Normal Rabbit Kidney Research Articles

Temperature sensitive nanogels for real-time imaging during transcatheter arterial embolization

ABSTRACTSeveral vascular embolization materials are commonly used in clinical practice, however, having application defects of varying degrees, such as poor intraoperative imaging and easy recanalization of embolized blood vessels, they are challenging for application during Transcatheter arterial embolization (TAE). Thus, an intraoperative visible vascular embolization material with good embolization effect and biocompatibility can improve transcatheter arterial embolization clinical efficacy to some extent. Our study aimed to synthesize a novel vascular embolization material that can achieve complete embolization of arterial trunks and peripheral vessels, namely poly (N-isopropyl acrylamide)-co-acrylic acid nanogel (NIPAM-co-AA). Iohexol 200 mg/mL was co-assembled with 7 wt% NIPAM-co-AA nanogel to create an intelligent thermosensitive radiopaque nanogel (INCA), which achieves a good intraoperative imaging effect and is convenient for transcatheter arterial bolus injection due to its good fluidity and temperature-sensitive sol-gel phase transition. The normal rabbit kidney embolism model further confirmed that INCA could effectively use Digital subtraction angiography (DSA) to achieve intraoperative imaging, and real-time monitoring of the embolization process could avoid mis-embolization and leakage. Meanwhile, in a 42-day study, INCA demonstrated an excellent embolization effect on the right renal artery of New Zealand white rabbits, with no vascular recanalization and ischemic necrosis and calcification remaining. As a result, this radiopaque thermosensitive nanogel has the potential to be an intelligent thermosensitive medical vascular embolization material, providing dual benefits in TAE intraoperative imaging and long-term postoperative embolization while effectively addressing the shortcomings and challenges of commonly used clinical vascular embolization agents.

MMP7 damages the integrity of the renal tubule epithelium by activating MMP2/9 during ischemia-reperfusion injury.

Renal ischemia-reperfusion (IR) injury is a common issue in urological surgery, and the renal tubules, particularly the proximal tubules, are extremely vulnerable to IR injury. In this work, we detected the differently expressed genes (DEGs) between normal rabbit kidneys and IR kidneys by RNA-sequencing, then identified that matrix metalloproteinase-7 (MMP7) played an important role in the progress of IR injury. Indeed, A time-dependent promotion of renal injury was detected in rabbit model, as demonstrated by the increased levels of MMP2/7/9, and the decreased of tight junction protein-1 (TJP1). Furtherly, similar results were confirmed in human renal proximal tubule epithelial (HK-2) cells model. Notably, downregulation of MMP7 affected the activity of MMP2/9 by suppressing expression of cleaved-MMP2/9 not the pro-MMP2/9 protein, which directly alleviated the degradation of TJP1 in HK-2 model. On the contrary, MMP7 had not been affected by inhibiting MMP2/9. In addition, coimmunoprecipitation assay showed that knockdown MMP7 restrained the interaction between MMP2/9 and TJP1. Collectively, this study suggested that MMP7 could serve as early biomarkers for renal tubular injury, and revealed that MMP7 could destroy the integrity of tubular epithelium through degrading TJP1 by activating MMP2/9.

Experimental research Contrast-enhanced ultrasonography follow-up after radiofrequency ablation in normal rabbit kidney

IntroductionThe aim of this study was to evaluate the diagnostic performance of SonoVue-enhanced ultrasonography in the follow-up of rabbit kidney lesions induced by percutaneous radiofrequency ablation.Material and methodsNew Zealand rabbits (28) underwent percutaneous radiofrequency ablation (RFA) to generate renal lesions. Lesions were evaluated by conventional 2D ultrasound and contrast-enhanced ultrasonography (CEUS) at 1 day, 1 week, 1 month and 3 months after RFA, and the results were compared to gross pathology.ResultsOne day and 1 week after RFA, renal lesions were wedge-shaped in the gross dissection cross-sectional profiles. Conventional ultrasound could not indicate the extent of the lesions; however, CEUS could exactly delineate the lesion shape and size. At 1 and 3 months, lesions were observed as hyperechogenic areas on conventional ultrasound, and as small perfusion defects on CEUS. The differences in the lesion measurements obtained by CEUS and in pathological specimens were not statistically significant (p > 0.05).ConclusionsThe conclusion could be deduced from the study that SonoVue-enhanced ultrasonography was effective for the follow-up of normal rabbit kidney percutaneous radiofrequency ablation.

