Using vital microscopy (tscm) to evaluate living kidneys.

Tandem Scanning Confocal Microscopy (TSCM) is a non-invasive form of vital microscopy which can be used to evaluate superficial uriniferous tubules in living kidneys. Because TSCM has a number of advantages over conventional microscopic examination of renal biopsies, the present study was undertaken to determine whether the histopathological images obtained by TSCM can be correlated with post-transplant renal function. The kidneys of New Zealand male rabbits were harvested, flushed in Euro-Collins solution, and stored at 0-2 degrees C for periods of 24, 48, 67 and 72 hours prior to transplantation. As expected, there was a significant deterioration in post-transplant renal function as the kidneys were stored for longer periods of time. TSCM observations of the kidneys prior to their transplantation revealed characteristic histopathological changes of the proximal convoluted tubules which correlated closely with post-transplant renal function. These observations support the proposed use of TSCM in evaluating human donor kidneys prior to their transplantation.

Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells.

We investigated the effects of hyperosmolality on survival and proliferation of subconfluent cultures of mIMCD3 mouse renal collecting duct cells. High NaCl and/or urea (but not glycerol) reduces the number of viable cells, as measured with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Raising osmolality from a normal level (300 mosmol/kg) to 550-1,000 mosmol/kg by adding NaCl and/or urea greatly increases the proportion of cells in the G(2)M phase of the cell cycle within 8 h, as measured by flow cytometry. Up to 600 mosmol/kg the effect is only transient, and by 12 h at 550 mosmol/kg the effect reverses and most cells are in G(1). Flow cytometry with 5-bromodeoxyuridine (BrdU) pulse-chase demonstrates that movement through the S phase of the cell cycle slows, depending on the concentrations of NaCl and/or urea, and that the duration of G(2)M increases greatly (from 2.5 h at 300 mosmol/kg to more than 16 h at the higher osmolalities). Addition of NaCl and/or urea to total osmolality of 550 mosmol/kg or more also induces apoptosis, as demonstrated by characteristic electron microscopic morphological changes, appearance of a subdiploid peak in flow cytometry, and caspase-3 activation. The number of cells with subdiploid DNA and activated caspase-3 peaks at 8-12 h. Caspase-3 activation occurs in all phases of the cell cycle, but to a disproportionate degree in G(0)/G(1) and S phases. We conclude that elevated NaCl and/or urea reduces the number of proliferating mIMCD3 cells by slowing the transit through the S phase, by cell cycle delay in the G(2)M and G(1), and by inducing apoptotic cell death.

Organic osmolyte distribution and levels in the mammalian urinary bladder in diuresis and antidiuresis.

Inositol, sorbitol, glycerophosphocholine (GPC), and betaine are organic osmolytes that are accumulated by renal medullary cells in response to hyperosmotic stress. Previous screening studies, using nuclear magnetic resonance spectroscopy, have shown some of these same compounds to be present in extracts of whole urinary bladder from rabbits and rats. In the present study, we used high-performance liquid chromatography to quantify levels of these compounds in the separated epithelium and muscle of bladders taken from normal rabbits as well as diuretic and thirsted rats. We find that 1) high concentrations of organic osmolytes, namely inositol, GPC, and sorbitol, are present in urinary bladder; 2) levels of these osmolytes in the bladder epithelium are higher than in the muscle; 3) increased urinary osmolality due to antidiuresis is associated with a 2.4-fold increase in total osmolyte levels in rat bladder epithelium and a lesser (1.5-fold) increase in the muscle, compared with corresponding levels in tissues from diuretic animals; and 4) these increases in total osmolyte amounts in the epithelium are due to increased levels of GPC, sorbitol, and perhaps inositol (P = 0.07), whereas only GPC increases in the bladder muscle.

The spatial organization of corneal endothelial cytoskeletal proteins and their relationship to the apical junctional complex.

