Sequential analysis of kidney stone formation in the Aprt knockout mouse.

BACKGROUND: We have previously shown that, as in human adenine phosphoribosyltransferase (APRT) deficiency, Aprt knockout mice form 2,8-dihydroxyadenine (DHA) renal stones. The disease develops earlier and is more severe in male than in female mice. To examine the biological bases for these differences, the area occupied by DHA crystals was quantified in kidney sections from male and female mice (strain 129) aged one day to eight months and this parameter was correlated with changes in renal histopathology. Aprt heterozygous and wild-type mice were used as controls. METHODS: Following anesthesia, the left kidney was removed and immediately frozen in dry ice. Unstained cryosections were examined by polarized light to determine total area of birefringent particles. The right kidney was perfused and embedded in plastic, and stained sections were viewed by light microscopy to examine the histopathology and to determine the location of the birefringent particles. A pathological score was assigned to the histological findings. The scores from the right kidney were compared with crystal/particle area in the left kidney, and the data were analyzed using two-way analysis of variance. The chemical composition of the particles was determined by x-ray diffraction analysis. Several stone fragments from the bladder were also examined by scanning electron microscopy (SEM). RESULTS: Crystals were detected in kidney sections from one- to two-day-old Aprt knockout mice. The crystal burden remained low in both sexes throughout the study except in males at the 120- to 240-day period. Furthermore, there was a substantial degree of renal pathology, primarily seen as interstitial fibrosis, in those males with a very high level of stone formation. The crystalline material was identified as 6-amino-2,8(3,9)-purine dione, a tautomeric form of DHA. SEM indicated that the crystals were spherical, with a diameter of 10 to 20 microm. Tissue staining and fixation procedures dramatically reduced the amount of birefringent material in kidney sections. Aprt heterozygotes of both sexes had low levels of crystalline material in the kidneys and no pathology. Birefringent material or pathological changes were not seen in kidneys from wild-type mice. CONCLUSIONS: Both male and female Aprt knockout mice accumulate DHA. However, the area occupied by DHA crystals was significantly greater in 120- to 240-day-old males compared with the females of similar age. Also, substantial renal pathology was detected in kidneys of male mice that had very high levels of stone material.

Shear level influences resistance artery remodeling: wall dimensions, cell density, and eNOS expression.

The magnitude of shear stimulus has been shown to determine the level of growth factor expression in cell culture. However, little is known regarding what effect shear level has on specific arterial wall remodeling events in vivo. We have hypothesized that the rate of luminal diameter change and specific remodeling events within the arterial wall layers are dependent on shear level. Selective ligations were made to alter the number of microvascular perfusion units of mesenteric arteries within the same animal to approximately 50%, 200%, and 400% of control. Arterial blood flow and wall shear rate were correlated with the degree of alteration in perfusion units. Luminal diameters were decreased in 50% arteries by day 2 and increased approximately 17% and 33% respectively, in 200% and 400% arteries at day 7. The rate of diameter change was greatest in 50% and 400% arteries. Wall areas (medial +37%; intimal +18% at day 2) and cell densities (intimal +26%; adventitial +44% at day 2) were altered only in the 400% arteries. A positive correlation existed by day 2 between endothelial staining for endothelial nitric oxide synthase and shear level. The results demonstrate that shear level influences the rate of luminal expansion, specific remodeling events within each wall layer, and the degree of endothelial gene expression. A greater understanding of how shear level influences specific remodeling events within each wall layer should aid in the development of targeted therapies to manipulate the remodeling process in health and disease.

Renal disease potentiates the injury caused by SWL.