Pure Ethiodized Oil-based Transcatheter Ablative Therapy in Normal Rabbit Kidneys and Kidneys Inoculated with VX-2 Carcinoma

To evaluate the efficacy of ablation with selective arterial injection of pure ethiodized oil followed by arterial occlusion with 9:1 ethanol-Ethiodol mixture (EEM) and coil placement in normal rabbit kidneys and kidneys inoculated with VX-2 carcinoma. All experiments were conducted with Animal Care and Use Committee approval. In six rabbits (group 1), one kidney was embolized with pure Ethiodol until capillary stasis, followed by injection of 9:1 EEM until arterial stasis and then coil placement into the main renal artery. In 12 other rabbits, one kidney was inoculated with VX-2 tumor. Ethiodol and EEM embolization and coil placement followed 7days later (group 2, n=6) or 11-14 days later (group 3, n=6). Kidneys were evaluated (angiography, computed tomography, macro- and microscopy) 7days after treatment. Capillary stasis was achieved in groups 1, 2, and 3 with (mean±standard deviation) 0.47±0.03, 0.53±0.02, and 0.56±0.04 ml of pure Ethiodol, followed by 0.47±0.05, 0.42±0.03, and 0.38±0.04 ml of EEM, respectively, which caused complete arterial occlusion in 17 of 18 kidneys. In group 1, all but one kidney showed at least 95% generalized coagulative necrosis. In group 2, all six kidneys exhibited 100% coagulative necrosis, with no viable tumor present. In group 3, 100% coagulative necrosis was present in all kidneys, with a small viable tumor in one. In the rabbit, selective arterial injection of pure Ethiodol can cause complete renal parenchyma and tumor ablation when it is followed by prompt, contiguous, and permanent occlusion of the arterial compartment.

Pure Ethiodized Oil as a Capillary Embolic Agent with and without Ethanol-ethiodol Mixture in the Rabbit Kidney: Embolic Efficacy and Temporal Histopathologic Findings

To determine the extent of ablation and the temporal histopathologic findings associated with selective arterial injection of pure Ethiodol in the normal rabbit kidney, with or without arterial occlusion of the main renal artery. In 19 rabbits, 27 kidneys were embolized by injecting 0.6 mL of pure Ethiodol into the main renal artery to achieve capillary stasis. A 9:1 ethanol-ethiodized oil mixture was then injected into 17 of the 27 kidneys until complete arterial stasis was accomplished. Macro- and microscopic evaluation was performed 10 minutes to 6 weeks and 60 minutes to 1 week, respectively, for kidneys with and without arterial occlusion. Ethiodol followed by ethanol-Ethiodol mixture (mean +/- standard deviation, 0.37 mL +/- 0.03) caused complete and permanent arterial stasis in all 17 kidneys. Thrombosis of the large arteries occurred initially. Ischemic coagulative necrosis of renal tubules and damage to glomeruli were detected 2 hours after embolization. Within 24 hours, the glomeruli and most tubules of the cortex and medulla were necrotic. Without arterial occlusion, the arteriocapillary bed of the kidneys was completely patent, with normal contrast medium excretion. Ethiodol was observed in glomeruli and interstitial capillaries from 60 minutes to 1 week and caused mild acute glomerulitis from day 1. The lesions were confined to the glomeruli, and no significant parenchymal changes were observed. In the rabbit, selective arterial injection of pure Ethiodol produces complete renal ablation within 24 hours if prompt and permanent occlusion of the arterial compartment guarantees its permanent capillary retention.

