kidney during, and for 5 min after, the administration of streptozotocin. We found that, in contrast to alloxan, streptozotocin caused no detectable renal injury at the dose employed. Previously described renal epithelial papillomas were identified, and were not influenced by altering the renal tissue kinetics of the drug. We conclude that no ‘protection’ procedure is necessary for the kidney when the streptozotocin model of diabetes is employed. In this regard streptozotocin may have greater utility than alloxan.

To elucidate morphologic changes in glomeruli of rats made diabetic with alloxan 40 mg/kg, we examined the filtration barrier with light microscopy, transmission (TEM), and scanning electron microscopy (SEM). The rats were killed at 10 or 20 weeks. The presence of diabetes was verified by blood and urine glucose measurements. In vivo perfusion was carried out through the left ventricle with gluteraldehyde in cacodylate buffer. SEM and TEM were performed by conventional techniques. SEM revealed striking changes in both the epithelial and endothelial surfaces which were focal and progressive with time. The epithelial surfaces showed podocyte fusion with areas of frank necrosis. Occasional areas of regeneration were seen. The endothelium showed a loss in number and decrease in size of fenestrae, proliferation of microvilli, and appearance of irregular cellular processes. TEM confirmed the presence of cellular changes. The basement membrane was irregularly thickened and the mesangium was more prominent than in control animals. We conclude that not only is the basement membrane affected by diabetes, but also important endothelial and epithelial cell changes occur as well. These changes are best appreciated by SEM and have not been previously described.

The ependyma lining the lateral recess of the third ventricle of the teleost inferior lobe has been studied by light and electron microscopy, including Golgi impregnation methods. As many as five different cell types appear to line the ventricle, but some of these may be similar cells in different stages of activity. One cell type contains small dense-cored vesicles and appears to have processes extending into deeper portions of the lobe. Golgi preparations reveal subependymal cells with apical processes extending to the ventricle and basal extensions which may reach the pial surface. The present observations are discussed in relation to similar studies in other fishes, amphibians and mammals. Possible functions for the various cells observed are suggested.

The present study utilizes the scanning electron microscope (SEM) to reveal the surface morphology of proximal tubular cells and the parietal cells of Bowman's capsule of the adult rabbit nephron. To facilitate the examination of the basal surface of these cells, proximal tubules were dissected free and treated with collagenase to remove the basememt membrane. Other blocks of tissue were cryofractured to expose the lateral cell surfaces of the proximal tubules. Our investigation has shown that the lateral and basal surfaces of both the convoluted and straight segments of the proximal tubule have numerous processes. However, the arrangement and degree of branching is distinctly different in the two segments. The convoluted segment has large lateral ridges which form at the base of the microvilli and fan out to divide into lateral-basal processes. Many of the lateral-basal processes reach the basement membrane intact, interdigitating with complementary processes from adjacent cells. However, some of the lateral-basal processes branch into short, knobby projections (basal villi) which may also reach the basement membrane. Patches of basal villi are interspersed between broad regions of interdigitating lateralbasal processes. Therefore, in the convoluted segment, the lateral-basal processes form the major part of the basal cell surface. In tubular cells of the pars recta, unlike convoluted tubular cells, the majority of the ridges remain unbranched and pass directly to the basal surface where they divide into elaborate basal villi. Thus the basal surface of the pars recta cells is highly complex, appearing leaf-like, being a composite of numerous basal villi with a few lateral ridges. The basal surface of some parietal cells of Bowman's capsule have parallel ridges, which results in patches of striations.