PURPOSE: To determine the spatial organization of the major cytoskeletal proteins and their relationship to the apical junctional complex (AJC) in the normal rabbit corneal endothelium. METHODS: Normal endothelial cytoskeletal structure in three dimensions was studied in rabbit eyes by laser scanning confocal microscopy after en bloc immunocytochemical staining of whole corneal tissue with various antibodies and fluorescent probes; specificity of antibodies to rabbit corneal endothelial cell proteins was established by Western blot analysis. RESULTS: Normal actin microfilament network organization was seen predominantly as a complex apical array forming a circumferential bundle. The tight junction-associated protein ZO-1 was positive at the apical junctions, forming a hexagonal pattern that was localized between and just proximal to the circumferential actin microfilament bundles. The distribution of ZO-1 was discontinuous around the cell, with the largest gaps (1 micron in diameter) occurring at the Y-junction between adjacent endothelial cells; transmission electron microscopy of the apical face of the endothelium confirmed the existence of 1-micron diameter gaps in the adherens junctions located at the Y-junction. Antivimentin antibodies showed a ring of intermediate filaments located just below the circumferential actin microfilament band. This ring appeared to be continuous with a basal mat of filaments, which together formed a basketlike structure within endothelial cells. An intricate cytoplasmic, perinuclear network of microtubules was observed by antitubulin antibodies that appeared unrelated either to the apical circumferential actin microfilament bundle or to intermediate vimentin filament ring. Staining of endothelial cells with NBD-ceramide identified a prominent, perinuclear Golgi complex suggesting an association between microtubules and Golgi. CONCLUSIONS: The organization of cytoskeletal elements and the tight junction-associated protein ZO-1 is similar to the classical AJC of transporting epithelia, comprised of a zonulae occludens (ZO) located apical to a zonulae adherens (ZA) and desmosomes. The organizational pattern seen in corneal endothelial cells, however, is distinct from transporting epithelia in that the ZO and ZA are discontinuous, with large gaps in the ZO-1 distribution at the Y-junction between adjacent endothelial cells. The authors propose that the structural differences in the AJC underlie the functional differences between classical transporting epithelia, which actively pump fluid from the lumen to the mucosa, and the corneal endothelium, which has a "pump-leak" fluid transport mechanism.

Early lead challenge and subsequent hypertension in Sprague-Dawley rats.

OBJECTIVE: The consequences of chronic, low grade lead (Pb) burden from earlier exposure on development of hypertension (HT) and cardiovascular disease is, at best, controversial, even though many epidemiological studies suggest the possibility. Accordingly, we examined ability of a short-term Pb challenge to cause later developing HT in rats. METHODS: We gave 12 newly weaned Sprague-Dawley rats (SD) a 1% Pb acetate solution to drink for 6 weeks, while 12 control rats drank water. The rats were further subdivided into groups consuming high and low amounts of sugar. All rats were followed for 4 months after cessation of the Pb challenge. RESULTS: Early Pb challenge caused no significant changes in body weight (BW) from controls; however, systolic blood pressures (SBP) of rats initially receiving Pb continued to rise significantly above their respective dietary controls for months after cessation of challenge. While a high sugar diet alone was associated with elevated SBP, high sugar consumers also challenged with Pb had the highest SBP. Protein excretion did not increase, suggesting, along with other evidence, a lack of significant renal damage. CONCLUSIONS: Previous exposure to Pb can cause subsequent chronic elevations in SBP.

An evaluation of the ability of dextrans to reduce acute tubular necrosis during cold storage preservation.

In this study, the ability of low molecular dextrans to prevent morphologically detectable acute tubular necrosis during cold storage was evaluated. Rat kidneys were flushed with a sodium phosphate buffer (pH 7.2) containing different concentrations of dextran 10 (m.w. of 10,000 or less) and stored at 0-2 degrees C for up to 5 days (samples taken at 24-hr intervals). It was found that solutions containing 20% or more of dextran 10 provided significantly improved morphological preservation of kidney nephrons when compared with currently popular kidney cold storage preservation solutions (i.e. University of Wisconsin and Euro-Collins solutions). Adding smaller amounts (i.e., 15%) of dextran 10 to a cold storage solution already containing another effective osmotic agent (i.e., sucrose) also resulted in superior morphological preservation, indicating a beneficial additive effect of using more than one osmotic agent. Dextran 40 (m.w. 40,000) did not provide as good morphological preservation as did a similar concentration of dextran 10. It is concluded that the use of the proper kind and proper amount of low molecular weight dextrans in preservation solutions can significantly reduce the morphologically detectable acute tubular necrosis during cold storage.