The preparation of sulfur hexafluoride lipid-coated microbubbles

Objective To prepare phospholipid-based sulfur hexafluoride gas-filled microbubbles, which were designed for targeting microbubbles. Methods The microbubbles composed of DSPC and mPEG2000-PE were produced by film-sonication method,and were compared with SonoVue. In the physical chemistry characteristics studies, the morphology, particle diameter, concentration, pH value and osmotic pressure were investigated. In the echogenicity studies, contrast harmonic imaging technique was used to investigate the enhancement of vitro water sac and normal rabbit kidney parenchyma. Results The lipid microbubbles were well-distributed, round with air holes. The average diameter of self-made microbubbles and SonoVue were 2.25 μm and 2.50μm respectively. The average diameter of self-made microbubbles and SonoVue distributed from 0.4 μm to 10 μm and 0.2 μm to 10 μm,and 90% were under 6 μm and 8 μm respectively,the concentration were 5 x 108～10 x 108/ml and 1 x I08～5 x 108/ml respectively with a stability of 6 hours. In vitro water sac, the gray scale of self-made lipid microbubbles and SonoVue were 121.67±6.76 and 122.33 ± 4.53 respectively( P>0.05). In normal rabbit kidney parenchyma, the peak video intensity of normal rabbit kidney parenchyma of self-made lipid microbubbles and SonoVue were 72.00 ± 7.21 and 74. 65± 10.93 respectively(P>0.05). Conclusions The lipid microbubbles have satisfactory physical chemistry characteristics and echogenicity. Key words: Ultrasonography; Microbubbles

Property and contrast-enhancement effects of lipid ultrasound contrast agent: A preliminary experimental study

This work investigated the influence of some factors on the property in vitro of a self-made lipid ultrasound (US) contrast agent (LCA) and evaluated the relationship of acoustic pressure and enhancement effect in normal rabbit kidney parenchyma. In the in vitro studies, filling gas, solvent and concentration of LCA solution were investigated. Morphologic characteristics, concentration and mean diameter of microbubbles were considered as indices. In the in vivo studies, contrast-tuned imaging (CnTi) technique was used to investigate the enhancement effects in kidney parenchyma under nine acoustic pressure levels. Among the samples saturated with different filling gases, perfluoropropane (C 3F 8) resulted in the highest concentration of microbubbles and air, the lowest. Microbubbles filled with C 3F 8 or sulfur hexafluoride (SF 6) were quite stable and remained at a high level of concentration (above 2 × 10 9 microbubbles per mL) much longer than did air-filled microbubbles. Among the four solutions tested, 5% glucose solution and 0.9% saline solution showed higher initial concentrations and greater longevity than dextran 40 glucose solution (6%) or distilled water. The concentration of LCA solution had a positive correlation with the microbubble concentration. All microbubble samples under different test conditions remained shape-complete and no aggregation or fusion was observed. The mean diameter of microbubble samples was about 3.4 μm. Contrast intensity and longevity of CnTi enhancement in vivo showed an acoustic-pressure-dependent decrease. At 1 kPa, contrast intensity increased 224-fold (4.47/0.02) and the longevity of CnTi enhancement in the kidney parenchyma remained longer. (E-mail: lctuua@yahoo.com.cn)

Ethiodized oil-ethanol capillary embolization in rabbit kidneys: temporal histopathologic findings.

To determine the temporal histopathologic findings associated with selective arterial injection of a 1:1 ethiodized oil-ethanol mixture (EEM) in normal rabbit kidney followed by administration of pure ethanol into the main renal artery. In five rabbits, the EEM was injected sequentially into each segmental renal artery of the right kidney until capillary stasis occurred. Pure ethanol was then injected into the main renal artery to achieve complete arterial stasis. Before sacrifice, the left kidney in each animal was acutely (ie, with a short follow-up period) embolized by using the same technique. The 10 kidneys of the five rabbits were evaluated microscopically at 1 (n = 3), 1(1/2) (n = 1), and 3 hours (n = 1) and 1 (n = 1), 3 (n = 1), 5 (n = 1), 7 (n = 1), and 14 days (n = 1) after embolization. Injection of the EEM (mean volume, 0.46 mL +/- 0.14 [SD]) followed by ethanol alone (mean volume, 0.25 mL +/- 0.09) led to complete stasis in all kidneys. There was no recanalization in the chronically (ie, with a longer follow-up period) embolized kidneys. Microscopically, uniform distribution of the EEM was evident in all slices at all time points. From 1 to 3 hours, sloughing of endothelium, formation of thrombi, and deposition of eosinophilic material along the renal, interlobar, and arcuate arteries were observed, without evidence of parenchymal damage. Within 24 hours, complete coagulative necrosis of the entire kidney occurred as a result of an occluding thrombus in the main renal artery. Analysis at subsequent time points revealed liquefaction of necrotic tissue and replacement with granulation tissue. In the rabbit, selective renal arterial injection of EEM followed by administration of ethanol produces vascular endothelial damage initiating thrombosis that results in renal infarction and ablation within 24 hours.