PURPOSE: The present study tested the hypothesis that renal disease potentiates the structural/functional changes induced by a clinical dose of shockwaves. MATERIALS AND METHODS: Experimental pyelonephritis was induced in 6- to 8-week-old pigs before treatment with 2,000 shocks at 24 kV. These pigs were divided into two groups according to whether they were infected with a highly virulent (Group 1) or less virulent (Group 2) inoculation of E. coli. All animals were imaged by MR prior to SWL as a means of documenting the extent of pyelonephritis and immediately after SWL to examine the lesion produced by the shockwaves. The glomerular filtration rate (GFR), renal plasma flow (RPF) and para-aminohippurate (PAH) extraction were determined bilaterally on day 30 (Group 1) or day 80 (Group 2). RESULTS: In group 2, urine flow and sodium excretion were reduced by 50% from baseline in the shocked kidneys at both 1 and 4 hours post-SWL. A sustained reduction in RPF through 4 hours post-SWL was noted in the shocked kidneys in Group 1, but RPF was significantly reduced only at the 1-hour determination in Group 2. Large, consistent reductions in GFR were evident at 1 and 4 hours post-SWL in shocked and unshocked kidneys of Group 2 and in the shocked kidneys of Group 1. No significant changes were noted in PAH extraction. CONCLUSION: Acute pyelonephritis exaggerated the effect of a clinical dose of shockwaves on renal hemodynamics. This effect suggests that renal disease may be risk factor for SWL-induced injury.

Relationship between kidney size, renal injury, and renal impairment induced by shock wave lithotripsy.

The relationship between kidney size and impaired renal function induced by shock-wave lithotripsy (SWL) was examined in 6- and 10-wk-old anesthetized pigs. Each pig received 2000 shock waves, 24 kV, or sham SWL to the lower pole calyx of one kidney. Bilateral GFR, renal plasma flow (RPF), and para-aminohippurate extraction was measured 1 h before and 1 and 4 h after SWL. The kidneys were then removed for morphometric analysis. Mean kidney weights were 66.1+/-2.7 g (n = 9) and 103.1+/-3.3 g (n = 8) in the SWL groups, and 60.1+/-2.6 g (n = 9) and 82.3+/-4.0 g (n = 9) in the sham-SWL groups. SWL-induced lesions occupied a significantly greater volume of the small kidneys (6.1+/-1.7 vol % versus 1.5+/-0.2 vol% in the large kidneys). RPF was significantly reduced by SWL in small and large kidneys, but to a significantly greater extent in small kidneys. RPF was also significantly reduced in the contralateral kidneys of both groups, but only at 1 h after SWL. SWL significantly reduced GFR to similar degrees in both kidneys of both groups, regardless of kidney size. Para-aminohippurate extraction was likewise reduced to similar degrees in both groups, but this effect was evident only in the SWL-treated kidneys, and only in the pole to which the shock waves had been applied. The injury induced by SWL affected a larger fraction of small kidneys than large ones, and the renal vasoconstriction induced by SWL was greatest in small kidneys.

In vivo pressure measurements of lithotripsy shock waves in pigs.

Stone comminution and tissue damage in lithotripsy are sensitive to the acoustic field within the kidney, yet knowledge of shock waves in vivo is limited. We have made measurements of lithotripsy shock waves inside pigs with small hydrophones constructed of a 25-microm PVDF membrane stretched over a 21-mm diameter ring. A thin layer of silicone rubber was used to isolate the membrane electrically from pig fluid. A hydrophone was positioned around the pig kidney following a flank incision. Hydrophones were placed on either the anterior (shock wave entrance) or the posterior (shock wave exit) surface of the left kidney. Fluoroscopic imaging was used to orient the hydrophone perpendicular to the shock wave. For each pig, the voltage settings (12-24 kV) and the position of the shock wave focus within the kidney were varied. Waveforms measured within the pig had a shape very similar to those measured in water, but the peak pressure was about 70% of that in water. The focal region in vivo was 82 mm x 20 mm, larger than that measured in vitro (57 mm x 12 mm). It appeared that a combination of nonlinear effects and inhomogeneities in the tissue broadened the focus of the lithotripter. The shock rise time was on the order of 100 ns, substantially more than the rise time measured in water, and was attributed to higher absorption in tissue.

Effects of SWL on glomerular filtration rate and renal plasma flow in uninephrectomized minipigs.