In vivo osmotic pertubation of intercellular fluid channels in the rabbit corneal endothelium.

An in vivo rabbit corneal model was used to evaluate morphological changes in the corneal endothelium associated with osmotically increasing fluid movement from the anterior chamber into the stroma. When the corneal stroma is rendered more hypertonic than normal by immersing the scraped epithelial side of the cornea in a hypertonic sucrose solution, intercellular channels and apical pores at the Y-junctions between endothelial cells become greatly enlarged. The foregoing changes are reversible and do not appear to result in damage to the corneal endothelium. These observations suggest that specific intercellular channels in the corneal endothelium may provide pathways for the movement of fluid from the aqueous humor into the stroma.

The histopathology of kidney uriniferous tubules as revealed by noninvasive confocal vital microscopy.

Tandem scanning confocal microscopy (TSCM) permits the noninvasive microscopic viewing of unstained, living, solid organs in real time. This article provides an overview of recent studies conducted by the author and other scientists, using TSCM and demonstrating its advantages in evaluating the histopathology of uriniferous tubules associated with normothermic renal ischemia, the nephrotic syndrome, and extracorporeal cold-storage preservation of kidneys.

Noninvasive vital microscopy to monitor tubular necrosis of cold-stored kidneys.

This study evaluated the ability of a new form of vital microscopy, termed tandem scanning confocal microscopy (TSCM), to determine the histopathological status of kidneys while they are being preserved in cold storage preservation solutions. The TSCM observations were performed on rat kidneys stored at 0-2 degrees C in one of several cold-storage preservation solutions (Euro-Collins, UW solution, phosphate-buffered dextran), and correlated with conventional light microscopic observations of these same kidney samples. The present investigation provides histopathological guidelines for the use of TSCM in determining the extent of necrosis that a kidney suffers during its cold-storage preservation, and demonstrates that TSCM can be used to observe cold-stored living kidneys repeatedly over time and provide immediate and artifact-free informative images regarding their histopathological status. The combined TSCM and conventional light microscopic observations also provided information regarding the ability of the various cold storage solutions being studied to preserve the normal histology status of cold-stored kidneys.

In vivo confocal microscopic studies of endothelial wound healing in rabbit cornea.

Corneal endothelial wound healing in living rabbit eyes after mechanical scrape (MS) and transcorneal freeze (TCF) injury was studied using tandem scanning confocal microscopy (TSCM). MS injury was created on the central corneal endothelium with an olive tip cannula; TCF injury was created using a 3-mm-diameter stainless steel probe cooled with liquid nitrogen. In vivo observation of wound healing using TSCM was correlated with scanning electron microscopy (SEM) for fixed tissues. At 6 h after MS, migrating endothelial cells at the leading edge showed lamellipodial processes on in vivo TSCM and SEM. After 24 h, the denuded area was almost fully resurfaced by migrating endothelial cells showing wide spaces between nuclei by TSCM. After 28 days, resurfaced endothelial cells showed normal hexagonal mosaic appearance with enlarged cells by TSCM and SEM. TCF injury produced fibroblastic changes in the endothelial cells with elongation and spreading by 24 h after injury. After 3 days, the wounded area was resurfaced with two cell types: (a) migrating endothelial cells at the peripheral area, which appeared polygonal in shape with wide intracellular spaces and (b) fibroblast-like cells at the center of the wound, which formed a retrocorneal fibrous membrane (RCFM). The RCFM was posteriorly covered with normal endothelium after 28-60 days. TSCM of the stroma demonstrated spindle-shaped, activated keratocytes migrating into the wounded stroma at 3-14 days. In conclusion, TSCM allows viewing of dynamic four-dimensional morphologic changes (x, y, z, and time) during in vivo cellular repair of corneal wound healing after either MS or TCF injury.

Actin filament organization during endothelial wound healing in the rabbit cornea: comparison between transcorneal freeze and mechanical scrape injuries.