Viscoelastic properties of tubule basement membranes in experimental renal cystic disease. Hereditary and acquired renal cysts develop from tubule segments that enlarge progressively. We measured the deformability of basement membranes surrounding individual normal tubules and cysts to determine if cysts might develop by simple extension of abnormally-deformable basement membrane in response to normal or increased transtubule hydrostatic pressures. Deformability (cm/dyne) was measured in individual tubules and cysts in vitro by a micropipet aspiration technique that related negative pressures within the pipet to the distance the tubule or cyst wall was aspirated into the pipet. Viscoelastic creep was determined from the time-dependent effect of pipet aspiration on membrane deformation. Proximal and collecting tubules, glomerular capsules and cysts were microdissected from controls and animals with acquired (Diphenylthiazole [rats], Nordihy-droguaiaretic acid [rats]), hereditary (C57 BL/6J [cpk/cpk] mice) and spontaneous (CFW w mice) renal cystic diseases. The major resistance to deformation was localized to the basement membrane since collagenase destroyed the elasticity of tubule and cyst walls. Tubule basement membranes adjacent to cysts appeared abnormal by electron microscopy in the animals fed DPT, but measurements of deformability and viscoelastic creep showed no differences between normal and cystic tubules in any animal model. Deformability values of cysts (7.7 ± 1.1, 10.9 ± 1.1, 11.2 ± 0.6, 9.4 ± 0.8 × 10 -3 cm/dyne in DPT, NDGA, C57 BL/6J and CF w , respectively) are consistent with the interpretation that high transtubule pressures ranging from 39 to 134 cm H 2 0 would be required if cysts form by simple stretching of the basement membrane secondary to a transepithelial hydrostatic pressure-gradient. Since in vivo measurements of hydrostatic pressures across cyst walls are not high enough we conclude that cysts do not enlarge due to increased deformability of tubule basement membranes.

Studies performed in several animal species have demonstrated that glomerulotubular balance is maintained throughout development despite the many changes that occur in the factors known to control it. In an attempt to understand the nature of this phenomenon we quantified the magnitude and described the profile of these changes in guinea pigs. The changes in physical forces were assessed from measurements of hydrostatic and oncotic pressures, whereas those in the permeability characteristics of the proximal tubule epithelium were estimated from permeance to macromolecules of graded radii, histologic measurements of the intercellular channels, and measurements of end-proximal ratio of tubular fluid-to-plasma osmolality (TF/Posm). Between 1 and 50 days of age the net pressure for reabsorption increased from 15.0 to 30.9 mmHg (P less than 0.01, n = 15) with the major change occurring during the first 2-3 wk of postnatal life. The urinary recovery of inulin, sucrose, and creatinine, injected in the early segment of proximal tubules did not vary with age. The urinary recovery of mannitol (MW 180 daltons, Stokes-Einstein radius 4.0 X 10(-10) m) increased from 92% at birth to 100% at 49 days of age (P less than 0.001, n = 24), consistent with a decrease of approximately 0.5 X 10(-10) m in the luminal openings of the paracellular channels. The length of the zonulae occludens and the width of the intercellular channels did not change during this period; however, the length of the channels increased from 5.0 +/- 0.17 to 8.9 +/- 0.48 micron (P less than 0.01, n = 16). These changes should result in an increase in resistance across the intercellular channels. Consistent with this assertion is the observation that the mean TF/Posm of the fluid collected toward the end of the proximal convoluted tubule decreased as a function of age from 1.05 at day 2 to 0.98 at day 80 (P less than 0.001, n = 24). The findings support the hypothesis that during early postnatal life glomerulotubular balance is made possible by a high permeability of the proximal tubule, which compensates for the low net reabsorptive pressure. As the animal matures and the proximal tubule epithelium becomes tighter, for glomerulotubular balance to be maintained, an increase in the number of intercellular channels and in the active transport of sodium need to be postulated.

