Induction of intramembranous particle clusters in mice with nephrogenic diabetes insipidus.

In mice with hereditary nephrogenic diabetes insipidus (NDI), the inability of vasopressin to increase hydraulic water permeability is reflected in a lack of intramembranous particle (IMP) clusters in apical membranes of inner medullary collecting ducts. The lack arises from anomalously high activity of one or two isozymes of adenosine 3',5'-cyclic monophosphate-phosphodiesterase (cAMP-PDE). We asked whether inhibition of these isozymes with rolipram and cilostamide would raise not only the tissue content of cAMP but also and simultaneously restore IMP clusters. Inner medullary collecting ducts from NDI mice were incubated in vitro. Tissue content of cAMP (fmol of cAMP per bundle) and number of IMP clusters (per 100 microns 2 of principal cell apical membrane) were, respectively: control, 44.8 +/- 13.0 and 4.16 +/- 1.49; arginine vasopressin (AVP), 31.7 +/- 8.0 and 3.98 +/- 1.56; rolipram and cilostamide, 109.7 +/- 21.0 and 58.09 +/- 15.74; and AVP plus rolipram and cilostamide, 305.7 +/- 75 and 48.63 +/- 11.03 (with the last four values showing significant difference from control and AVP only, respectively). In addition, treating NDI mice with rolipram and cilostamide in vivo reduced their high fluid turnover. We conclude that failure by AVP to increase cAMP in cells of collecting ducts, which results from anomalously high activity of one or two specific isozymes of cAMP-PDE, is the major or sole cause for the excretion of hypotonic urine in NDI mice (DI +/+ Severe strain).

Causes of the urinary concentrating defect in mice with nephrogenic diabetes insipidus.

In a strain of mice called DI +/+ Severe, nephrogenic (or vasopressin-resistant) diabetes insipidus is caused by an inability of the antidiuretic hormone (ADH, or vasopressin) to increase the water permeability of the renal collecting system. That inability, in turn, arises from abnormally high activity of the enzyme cAMP-phosphodiesterase, specifically of the isozyme type III (PDE-III), which hydrolyzes cAMP and prevents the intracellular buildup of this second messenger. Two rather specific inhibitors of PDE-III, rolipram and cilostamide, used either in vitro or in vivo, reverse the deficiencies in DI +/+ Severe mice by increasing intracellular cAMP and water permeability toward or to their normal values. These results have implications for the treatment of nephrogenic diabetes insipidus in human patients.

Heparin inhibits mesangial cell proliferation in habu-venom-induced glomerular injury.

The authors have investigated the ability of anticoagulant heparin and nonanticoagulant heparin to inhibit mesangial-cell proliferation after the administration of habu (Trimeresurus flavorivids) snake venom to rats. Rats given injected habu venom exhibited glomerular capillary cystic lesions 6 to 24 hours later, and marked mesangial proliferation was noted within the cyst after 3 days. At 7 days 87% of these lesions (nodules) contained primarily mesangial cells embedded in a dense matrix and fibrin. A decrease in the frequency of nodules and the persistence of cysts indicate effective antiproliferative treatment. When anticoagulant heparin treatment extended from 18 hours after venom administration until sacrifice at 7 days, the percentage of nodules was reduced to 40%. Nonanticoagulant heparins resulted in some, but inconsistent, inhibition of mesangial-cell proliferation. The mechanism of the antiproliferative action of heparin on mesangial cells is not known but may be similar to that for vascular smooth muscle growth regulation. The authors suggest that endogenous heparin in the glomerular basement membrane and mesangial matrix may exert an antiproliferative effect under normal conditions. Loss of this inhibition due to glomerular damage might be reversed by the addition of exogenous heparin.

The influence of age on acute renal toxicity of uranyl nitrate in the dog.