PURPOSE: To compare and contrast the in vivo mechanism of wound healing after mechanical scrape and transcorneal freeze (TCF) injury in a rabbit eye model by examining changes in the cytoskeletal organization of contractile, filamentous actin (f-actin) microfilaments as relates to differences in cell migration or translocation during endothelial repair. METHODS: Endothelial wound healing after mechanical scrape and transcorneal freeze injury was studied in rabbit eyes using laser scanning confocal microscopy (LSCM). Central corneal mechanical scrape injury was made using an olive tip cannula, and TCF injury was made using a 3-mm diameter stainless steel probe cooled with liquid nitrogen. Cytoskeletal changes in f-actin stained with phalloidin-FITC were observed during wound healing using LSCM. RESULTS: At 6 hours after mechanical scrape, the leading edge of the migrating sheet showed a decrease in the intensity of phalloidin-FITC staining, suggesting a decrease in cortical f-actin. Migrating endothelial cells in vivo did not appear to develop stress fibers after mechanical scrape, which is consistent with an in vitro cell spreading mechanism of endothelial wound healing. By 24 hours, the denuded area was almost fully resurfaced by migrating endothelial cells. On the other hand, TCF injury produced fibroblastic changes in the endothelial cells with extension and elongation of spindle-shaped endothelial cells at the leading edge by 24 hours after injury. Fibroblastic endothelial cells developed prominent actin stress-fibers, which is consistent with an in vitro cell migration mechanism of endothelial wound healing. Three days after TCF, the wounded area was resurfaced with two cell types: rough, fibroblast-like cells forming a retrocorneal fibrous membrane having prominent f-actin bundles or stress fibers with few cell-cell junctions, and smooth, polygonal-shaped endothelial cells having tight cell junctions with a cortical distribution of f-actin. After 28 days the retrocorneal fibrous membrane was posteriorly covered with normal endothelium. CONCLUSIONS: These data support the hypothesis that endothelial wound healing involves two separate, injury-dependent, mechanisms--cell spreading and cell migration.

Noninvasive microscopic evaluation of the intact living nephrotic kidney.

BACKGROUND: Tandem Scanning Confocal Microscopy (TSCM) is a new form of microscopy that allows one to noninvasively "optically section" into intact structures and record microscopic images in real-time. In this study, we have evaluated the ability of this new technology to distinguish histopathologic changes in unstained living kidneys that occur coincident with the onset of puromycin aminonucleoside-induced nephrosis. EXPERIMENTAL DESIGN: The rats were anesthetized and a laparotomy was performed to expose the kidneys. Using a TSCM equipped with a 24x water-immersion objective, we viewed and recorded real-time images of subcapsular living uriniferous tubules and glomeruli of puromycin aminonucleoside-induced nephrosis rats exhibiting different levels of proteinuria. RESULTS: TSCM revealed a variety of histopathologic changes in the puromycin aminonucleoside-induced nephrosis uriniferous tubules including dilation of proximal tubular lumens, loss of microvillous brush border, cellular debris in the tubule lumens, and focal regions of extensive tubular necrosis. Using the fluorescent probe carboxyfluorescein, we were able to demonstrate significant heterogeneity in the movement of this fluorescent probe through the uriniferous tubules (e.g., some tubules were completely blocked), and irregular patterns in flow of carboxyfluorescein through nephrotic glomeruli. CONCLUSIONS: These observations indicate that TSCM is capable of revealing a variety of histopathologic changes in unstained, intact, living kidneys. In addition, many of the histopathologic features of the uriniferous tubules revealed by TSCM are difficult or impossible to distinguish in biopsied samples of renal tissue.

Effects of dietary protein on renal ischemia induced tubular necrosis.

Previously we have shown that the amount of protein in a dietary regimen prior to the induction of renal ischemia will significantly affect the degree of postischemic acute renal failure (Andrews PM, Bates SB: Kidney Int 30:299-303, 1986). The present investigation was undertaken to determine what effect dietary protein regimen has on the histopathology of renal ischemia. Rats were pair-fed for 2 weeks on either 0% or 5% (restricted), 20% (normal), or 60% (high) purified protein isocaloric diets. Ischemia was induced by 45 min of renal pedicle clamping. Light and electron microscopic evaluation of kidney morphology immediately following renal ischemia (prior to blood reflow) revealed that the extent of morphological damage to cells lining proximal convoluted tubules and the thick ascending segments of Henle's loop increased with increasing concentration of dietary protein. However, all dietary protein groups also exhibited heterogeneity in the extent of damage to different nephrons within the same kidney.