Nephron obstruction in nordihydroguaiaretic acid-induced renal cystic disease. Studies were performed to characterize conditions in rat kidneys whose nephrons were made cystic by feeding 2% nordihydroguaiaretic acid (NDGA) to the animals. Using two micropipettes, we monitored intratubular hydrostatic pressures while perfusing single surface nephrons in NDGA-exposed (5 to 7 weeks) and normal rat kidneys. The introduction of 50 nl of Ringers solution labeled with 3H-inulin at a flow rate of 25 nl/min was associated with a significant mean (± SEM) increase (167 ± 61%; P < 0.02) in pressure in cystic but not in nondilated (-0.5 ± 27.2%) or normal (31 ± 23%) nephrons, respectively. The relative amount of 3H-inulin excreted in 40 min from cystic (4.0 ± 2.0%) was less than that excreted from either nondilated (19 ± 7%; P < 0.05) or normal (105 ± 26%; P < 0.01) nephrons. Intralumenal pressures in nondilated but not other nephron groups correlated with urinary flow rates (r = 0.51; P < 0.02). Single nephron filtration rates and tubular-fluid-to-plasma 3H-inulin ratios in additional rats were similar among all groups of tubules. Concluding that these data reflected increased resistance to outflow from cystic nephrons, we examined these and additional NDGA-exposed (1 to 24 weeks) kidneys. 3H-thymidine radio-autography demonstrated maximum collecting tubular cell hyperplasia (13% labeling) at 2 to 3 weeks of NDGA-exposure. Microscopy and microdissection demonstrated tiny mural polyps along outer medullary segments of collecting tubules. Thirteen tubules were traced to their outlets; polyps impinging on outflow lumens were found in all 13 instances. We conclude that partial nephron obstruction exists in NDGA-exposed kidneys and that obstruction is a likely contributor to cyst formation in this model.Obstruction néphronique dans la maladie kystique du rein déterminée par l'acide nordihydroguaiarétique. Cette étude a porté sur des reins de rats dont les néphrons ont été rendus kystiques par l'addition à l'alimentation d'acide nordihydroguaiarétique (NDGA) à 2%. Au moyen de deux micropipettes les pressions hydrostatiques intratubulaires ont été enregistrées pendant la perfusion de néphrons superficiels chez des rats exposés à NDGA (5 à 7 semaines) et des rats normaux. L'introduction de 50 nl de Ringer contenant de l'inuline 3H au débit de 25 nl/min est associée à une augmentation significative (167 ± 61%; P < 0,02) de la pression dans les néphrons kystiques mais pas dans les néphrons intacts (-0,5 ± 27,2%) ou normaux (31 ± 23%). La quantité relative d'inuline 3H excrétée en 40 min par les néphrons kystiques (4,0 ± 2,0%) est inférieure à celle excrétée par les néphrons non dilatés (19 ± 7%; P < 0,05) ou normaux (105 ± 26%; P < 0,01). Les pressions luminales des néphrons non dilatés, mais non celles des autres groupes, sont correlées aux débits urinaires (r = 0,51; P < 0,02). Les débits de filtration glomérulaires des néphrons individuels et le rapport de concentration de l'inuline tube/plasma, étudiés chez d'autres rats, sont semblables pour tous les groupes de tubules. Puisque la conclusion était que ces observations reflètent une augmentation de la résistance à l'écoulement dans les néphrons kystiques nous avons étudié d'autres reins exposés à NDGA pendant 1 à 24 semaines. Des autoradiographies avec de la thymidine 3H ont montré une hyperplasie maximale (13% de marquage) des cellules tubulaires des collecteurs entre 2 et 3 semaines d'exposition à NDGA. La microscopie et la microdissection ont montré des petits polypes muraux le long des segments médullaires externes des tubes collecteurs. Treize tubules ont été suivis jusqu'à leurs terminaisons et dans les 13 cas des polypes gênant l'écoulement ont été trouvés. Nous concluons qu'il existe une obstruction néphronique partielle dans les reins exposés à NDGA et que cette obstruction contribue vraisemblablement à la formation de kystes dans ce modèle.

Although a number of morphological studies have investigated the vascular system of the rat kidney, minimal data are available on the detailed anatomy of the efferent arterioles located throughout the cortex of the kidney. In the present study, the renal vascular system was filled with Microfil and the various efferent arteriole patterns were examined extensively. The efferent vessels of the entire cortex appear to form three major patterns which in turn divided the cortex into three separate regions: the outer, middle and inner cortex. The efferent arterioles of the outer cortex leave the glomerulus and run perpendicular to the kidney capsule. However, as the efferent arterioles ascend, they may show three variations in the way they branch: (1a) the efferent arteriole does not branch until directly beneath the capsule, (1b) the efferent vessel begins to divide into its major branches 100-200 mum below the surface of the kidney and (1c) the efferent vessel has only a short course before giving off many side branches. In the middle cortical area, the branches of the efferent arteriole run lateral to the glomerulus. However, the efferent arterioles of the inner cortex have a few branches which run lateral to the glomerulus while most of them descend into the medulla as vasa rectae. The unique morphological features of the efferent arterioles of the outer cortex are of particular interest in light of the functional data which suggests that the reabsorption of fluid by peritubular capillaries may indeed regulate the rate of net tubular sodium reabsorption.