Effects of Nonsteroidal Anti-Inflammatory Drugs on Prostacyclin and Thromboxane in the Kidney

Dose-response curves were obtained relating the effects of increasing amounts of aspirin, a nonselective cyclooxygenase (COX) inhibitor, and celecoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on the concentrations of prostacyclin and thromboxane in renal cortex and medulla of rabbits. The concentrations of the two agonists (aspirin and celecoxib) which elicit a half-maximal response on the prostanoid concentration (EC₅₀) were compared. Additionally, controls for prostacyclin and thromboxane were related to values for the experimental groups. The EC₅₀ values for celecoxib were considerably lower than those for aspirin, indicating that celecoxib was more effective in suppressing prostanoid production. There were also significant differences between the majority of experimental groups and their respective controls, further evidence for the greater inhibitory activity of celecoxib on prostacyclin. Celecoxib lowered the ratio prostacyclin/thromboxane in the renal medulla; mercuric chloride further diminished the concentration of prostacyclin in the renal medulla. The results confirm that in the normal rabbit kidney, both nonselective and specific COX inhibitors interfere with renal prostanoid synthesis, but that a selective COX-2 inhibitor is more effective.

Renal expression of tissue factor pathway inhibitor and evidence for a role in crescentic glomerulonephritis in rabbits.

Tissue factor pathway inhibitor (TFPI) was demonstrated in the kidneys of normal rabbits and in a crescentic model of glomerulonephritis (GN), where fibrin is a key mediator of injury. In normal kidneys, TFPI was expressed in glomeruli, in intrarenal arteries and the interstitial capillary network. Evidence for TFPI synthesis in vivo was provided by in situ demonstration of TFPI mRNA in glomeruli and intrarenal vessels and by biosynthetic labeling of TFPI released from glomeruli in vitro. In fibrin-dependent crescentic GN, glomerular TFPI synthesis and expression was initially decreased (TFPI antigen at 24 h, 7.5 +/- 0.7 ng/10(3) glomeruli; normal, 11.1 +/- 0.9 ng/10(3) glomeruli, P < 0.02) and subsequently returned to normal values. Plasma TFPI levels increased progressively throughout the evolution of disease. In vivo inhibition of TFPI using an anti-TFPI antibody during the development of GN significantly increased glomerular fibrin deposition (GFD) and exacerbated renal impairment. Infusion of recombinant human TFPI significantly reduced development of GFD (fibrin scores, TFPI treated 0.82 +/- 0.11, control 1.49 +/- 0.14, P < 0.01), proteinuria and renal impairment. This data indicates that TFPI is synthesized and expressed in normal glomeruli and is down regulated in the early response to glomerular injury. Endogenous glomerular TFPI and treatment with recombinant TFPI reduces GFD and injury in fibrin dependent GN. TFPI has the potential to be of therapeutic benefit in the management of fibrin dependent human GN.

Phospholipids regulate growth and function of MDCK cells in hormonally defined serum free medium.