Digital image acquisition in in vivo confocal microscopy.

A flexible system for the real-time acquisition of in vivo images has been developed. Images are generated using a tandem scanning confocal microscope interfaced to a low-light-level camera. The video signal from the camera is digitized and stored using a Gould image processing system with a real-time digital disk (RTDD). The RTDD can store up to 3200 512 x 512 pixel images at video rates (30 images s-1). Images can be input directly from the camera during the study, or off-line from a Super VHS video recorder. Once a segment of experimental interest is digitized onto the RTDD, the user can interactively step through the images, average stable sequences, and identify candidates for further processing and analysis. Examples of how this system can be used to study the physiology of various organ systems in vivo are presented.

Tandem scanning confocal microscopy (TSCM) of normal and ischemic living kidneys.

Tandem Scanning Confocal Microscopy (TSCM) allows one to section optically into and record real-time images of living organs and tissues in a noninvasive fashion. In this paper, we will present some initial TSCM observations of subcapsular nephrons in the living, intact kidneys of Munich-Wistar rats and evaluate the nephron's responses to temporary ischemia and to intravenous infusion of mannitol. The rats were anesthetized with Inactin and a laparotomy performed to expose the kidneys. Using a TSCM equipped with a 20 x water-immersion objective, we optically sectioned through the intact kidney capsule and recorded real-time images of living subcapsular glomeruli and uriniferous tubules. The proximal tubule brush border was highly reflective and allowed us to distinguish between the first and second segments of the proximal tubules as well as the distal tubules. Cellular elements of the blood could be seen passing rapidly through peritubular capillaries and individual glomerular capillary loops. With fluorescent filters in place, intravenously injected carboxyfluorescein was seen to pass through the glomerular capillary loops and then progressively through the different segments of the uriniferous tubules. Ligation of the renal artery resulted in rapid swelling of proximal tubule cells into the tubular lumens, loss of reflectiveness of the microvillous brush borders, and closure of the peritubular capillary spaces. Upon release of the ligature, the proximal tubule lumens again became patent, often opening up abruptly and in a zipper-like fashion down the length of the tubules. Increasing the glomerular filtration rate by intravenous infusion of mannitol resulted in increases in tubular luminal and perimeter dimensions. Mannitol also acted as an effective impermeant osmotic agent and prevented most of the cellular swelling which was otherwise seen in response to renal ischemia.

In vivo, real-time confocal imaging.

We have adapted a tandem scanning confocal microscope for real-time, non-invasive imaging of cells under in vivo conditions. This form of in vivo confocal imaging relies on the optical sectioning abilities of the confocal microscope to obtain en face, sequential, reflected light images of cells at various depths, up to 1 mm, within opaque organs in living animals. Of major consideration in the design of an in vivo confocal microscope is maximizing the real-time detection of signals reflected from low contrast structures which can be affected by the microscope design, objective, and image detector systems. Using an in vivo confocal microscope design with a 20 x BioOptics surface contact objective we have obtained live cellular images from selected tissues including cornea, kidney, liver, adrenal, thyroid, epididymis, and muscle and connective tissue of rabbits and rats. Images were captured, digitized, and processed using a DAGE Mti low light level SIT camera coupled to a Gould IP9527 image processor. In vivo images were also compared with conventional bright field light and scanning electron microscopic images of "dead," fixed tissues. Overall, in vivo confocal imaging can provide remarkable detail of living cells comparable to that of conventional microscopic images of "dead," fixed, and stained tissue. A more unique feature of in vivo confocal imaging is the ability to study cellular structure and function sequentially over time in the same organ or tissue and represents a fundamentally new paradigm in microscopy. With continued refinements in the microscope, objective and detection system designs and their consequent improvements in lateral and axial resolution, in vivo confocal microscopy will enable us as observers to see what no one has been able to see before.

High dietary protein regimens provide significant protection from mercury nephrotoxicity in rats.