The present study describes the development and maturation of the glomerular visceral epithelium and capillary endothelium in an attempt to clarify kidney function in the neonate. The puppy kidney undergoes nephrogenesis for at least two weeks after birth, and thus possesses nephrons of different ages and levels of maturation. Since nephron development varies considerably from inner to outer cortex, renal corpuscles were examined within three arbitrary zones (outer, middle and inner cortex). The visceral epithelium of the smallest glomeruli, i.e., those of the outer cortex, is composed of cuboidal cells which are densely clustered and rests directly upon the limited number of small capillaries. More deeply within the outer cortex, broad, flat epithelial processes extend around the capillary, but lack any visible pedicels. Mid cortical glomerular capillaries undergo an increase in diameter and length, and at the same time the podocytes flatten and separate from one another. Pedicels begin to appear in this zone as thickened, disorganized processes. Juxtamedullary glomeruli are the largest, due primarily to their extensive capillary branching (looping). Podocytes are nearly adult-like in appearance, with many long primary processes and pedicels. The endothelial morphology of glomerular capillaries varies from one loop to another, as well as within a single loop. The immature endothelium of outer glomeruli is initially characterized by extensive expanses lacking pores, multiple cellular layers and small, scattered pinholes. Greater capillary maturity is apparent in the mid cortex, due to an increase in pore number and diameter as well as greater attenuation of endothelial cells. However, evidence of remodeling and a lack of pores still exists in some capillaries. The glomerular capillaries of the inner cortex appear the most mature, but possess regions that are still immature when compared to the adult.

To determine whether vascular smooth muscle cells around intestinal arterioles of various sizes undergo comparable changes in spontaneously hypertensive rats, 4- to 6-week-old (n = 10) and 17- to 19-week-old (n = 10) rats from the Wistar-Kyoto and the spontaneously hypertensive strains were used to study the external morphology of vascular smooth muscle cells by scanning electron microscopy and the vessel wall cross-sectional characteristics by light microscopy. At the time of fixation all vascular tone had been abolished. Scanning electron microscopy analysis revealed that all Wistar-Kyoto and spontaneously hypertensive rats at a given age have spindle-shaped vascular smooth muscle cells of comparable length and longitudinal width for a given branching order of arterioles. However, normal maturation is associated with elongation and widening of the vascular smooth muscle cells. Light and scanning electron microscopy indicated that a monolayer of vascular smooth muscle cells, wrapped at almost 0 degree to the vessel's radial axis, is maintained in adult spontaneously hypertensive rats. The radial thickness of this vascular smooth muscle cell monolayer was significantly (p less than 0.025) increased for only the largest arterioles of young and adult spontaneously hypertensive rats. This radial thickening of individual vascular smooth muscle cells increased the muscular component of the wall area for the largest arterioles by about 50% in adult spontaneously hypertensive rats. Other smaller submucosal arterioles of young and adult spontaneously hypertensive rats had normal vessel wall and vascular smooth muscle cell characteristics. These data indicate that hypertrophy in the smooth muscle cell's radial dimension is the primary morphological change in intestinal arterioles of spontaneously hypertensive rats. However, the vascular smooth muscle cell hypertrophy is confined to the largest arterioles such that the remaining smaller arteriolar vessels in the spontaneously hypertensive rat retain a normal smooth muscle cell and overall wall morphology.

The present study defined the ultrastructural features of peritubular capillary development. Two-day-old beagle puppies and adult dogs were perfused with 2.5% glutaraldehyde and routinely prepared for light and transmission electron microscopy. Some of the fixed tissue was subsequently used to make freeze-fracture replicas. The outer cortex of the puppy kidney possessed large, thick-walled vessels best termed sinusoidal capillaries instead of the small caliber vessels (peritubular capillaries) noted in the adult. These sinusoidal vessels showed extensive overlapping of the endothelium with isolated patches of fenestrae. Their luminal surfaces were irregular, owing to prominent ridges and sporadic bulges of endothelium. The basement membrane of most vessels was not present. Interstitial spaces were filled with mesenchymal cells and cells closely resembling pericytes. The diameter of the fenestrae of vessels throughout the cortex was similar; however, the number of fenestrae per micrometer of endothelium increased significantly from outer to inner cortex. Vessels of the inner cortex were also immature when compared to the adult. From these morphological findings, it was apparent that a true peritubular capillary system does not exist in the two-day-old puppy. Ultrastructural features of these vessels suggested reduced permeability characteristics.