The effects of the simple phospholipids phosphatidic acid (PA) and lysophosphatidic acid (LPA) on the growth and function of Madin Darby Canine Kidney (MDCK) cells has been studied. We observed that PA and LPA not only stimulated the growth of MDCK cells (at 20 microM), but also stimulated the growth of normal rabbit kidney cells in serum free medium (albeit at a lower dosage of 5 microM). Evidence was obtained that PA interacts synergistically with insulin so as to elicit a growth stimulatory effect. Recently, extracellular PA and LPA were proposed to stimulate mitogenesis in several types of animal cells by binding to particular sites on the plasma membrane which are coupled to signaling mechanisms such as adenylate cyclase via a pertussis toxin sensitive, inhibitory guanosine triphosphate binding protein (Gi protein) (15). However, even when the pertussis toxin dosage was increased to 50 ng/ml, LPA still had a dramatic growth stimulatory effect on MDCK cells. In the absence of LPA pertussis toxin was slightly growth stimulatory to MDCK cells. Phospholipids such as PA and LPA have been observed to prevent prostaglandin-induced increases in adenylate cyclase activity in other cell types via their effects on such a pertussis toxin sensitive Gi protein. If PA and LPA act on MDCK cells in this manner, then these phospholipids may possibly prevent the effect of PGE1 on the growth of normal MDCK cells. However PGE1 was still growth stimulatory to normal MDCK cells. The effects of PA on PGE1 independent variants of MDCK cells, which have elevated intracellular cyclic AMP levels (22), were also examined. In the presence of PA, PGE1 remained growth inhibitory, rather than growth stimulatory to the PGE1 independent cells. However, the PA dosage required to elicit an optimal growth response (5 microM) was dramatically reduced, as compared with normal MDCK cells (20 microM). This altered dosage requirement could be explained by the elevated intracellular cyclic AMP levels in the PGE1 independent variants. Like PGE1 and 8-bromocyclic AMP, PA and LPA also significantly increased the initial rate of Rb+ uptake by confluent monolayers of MDCK cells. The increase in the initial rate of Rb+ uptake could be explained by an increase in the ouabain-sensitive component of Rb+ uptake. An increase in the initial rate of ouabain-insensitive Rb+ uptake was also observed in LPA treated MDCK cell cultures.

H-K-ATPase immunoreactivity in cortical and outer medullary collecting duct

Biochemical and physiologic studies in individual segments of the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) have provided evidence for the presence of an H-K-ATPase which is involved in the reabsorption of potassium in exchange for protons. The present study was designed to determine the cellular distribution of H-K-ATPase immunoreactivity in the CCD and OMCD of the rat and rabbit using mouse monoclonal antibodies against hog gastric H-K-ATPase. Kidneys of normal rats and rabbits were preserved for light microscopic immunohistochemistry and embedded in paraffin. Sections were incubated with the primary antibody followed by the avidin-biotin-horseradish peroxidase procedure. Sections incubated without primary antibody or with a non-specific mouse Ig served as controls. Light microscopy revealed diffuse cytoplasmic staining indicating H-K-ATPase immunoreactivity in intercalated cells in the CCD and OMCD in both rat and rabbit. In all segments studied except the rat CCD, the percentage of H-K-ATPase immunoreactive cells corresponded to the percentage of intercalated cells. In the rat CCD only 23% of the cells were reactive with H-K-ATPase antibodies, which is less than the percentage of intercalated cells in this region. It is possible that only type A intercalated cells possess H-K-ATPase immunoreactivity or that some intercalated cells did not have sufficient activity to be detected by our method. These results demonstrate H-K-ATPase immunoreactivity in the intercalated cells of the CCD and OMCD of rat and rabbit, suggesting that these cells are involved in potassium reabsorption in exchange for proton secretion in the mammalian collecting duct.

Tubulostromal relations in the normal rabbit and human kidney

Tubulostromal relations in the normal rabbit and human kidney

Nonsaturable amino acid reabsorption in kidneys of normal and mercury-poisoned rabbits.

At high plasma concentrations, a high-capacity, low-affinity or nonsaturable flux (Jhc) accounts for a residual fractional reabsorption of cycloleucine, aspartate, and AIB of approximately 50% of the filtered load in rabbits; Jhc in micromoles per milliliter glomerular filtrate is reduced in Hg-poisoned animals. The nonsaturable flux of cycloleucine is characterized by a transepithelial transit time (TET) of approximately 2 min in control animals; it was consistently much longer in Hg-poisoned animals. The clearance ratio of creatinine/inulin averaged 1.0, and no Jhc could be demonstrated for glucose. We conclude that Jhc is a high-capacity, low-affinity amino acid flux which passes through an intracellular solute pool, and which is sensitive to Hg at both the brush border and the basolateral cell membrane. If calculation of the saturation constants of aspartate reabsorption is restricted to experiments in which U/P less than 1.0, i.e. where Jhc is unlikely to contribute greatly to reabsorption, values some 20% lower than those previously reported are obtained; the Hg inhibition still is apparently uncompetitive in nature.