The effects of high protein dietary regimens prior to the administration of inorganic mercury were investigated. Male Sprague-Dawley rats were pair-fed on purified test diets containing either normal (20%) or high (60%) concentrations of protein. Mercury was administered as a single intravenous injection of mercuric chloride (1 mg/kg). All rats maintained on normal dietary protein prior to and following mercury injection exhibited severe kidney dysfunction, extensive necrosis of both second (S2) and third (S3) segments of the kidney proximal tubules, and 100% mortality. In contrast, rats maintained on high dietary protein for 48 hr or longer just prior to mercury injection and returned to normal dietary protein immediately following mercury administration all survived and exhibited normal serum creatinine and BUN values within 4 days following mercury administration. The kidneys of this latter group took up significantly less radiolabeled mercury during the first 12 hr following mercury injection, and exhibited relatively little damage to the second segments (S2) of the proximal tubules. The third segments (S3) of the proximal tubules, however, exhibited the same degree of necrosis as that observed in the control group. Maintaining rats on high dietary protein regimens for shorter periods of time prior to mercury infusion (i.e., 12 or 24 hr) also dramatically reduced subsequent acute renal failure and improved survival, although not to the extent noted following 48 hr or longer on these diets. These observations suggested that high dietary protein regimens may protect from mercury nephrotoxicity by reducing mercury uptake to the second segments (S2) of the proximal tubules during the initial period of exposure to intravenously administered mercury.

Dietary protein regimen prior to renal ischemia significantly affects the postischemic uremic response.

Recently we reported that maintaining rats on restricted dietary protein regimens prior to renal ischemia will significantly improve postischemic survival rates. This effect required a week or more of maintenance on a restricted protein diet prior to the renal insult and appeared to be independent of the postischemic dietary protein regimen. The present study was designed to evaluate the role of systemic toxicity in this protection. Adult male Sprague-Dawley rats were pair-fed by weight on restricted or high isocaloric protein diets for 8-10 days prior to 45 min of renal ischemia induced by renal pedicle clamping. When placed on a normal dietary protein regimen immediately following ischemia, the rats preconditioned to restricted dietary protein exhibited significantly less acidosis, less hyperkalemia, lower blood urea nitrogen values, and improved survival rates compared with rats preconditioned on a high dietary protein regimen. In order to separate the possible effects of prior dietary protein regimen on acute tubular necrosis suffered during renal ischemia from its effects on the uremic response, bilateral nephrectomies were performed on rats preconditioned for 14 days to low, normal, and high dietary protein regimens. Although all of the rats were placed on the same dietary protein regimen immediately following bilateral nephrectomy, those that had previously been on a lower dietary protein regimen exhibited a significantly reduced uremic response and lived longer. These findings indicate that dietary protein regimen prior to renal ischemia is a risk factor which can have a significant lingering effect on the severity of postischemic systemic toxicity.

Regional chemotherapy in an experimental model of Wilms' tumor in rats.

A s.c. experimental model of Wilms' tumor in rats was used to compare the effects of intratumoral treatment with vincristine plus actinomycin D to i.v. treatment with these chemotherapeutic drugs. The Wilms' tumor model is a fast-growing solid tumor that has been shown to be resistant to traditional clinical treatment procedures used for Wilms' tumor in man. Injection of the chemotherapeutic drugs directly into the tumor mass was found to be more effective than i.v. therapy in causing long-term remission of the tumor. Intratumoral therapy was also less toxic to the animals than i.v. therapy when measured by post-treatment survival rates and weight loss during the 1st week following treatment. However, intratumoral treatment caused an initial fibrosis of the tumor tissue, which resulted in a slower rate of absorption of the resultant fibrotic tumor mass than was seen in tumors treated i.v. Also, intratumoral injection resulted in necrosis of the overlying skin, which healed as the fibrotic tumor tissue was absorbed. Intratumoral treatment of a cervical tumor was found to cause the remission of a second major tumor mass located at some distance from the initial injection (i.e., in the lumbar region). No significant benefits were noted when dimethylsulfoxide (DMSO) was used in place of aqueous mannitol as a vehicle to deliver the chemotherapeutic agents. There was a significant correlation between the drug dose-to-tumor-size ratio (D/T ratio) and the effectiveness of the chemotherapy. When this ratio was high enough, a single treatment with a combination of vincristine and actinomycin D usually resulted in total remission of the experimental Wilms' tumor in response to either intratumoral or i.v. therapy.