This study tested the hypothesis that the effects of SWL on hemodynamics in solitary kidneys differ from those in kidneys of binephric animals. Five female miniature pigs (Pitman-Moore, 6 months of age, 30-35 kg) were anesthetized for unilateral nephrectomy. Seven pigs served as binephric controls. Two weeks later, each pig was anesthetized, prepared for unilateral or bilateral urine collections, and subjected to SWL (Dornier HM3, 2000 shocks, 24 kV). Clearances of inulin (glomerular filtration rate; GFR) and para-aminohippurate (renal plasma flow; RPF) were measured 1 hour prior to and 1, 4, and 24 hours after SWL. The GFR and RPF were higher in uninephrectomized than in intact pigs at all time points. In both groups, SWL reduced GFR and RPF. In the binephric pigs, RPF was reduced at all times post-SWL, but in the uninephrectomized pigs, RPF was returning toward baseline by 4 hours post-SWL and was not different from baseline at 24 hours. A comparison of whole-animal GFR and RPF (righ plus left clearances in binephric pigs v solitary renal clearances in uninephrectomized pigs) showed that whole-animal GFR and RPF did not differ between the groups before or after SWL. Compensatory renal hypertrophy and improved hemodynamics in solitary kidneys may acutely attenuate the renal vasoconstrictive effect of SWL. The long-term consequences of the compensatory changes are unknown.

Quantitation of shock wave cavitation damage in vitro.

Acoustic cavitation damage was quantitated using aluminum foil targets placed within 2-mL polypropylene cryovials. The vials contained various media tested for their potential to support cavitation and were exposed to shock waves using an unmodified Dornier HM3 lithotripter. Foil damage, expressed in terms of a "damage index," was measured from digitized light microscopy images by quantitating the spread of gray-scale histograms. Target sensitivity was demonstrated by reproducible dose-response curves over the range (1-200 shock waves) commonly used for in vitro cell injury studies. Increased shock wave repetition rate reduced the damage index. Untreated foils showed a very low damage index (0.001% +/- 0.001%), while treated foils submerged in Ringer buffer yielded significant damage (2.2% +/- 0.3%, p < 0.001). Degassing the buffer reduced damage to 0.3% +/- 0.1% (p < 0.001). Foils submerged in castor oil showed virtually no damage. These results implicate acoustic cavitation in target damage. Targets immersed in biological fluids (blood and urine) had significantly less damage than in Ringer. The effect of degassing was also evaluated in a red blood cell lysis assay. Hemoglobin release in degassed preparations was significantly reduced compared to nondegassed controls (p < 0.001) and correlated with reduced foil damage index in cell-free vials. These findings characterize a sensitive method to quantitate acoustic cavitation and implicate a role for cavitation in shock wave lithotripsy-induced cell lysis.

Quantitation of shock wave lithotripsy-induced lesion in small and large pig kidneys.

BACKGROUND: Shock wave lithotripsy (SWL) is known to cause injury to the kidney. However, it is not known how lesion size varies as the parameters of SWL treatment (number of shocks, kilovoltage, kidney size) are changed. This hypothesis could not be tested because there was no method available to quantitate accurately the SWL-induced renal lesion. METHODS: A dosage of 2,000 shocks at 24 kV delivered by an unmodified Dornier HM3 lithotripter was applied to the lower pole calyx of the right kidney of small and large pig kidneys. A new method was developed to embed a whole pig kidney for serially sectioning, recording, and digitization. Automated computer color recognition made it possible to discriminate regions of hemorrhage from undamaged tissue and allowed quantitation of the lesion in single sections and in the entire kidney. RESULTS: The new protocol resulted in an accurate identification of sites of hemorrhage and calculations of the volume fraction of injured renal tissue. Lesion size induced in small kidneys was significantly larger than that induced in the larger kidneys (7.6 +/- 1.2% and 1.6 +/- 0.7%, respectively). CONCLUSIONS: Computer segmentation of serially sectioned SWL-treated kidneys has determined that kidney size is a risk factor for enhanced renal injury.

Effects of extracorporeal shock wave lithotripsy to one kidney on bilateral glomerular filtration rate and PAH clearance in minipigs.