Acute Tubular Necrosis

Sequential fast magnetic resonance (MR) images (repetition time = 33 ms, echo time = 7 ms, alpha = 22 degrees, one image every 12 s) were acquired using gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) (10 or 100 mumol/kg) to study perfusion and concentrating ability in normal rabbit kidneys and in kidneys with HgCl2-induced acute tubular necrosis (ATN). In normal rabbits receiving 100 mumol Gd-DTPA/kg a concentric region of decreased MR signal was observed. In sequential images the dark ring pattern migrated centripetally through the kidney moving from the corticomedullary junction to the inner medulla. The decrease in MR signal intensity occurred as a consequence of T2 relaxation (magnetic susceptibility) due to high concentration of Gd-DTPA within the tubules. This suggests that the dark ring pattern may serve as a qualitative feature indicative of the ability of the kidneys to concentrate. With the onset of HgCl2-induced ATN the pattern of enhancement due to Gd-DTPA administration changed markedly. Although the kidneys with ATN did continue to be perfused, the concentric dark ring pattern seen in normal kidneys receiving 100 mumol Gd-DTPA/kg was not observed. These results suggest that Gd-DTPA and fast imaging MR may provide a method of assessing perfusion and concentrating ability within the healthy or diseased kidney.

Failure of anti-inflammatory steroids to inhibit prostaglandin release from the hydronephrotic rabbit kidney.

The release of prostaglandins E2, F2 alpha, I2 and thromboxane A2 from isolated perfused normal and hydronephrotic rabbit kidneys was investigated by extraction and radioimmunoassay. In both types of kidneys, basal PG efflux increased with time and was not altered by co-perfusion with dexamethasone or hydrocortisone. Several vasoactive substances at 1 to 4 micrograms (e.g., bradykinin, angiotensin II, substance P, noradrenaline and vasopressin) caused release of additional amounts of prostaglandins. PGE2 and 6-keto PGF1 alpha were the major prostanoids detected, but substantial amounts of PGF2 alpha were also found. Thromboxane A2 was not released from normal kidneys. In hydronephrotic kidneys there was greatly augmented release of prostaglandins E2 and I2, some increases in PGF2 alpha, and the appearance of substantial amounts of thromboxane A2 (measured as immunoreactive TXB2) when the kidneys were challenged with angiotensin, bradykinin and vasopressin, and smaller augmentation of the response to noradrenaline and substance P. There was no evidence that these evoked increases in renal PG output could be inhibited by dexamethasone or hydrocortisone. Some explanations for the failure of steroids to alter prostanoid metabolism from arachidonate in rabbit kidney are discussed, and it is proposed that there are clear exceptions to the concept that steroids inhibit prostaglandin generation in intact tissues.

Cellular aspects of rabbit Masugi nephritis V. Ultrastructural cytochemistry of monocytic cells.

Ultrastructural cytochemistry for nonspecific esterase (NSE) was performed on normal and nephritic rabbit kidneys. In normal glomeruli distinct reaction deposits appearing as electron-dense granules were present in visceral epithelial cells (podocytes) and epithelial cells of Bowman's capsule, but only a few small or minute deposits were seen in some endothelial and mesangial cells. In acute proliferative glomerulonephritis (Masugi nephritis), many reaction deposits occurred in mononuclear cells accumulating in glomerular tufts which presented the characteristic features of monocytic cells. Macrophages which had migrated into subendothelial space as well as epithelioid cells and multinucleated giant cells, all of which are known to be derived from monocytes, also exhibited the reaction product. The NSE granules in mesangial and endothelial cells were much smaller and fewer in number than those in monocytic cells. The present method may contribute to the more precise differentiation of monocytic cells from mesangial and endothelial cells in proliferative glomerulonephritis.