The ultrastructure of ependymal cells of the median eminence in young Pacific blacktip reef sharks is described. Three major cell types have been observed. The first type is an irregularly-shaped, electron dense cell with many long microvilli and occasional cilia projecting into the ventricle. A second type contains dense cytoplasmic granules in sizes ranging from 1000–2000 A, and may be morphologically equivalent to “liquor-contacting neurons” observed in ependymal linings elsewhere. The third type appears very similar to crown cells of saccus vasculosus of this and other species. Cells of the third type possess large, lipid-like inclusions, bulbous projections originating from basal bodies, and prominent smooth endoplasmic reticulum. The crown-like cells are compared to cells of saccus vasculosus of young and adult sharks, and their possible significance is discussed.

HCO-3 transport (JHCO-3) in early juxtamedullary proximal convoluted tubules isolated from infant rabbits during the 1st 3 wk of life is about one-third that in tubules obtained from adults. A rapid increase in transport ensues during wk 4 through 6, so that near-mature levels are attained by the end of this time. Because the pattern for development of glucose absorption was similar and because both HCO-3 and glucose absorption are driven by the lumen-to-cell Na+ flux, the activity of Na-K-ATPase (the Na+-extruding pump) was considered to be a critical mediator. A kinetic microassay (which couples ATP hydrolysis to NADH oxidation) allowed the measurement of Na-K-ATPase and ouabain-insensitive ATPase on the same tubular segment. Three to nine early juxtamedullary proximal convoluted tubules were obtained after collagenase treatment of the kidney and four to six rabbits were studied at each week of life. The mean activity of Na-K-ATPase during the 1st wk of life was 44.5 +/- 3.5 pmol X min-1 X mm-1, one-third of the adult level. During an interim period of development (2-6 wk), enzyme activity gradually reached 60% of adult levels (76.3 +/- 3.0 at 6 wk), while transport of HCO-3 and glucose, studied previously in other animals, attained mature rates. Only in the 7th wk did the enzyme activity reach that of the adult (106.8 +/- 6.8 in wk 7 vs. 128.4 +/- 14.0 in adult rabbits).(ABSTRACT TRUNCATED AT 250 WORDS)

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5'-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.

Female Sprague-Dawley rats were used to study effects of lithium carbonate on the ultrastructure of the rat kidney. Experimental rats received daily intraperitoneal injections of lithium carbonate in dosages of 10, 30 or 100 mg/kg/day. These animals were killed on day 12 to 60. Control rats were either non-injected or injected with either sodium chloride or sodium carbonate. Kidneys from control rats showed no abnormal changes. The 10 and 30 mg/kg lithium carbonate dosages caused progressive mitochondrial swelling, dilatation of cisternae of rough endoplasmic reticulum and occasional swelling of apical cytoplasm in tubular cells localized in the distal portion of the nephron. The 100 mg/kg lithium dosage produced damage in all portions of the nephron. However, the most severe damage, consisting of mitochondrial swelling, dilatation of the rough endoplasmic reticulum, apical cytoplasmic rarefaction and liquefaction, karyolysis and karyorrhexis was noted in the distal convoluted tubules and collecting ducts. The present study demonstrated that low dosages of lithium carbonate do affect the structure of the rat kidney.

In addition to being tubular nephrotoxins, the aminoglycosides are known to injure the glomerular endothelium by decreasing the diameter and density of endothelial fenestrae. To determine whether or not these changes correlate with the rank order of toxicity we compared in rats the effects of netilmicin, tobramycin, and amikacin on renal function, tubular and glomerular ultrastructure, at 10 and 25 times the recommended human dose. According to measurements of glomerular filtration rate their rank order of toxicity, in increasing severity was: netilmicin, tobramycin, and amikacin. The same order of effect was observed on glomerular ultrastructure. Thus glomerular structural changes correspond to glomerular functional changes in this model.