PURPOSE: This study examined the acute time course of effects of extracorporeal shock wave lithotripsy (ESWL) on renal hemodynamics in anesthetized minipigs with and without pretreatment with verapamil. MATERIALS AND METHODS: We applied ESWL (2000 shocks, 24 kV, unmodified Dornier HM3), to the right kidneys of isoflurane-anesthetized female pigs. Urine flow and renal hemodynamics were monitored from each kidney via ureteral balloon catheters. Arterial blood pressure and bilateral urine flow, glomerular filtration rate (GFR, insulin clearance) and renal plasma flow (RPF, para-aminohippurate clearance) were monitored for 45 minutes before ESWL, and at 1, 4 and 24 hours after ESWL. RESULTS: Treatment with ESWL consistently caused unilateral hematuria and subcapsular renal hematomas in the shocked kidneys and significantly reduced GFR and RPF in those kidneys at 1 and 4 hours after ESWL. Urine flow was reduced through 24 hours in the shocked kidneys. Renal plasma flow, but not GFR, was significantly reduced in the contralateral (unshocked) kidneys at 1 and 4 hours after ESWL to the other kidneys. Verapamil blunted the ESWL-induced reductions of urine flow, GFR and RPF in the shocked kidneys and eliminated the reduction of RPF in the unshocked kidneys. CONCLUSIONS: These experiments demonstrate that ESWL to 1 kidney acutely impaired hemodynamics in both kidneys and that verapamil attenuated the response in the shocked kidneys and eliminated it in the contralateral unshocked kidneys.

Branching patterns of the renal artery of the pig.

BACKGROUND: The pig kidney is similar in structure and function to the human kideny, thus making it a useful model in understanding the human kidney in health and disease. However, little is known about the branching pattern of the pig renal artery as compared with the human and other animals. METHODS: The right and left kidneys from 11 juvenile pigs were injected with either Mercox compound to form a vascular cast or contrast media to obtain a renal arteriogram. Branching patterns of the renal artery were then examined to the level of the interlobar arteries. RESULTS: Examination of all 22 kidneys showed the main stem renal artery branching into two predictable patterns, designated I and II. The renal artery in pattern I (17 of 22 kidneys) divides into upper and lower polar arteries that then form anterior and posterior segmental arteries. Pattern I has two variations. Pattern II (5 of 22 kidneys) has a variable blood flow to the upper pole. Not only do anterior and posterior segmental arteries pass to the upper pole, but additional vessels arising from the lower polar artery also supply the upper pole. CONCLUSIONS: The most common branching pattern of the main stem renal artery is pattern I, which divides the blood flow of the entire kidney into two distinct regions. Pattern II is less frequently seen and shows more variability in number and location of branches.

Role of obstruction in autosomal dominant polycystic kidney disease in rats.

Kidney micropuncture and microdissection studies were carried out on heterozygous 2- to 4-month-old female and male Han:SPRD rats with autosomal dominant polycystic kidney disease (ADPKD) and on normal controls, to determine whether cysts are obstructed. Pressures in proximal tubules and cysts were determined using a servo null device and were recorded before, during, and after intraluminal infusion of an isotonic equilibrium solution at 15 and 50 n1/min. Initial cyst pressures in nine cystic rats averaged 18.5 +/- 5.9 (SD) mm Hg, N = 49, significantly (P < 0.01) higher than in normal proximal tubules in four control rats, 14.3 +/- 1.6 mm Hg, N = 36. Pressures in non-cystic tubules in cystic rats, 16.8 +/- 4.4 mm Hg, N = 25, were not significantly different from pressures in control kidneys or in cysts. When proximal tubules were microinfused at 15 nl/min in control rats, tubule pressure increased by 3.8 +/- 1.2 mm Hg, N = 24. In cysts, the response was highly variable. Twenty out of 33 microinfused cysts (61%) showed responses similar to normal tubules and were considered to be nonobstructed; 13 (39%) showed large pressure increases upon microinfusion, sometimes to values over 100 mm Hg (obstructed cysts). Left kidney inulin clearance (in microliter/min. 100 g body wt) averaged 335 +/- 65 (N = 4) in control rats and 344 +/- 144 (N = 9) in cystic rats; at this early stage of the disease no decline in GFR was seen. Weights of cystic kidneys were twice those of normal animals. Microdissection and scanning electron microscopy revealed the presence of intraluminal casts and debris and constrictions between cysts that would impede fluid flow. We conclude that obstruction is a frequent, early event in PKD and, when present, promotes cyst enlargement. Since many cysts are not obstructed, we suggest that factors other than fixed obstruction initiate cyst formation.

Apical membrane permeability of MDCK cells.