Spontaneous glomerulonephritis in rabbits: Role of a glomerular capillary antigen

Overt glomerulonephritis, detected by abnormal proteinuria, occurred in 3.3% of young (2.5 kg) male New Zealand White (NZW) rabbits. Histologically, mild to moderate mixed membranous and proliferative glomerulonephritis was observed. Glomerular deposits of IgG and C3 and electron microscopic findings were not typical of circulating immune complex accumulation, nor did they suggest anti-glomerular basement membrane (GBM) antibody. Segmental and less intense glomerular deposits of IgG were found in up to 48% of nonproteinuric NZW rabbits of the same age; histologic changes were minimal. IgG antibodies in sera from proteinuric rabbits or eluted from their kidneys reacted by indirect immunofluorescence with antigens distributed (discontinuously) along the glomerular capillary walls, and in some cases within the walls of small arteries of normal rabbit kidney sections. By indirect immunoperoxidase electron microscopy, the reactive antigens were present at the surfaces of the epithelial cell foot processes where they abut the GBM. This spontaneous glomerulonephritis appears to involve fixation of antibodies to antigens distributed in a discontinuous pattern in the glomerular capillary wall. The mechanism would be much like that causing anti-GBM antibody glomerulonephritis, except that the glomerular antigen is different. Possibly some forms of glomerulonephritis develop in humans as a result of a similar process.

Heterogeneity of uridine incorporation along the rabbit nephron. II. Effect of DOCA.

Uridine uptake was investigated in microdissected segments from the entire length of tubules from normal rabbit kidneys and the effect DOCA treatment was studied. Uridine uptake was measured after in vitro incubation of kidney pyramids with [14C]uridine (0.25 mM). Determinations were done on intact segments, and also using TCA precipitation on both the acid-soluble fraction considered as representative of the pyrimidine nucleoside precursor pool and the acid-insoluble one containing the precipitated nucleic acids. In normal rabbits the fraction of uridine incorporated in the insoluble fraction represented about 5% of that in the soluble one for all segments. Maximal values were found in the distal convoluted tubule (DCT) and the connecting tubule (CNT), where uridine uptake was two- to fourfold higher than in other segments. After DOCA treatment, a significant increase in uridine uptake in the soluble fraction was present in the cortical collecting (CCT) (+90%, P less than 0.001) and medullary collecting tubules (MCT) (+63%, P less than 0.05) when values were corrected for the cellular swelling induced by DOCA in all segments. An even more marked rise in uridine incorporation was observed in the insoluble fraction of the DCT (+88%, P less than 0.05), CCT (+291%, P less than 0.001), and MCT (+422%, P less than 0.05). These results indicate that 1) the level of uridine uptake in soluble and insoluble fractions widely differs along the tubular length of the normal rabbit nephron; and 2) DOCA treatment induces an increase of uridine incorporation in both the pyrimidine precursor pool and nucleic acids of the collecting tubule, known to be the target site for corticosteroid hormones.

Culture of kidney tubular cells in vitro

The purpose of this study was to develop a system of culturing renal tubular epithelial cells to investigate the function of the kidney. Nephron segments were isolated from normal rabbit kidneys using a microdissection or a discontinuous Percoll gradient technique and cultured with five different culture media. The tubular cells were characterized by the enzyme activities and the receptor activities against hormones. The modified K-l medium supplemented with 5% fetal calf serum was most appropriate for the growth of the tubular cells. The outgrowth of epithelioid cells from proximal straight tubules, thick ascending limbs of Henle’s loop and collecting ducts was observed. The cultured cells from the denser fraction in Percoll gradients exhibited jr-glutamyltransferase activity, but neither alkaline phosphatase nor hexokinase activity. The cyclic AMP level in these cells increased about four-fold within 2 min with 1-34 synthetic parathyroid hormone (2 U/ml). In the presence of arginine vasopressin (5 mU/ml) and isoproterenol (10 μM), the cyclic AMP level was elevated slightly, but not influenced by calcitonin (50 mU/ml). The results suggest that the renal tubular cell culture retains the differentiated properties of the proximal tubular epithelial cells of origin.