The influence of age upon uranyl nitrate (UN) induced acute renal failure (ARF) was evaluated in 30 canine puppies 1-2 wk and 3-5 wk old. Renal function and morphologic studies were performed 2 h (initiation phase) and 24 h (maintenance phase) after UN administration. Age-matched controls received vehicle alone. Administration of UN to 1-2-wk-old puppies produced no changes in whole kidney glomerular filtration rate (GFR), despite a significant reduction in renal plasma flow (RPF) (P less than 0.01). In contrast, during the maintenance phase, GFR was 60% lower than in the control group (P less than 0.02) whereas values for RPF were nearly identical to control values. In 3-5-wk-old puppies the magnitude of response to the heavy metal was much greater and GFR was nearly completely suppressed during the maintenance phase. This major alteration of GFR was independent of changes in RPF, because RPF remained similar to control values. Morphologic alterations consistent with the nephrotoxic effects of UN were observed in the proximal tubules of the most differentiated nephrons. These age-related morphologic alterations correlated well with the functional response (GFR) observed after UN administration, i.e., a proportionately greater degree of both morphologic and functional alterations followed the administration of the heavy metal in the oldest group of puppies.

Cytoplasmic contractile elements in glomerular cells.

The presence and possible roles of cytoplasmic contractile elements in glomerular epithelial podocytes and the glomerular mesangium are briefly discussed. Glomerular podocytes contain actinlike filaments distributed throughout their cytoplasm and more concentrated filamentous material within their foot processes. Antibody labeling and nitrobenzoxadiazole-phallacidin labeling have confirmed the presence of especially high concentrations of actin within podocyte foot processes. Studies with cytochalasins have suggested that contraction of actin within podocytes leads to a flattening of foot processes and a loss of the intervening filtration slits. Conversely, relaxation of these contractile elements leads to a narrowing of the bases of foot processes and an increase in the number of fully open intervening filtration slits. These observations have led to the proposal that glomerular podocytes have the potential to regulate the glomerular filtration rate by changing the shapes of their foot processes and thereby decreasing or increasing the number of filtration slits available for solute efflux across the glomerular wall. There is also evidence to indicate the presence of contractile elements within the glomerular mesangium. These contractile elements may be represented in part by bundles of fine filaments associated with electron-dense bodies that are present within many processes that radiate from these cells. In vitro studies in particular have suggested that these cells may possess contractile properties. It has been proposed that contraction of the mesangial cells may lead to a shunting of blood within the glomerulus or to a decrease in glomerular size.

Protection of kidneys from acute renal failure resulting from normothermic ischemia.

Rat kidneys were flushed in situ with selected preservation solutions prior to clamping the renal vessels for 1 hour. Collins and Euro-Collins flushing solutions did not appear to protect the physiologic or morphologic status of rat kidneys when examined 2 days after the ischemic insult. These experimental groups exhibited serum creatinine levels similar to those seen in ischemic controls, correspondingly low urine creatinine levels, anuria, and significant deterioration of the uriniferous tubules as revealed by light and electron microscopy. In situ flushing with hypertonic Sacks or isotonic phosphate-buffered sucrose solutions, however, resulted in significant improvements in serum and urine creatinine levels, prevented anuria, and dramatically improved the morphologic integrity of the uriniferous tubules. Flushing with a phosphate-buffered sucrose solution that contained ATP-MgCl2 further improved the physiologic and morphologic status of ischemic kidneys to the point that they were indistinguishable from the nonischemic controls. The degree of protection obtained by flushing kidneys with the isotonic phosphate-buffered sucrose solution plus ATP-MgCl2 is greater than that provided by any other single pretreatment or posttreatment for ischemia that is currently available. We, therefore, believe that the use of this procedure can provide a valuable approach to surgical situations in which postischemic acute renal failure is a potential problem.

Ultrastructure of kidney preservation: varying the amount of an effective osmotic agent in isotonic and hypertonic preservation solutions.