The osmotic water permeability (Pf) and permeability to nonelectrolytes were determined for the apical membrane of clonal strain Madin-Darby canine kidney (MDCK) C12 cells cultured as cysts with the apical membrane facing the surrounding medium. Pf and solute permeabilities were calculated from the rate of volume change of cysts by digitizing images at 1-s intervals after instantaneous osmotic challenge. Image measurement was fully automated with the use of a program that separated the image of the cyst from the background by using adaptive intensity thresholding and shape analysis. Pf, calculated by curve fitting to the volume loss data, averaged 2.4 +/- 0.1 micron/s and was increased by addition of amphotericin B. The energy of activation for Pf was high (16.3 kcal/mol), and forskolin (50 microM) had no effect on Pf. Two populations of MDCK cysts were studied: those with two to three cells and those that appeared to be composed of only one cell. The Pf of multicell cysts was the same as single cell cysts, suggesting that paracellular water flow is not significant. Solute permeability was measured using paired osmotic challenges (sucrose and test solute) on the same cyst. Urea permeability was not different from zero, whereas the permeabilities of acetamide and formamide were consistent with their relative oil-water partition coefficients. Our data are similar to values from studies on the permeability properties of vesicles of water-tight epithelial apical membrane. The combination of the unique model of MDCK apical-out cysts and fully automated data analysis enabled determination of apical membrane permeability in intact epithelial cells with high precision.

Morphometric analysis of vascular smooth muscle cells by scanning electron microscopy.

The information presented in this chapter clearly shows the usefulness of SEM for analyzing the components of the wall of microvessels. The literature contains several methodologies that allow one to successfully expose single smooth muscle cells that line microvessels. It appears that chemical and enzymatic digestion steps will continue to be the principal elements of any methodology for exposing these cells. The combination of microdissection, prior to processing the specimen for SEM, with digestion has allowed better preservation of the tissues by reducing the amount of material obscuring the tissue of interest as well as reducing the length of time the tissue is exposed to chemicals. The next important improvement was the mobilization step of selected vessel segments so that they could be mounted "end on" for circumferential viewing and photographing. Finally, at least three different mathematical approaches have been developed and tested for the quantitation of single cell length and width determination.

Analysis of insulin-like growth factors (IGF)-I, and -II, type II IGF receptor and IGF-binding protein-2 mRNA and peptide levels in normal and nephrectomized rat kidney.

Immunocytochemistry, in situ hybridization, and radioimmunoassay were employed to examine the cellular distribution of mRNAs and proteins for IGF-I, II, IGF-II/M6P receptor, IGFBP2 as well as the levels of IGF-I and II in normal and unilaterally nephrectomized (Nx) adult rat kidneys. A similar distribution of immunoreactive IGF-I, and -II as well as IGF-II/M6P receptor was found in the principal cells of the cortical collecting duct and in all cells of the inner medullary collecting duct. In addition, immunostainable IGF-I and IGF-II/M6P receptor were noted in some inner medullary loops of Henle, while IGFBP2 was seen in the collecting ducts and loops of Henle of the inner medullar and the renal vasculature of all animals. By comparison, in situ hybridization revealed IGF-I mRNA only in the medullary thick ascending limbs while IGF-II mRNA was localized to the wall of the renal microvasculature in all kidneys. IGFBP2 mRNA was localized to the renal corpuscle and to inner medullary interstitial cells of all kidneys. These data suggest that renal IGF-I and IGFBP2 are synthesized at upstream sites along the nephron and then transported downstream for interaction with IGF receptors. Following nephrectomy, the renal levels of IGF-I peptide and mRNA were elevated at both 5 and 33 days post-nephrectomy, supporting a potential functional role for IGF-I in stimulating the structural and functional recovery in compensatory hypertrophy.

Prevention of experimental cyclosporin-induced interstitial fibrosis by losartan and enalapril.

The pathogenesis of renal scarring in chronic cyclosporin nephropathy is unknown. In this study, we evaluated the effects of renin-angiotensin system blockade by enalapril and losartan in a salt-dependent model of cyclosporin-associated chronic tubulointerstitial fibrosis (TIF). Rats kept on normal or low-salt diet were given cyclosporin, cyclosporin+enalapril, cyclosporin+losartan, cyclosporin+enalapril#losartan, or vehicle for 14 and 28 days. Cyclosporin reduced glomerular filtration rate (GFR) in rats fed either diet, but only salt-depleted animals developed significant TIF. Cyclosporin also impaired renal concentrating ability and caused tubular enzymuria. Renin-angiotensin system blockade decreased blood pressure (BP) and promoted afferent arteriolar vasodilatation. Losartan reduced plasma renin activity and prevented cyclosporin-induced increment of cortical alpha 1(I) procollagen mRNA. Renin-angiotensin blockade did not improve GFR and tubular function; however, it strikingly prevented TIF development, even in presence of very low BP. Rats treated with cyclosporin, hydralazine, and furosemide achieved BP values similar to losartan or enalapril groups, but there was no protection against interstitial fibrosis development. These results suggest that cyclosporin-related chronic interstitial injury is mediated by angiotensin II and that the mechanisms promoting the interstitial scarring can be dissociated from glomerular and tubular dysfunction in cyclosporin nephropathy.