Currently the level of effective (i.e., impermeant) osmotic agent necessary to prevent cell swelling during the cold storage of kidneys is not known. In the present investigation, we used light microscopy and transmission electron microscopy to evaluate the amount of the osmotic agent sucrose which is needed to protect the rat kidney parenchyma from damage over 24 and 48 hr of cold storage. Our observations indicate that when sucrose contributes 140 mOsmol or more to the total osmolality of phosphate-buffered solutions, most cell swelling and associated ultrastructural damage can be prevented over 48 hr of cold storage. We also found little difference between the quality of kidney ultrastructural preservation which results when kidneys are stored in isotonic (300 mOsmol) versus hypertonic (400 mOsmol) solutions that contain the same amount of sucrose. The overall quality of preservation seen with solutions which contain 140 or 200 mOsmol of sucrose is dramatically better than that which we previously observed with such clinically popular kidney preservation solutions as Collins, Euro-Collins, and Sacks.

Factors that improve the preservation of nephron morphology during cold storage.

Light and electron microscopy were used to evaluate the nephron morphology of rat kidneys that were perfused and stored at 0 to 2 degrees C. for up to 48 hours in preservation solutions which (1) contained different osmotic agents, (2) were adjusted to different levels of osmolality, and (3) which had different ionic compositions (e.g., intracellular-like, extracellular-like). Storage in an enriched phosphate-buffered isotonic culture medium that contained very little effective osmotic agent (i.e., medium 199) results in significant necrosis of glomeruli and uriniferous tubules with 12 to 24 hours. Storage in Collins solution that has an intracellular-like ionic composition and that contains dextrose as an osmotic agent results in significantly better preservation of kidney nephrons. When the dextrose in Collins solution is replaced with mannitol or sucrose, there is dramatic improvement in the preservation of glomeruli and uriniferous tubules. Overall, sucrose appears to be the most effective osmotic agent in preventing degeneration of the nephron during cold storage. Further improvement in nephron preservation is seen when the osmolality of Collins solution is raised (i.e., 400 to 600 mOsmoles) by adding more of the osmotic agents. Finally, storage in simple sodium phosphate-buffered solutions containing either dextrose, mannitol, or sucrose results in the same quality of morphologic preservation as seen with Collins intracellular-like solutions containing similar amounts of these osmotic agents. It appears, therefore, that the selection of an effective osmotic agent (e.g., sucrose) and making the preservation solution hypertonic with this osmotic agent are of primary importance in preserving nephron morphologic integrity during cold storage. The presence of an intracellular-like ionic composition in the preservation solution, however, does not appear to influence morphologic preservation. The effects of the above variables on the ultrastructural morphology of different segments of the uriniferous tubules and the renal corpuscles are described and illustrated.

In vitro studies of kidney glomerular epithelial cells.

An in vitro model system for studying the glomerular epithelium exposed on the surfaces of kidney slices is described. In the first 12 hours of incubation at 37 degrees C, glomerular podocytes exhibit an increase in number of free surface microprojections and number of cytoplasmic lipid inclusions. During the next several days these cells undergo a series of morphological alterations which resemble a dedifferentiation of the glomerular epithelium. By one week of incubation, the glomerular epithelium has been transformed into a compact group of rounded cells. These cells remain rounded but viable throughout several weeks of subsequent incubation. The glomerular endothelium becomes thickened but also remains viable throughout several weeks of incubation. Surface epithelial cells in both cortical and medullary regions become modified into a thin layer of viable cells. At lower incubation temperatures (e.g. 15 to 33 degrees C), the dedifferentiation of glomerular podocytes and formation of a viable surface layer of cells are significantly inhibited. Both undifferentiated and puromycin aminonucleoside nephrotic glomerular podocytes survive and adapt to the in vitro environment. The in vitro response of glomerular epithelial cells to various compounds added to the incubation media are briefly described. Compounds which induce a loss of cytoplasmic microtubules (e.g. vinblastine, colchicine), cause a loss of podocyte cell body and major process morphological integrity. Neuraminidase removal of the glomerular sialic acid surface coat inhibits the formation of free surface microvilli and results in an early loss of podocyte foot processes. The nephrotoxic agent puromycin aminonucleoside has no effect on the viability or morphology of glomerular cells except at very high concentrations (300 micrograms/ml). The distribution and effects of polycationized ferritin on living glomeruli are briefly described. Finally, in vitro treatment with cytochalasin B appears to inhibit cation-induced loss of podocyte foot processes.