Vascular endothelium and smooth muscle remodeling accompanies hypertrophy of intestinal arterioles in streptozotocin diabetic rats.

The purpose of this study was to document alterations in endothelial and smooth muscle cell morphology of first- and second-order intestinal arterioles after 6 months of streptozotocin-induced diabetes. Both light and scanning electron microscopic techniques were used to quantitate the changes in the microvasculature. After rendering the first- and second-order intestinal arterioles passive and processing the vessels, it was determined that these microvessels were significantly dilated in the diabetic animals. Further examination revealed that in the diabetic animals, the cross-sectional area of the endothelial layer was increased in both 1A and 2A vessels, and the smooth muscle layer cross-sectional area was significantly increased in 1A vessels. Individual smooth muscle cells were significantly increased in width in the diabetic animals, but not in length. These data suggest that in this model of diabetes in rats, intestinal arteriolar hypertrophy was accompanied by significant remodeling of the arteriolar wall.

On the probability that kidneys are different in autosomal dominant polycystic disease.

We hypothesized that highly variable cyst fluid sodium concentrations are a characteristic of every kidney in autosomal dominant polycystic kidney disease (ADPKD). We added our data on sodium concentrations in 124 fluids from ten ADPKD kidneys to data published by others of concentrations in 32 fluids from five kidneys. The values ranged from 3 to 207 mEq/liter; none fell between 59 and 74 mEq/liter. Fluids were designated as low (< 60 mEq/liter; 50 fluids) or high (> 60 mEq/liter; 106 fluids) sodium fluids. Transmission electron microscopy identified differences in the depths of apical tight junctions between cells from cyst walls of 12 of the low and 10 of the high sodium fluids from two kidneys (mean +/- SE depths of 2039 +/- 74 A vs. 386 +/- 18 A respectively; P < 0.0001). When fluids were grouped by kidney of origin, six of the 15 kidneys had only high sodium fluids. The probability that chance had led to the sampling of only high sodium fluids in these organs, given that 32% of all fluids were low sodium fluids, was calculated at < 0.00015. The possibility must be considered that all kidneys are not alike in ADPKD.

Ultrastructural features of the rabbit proximal tubules.

The unique ultrastructure of the epithelial cells that line the entire length of the proximal tubule have been described. In particular, scanning electron microscopy has been used to illustrate the varied nature of the apical, lateral and basal surfaces of cells located along P1, P2 and P3. The functional implication of the highly specialized microanatomy of the basal cell surface remains an interesting area of investigation.

Vascular smooth muscle structure and juvenile growth in rat intestinal venules.

The morphological structure of individual vascular smooth muscle cells from intestinal venules was evaluated with a combination of quantitative scanning (SEM) and transmission (TEM) electron microscopy techniques. In addition, growth of individual venular smooth muscle cells and of the overall vessel wall was compared from measurements of these variables during the rapid juvenile growth spurt from ages 4 to 6 and 10 to 12 weeks in Wistar-Kyoto rats. SEM revealed that smooth muscle cells of intestinal venules in weanling rats are very long (379 +/- 91 [SD] microns) and wide (6.0 +/- 1.3 microns) and very little further cell enlargement occurs during rapid juvenile growth. TEM studies indicated that passive inner vessel diameter and total muscle layer cross-sectional area of both the largest and intermediate diameter venules of young rats, as well as the percentage of the total wall area as muscle tissue in each venule type, did not significantly increase during body growth. These observations indicate that both the intestinal venules and their smooth muscle cells reach mature dimensions at a very early stage of life. Comparison of intestinal vascular smooth muscle cell dimensions indicates that venular smooth muscle cells are much larger in both cell length and volume than comparable arteriolar smooth muscle cells.