Characteristics of urate influx in the rat nephron.

Permeability of the tubular epithelium to urate presented on the peritubular side was studied in anesthetized rats during mannitol diuresis by capillary microinjections of [14C]urate and [3H]inulin, [14C]PAH and [3H]inulin, or [14C]urate and [3H]PAH. Recovery of isotopes was determined in urine collected serially from the injected and contralateral kidney. Appearance and peak excretion of urate in the experimental but not in the contralateral kidney preceded inulin and coincided with PAH, indicating proximal permeability to urate. Recovery of urate was higher from the injected than from the contralateral kidney. Urate precession and recovery did not change after addition of PAH (1.5-6.4 mm) to [14C]urate-[3H]inulin solutions, whereas they decreased significantly in the experimental kidney after pyrazinoate (1.6-3.2 mM) addition. On the other hand, no effect of urate on [14C]PAH-[3H]inulin injections was detectable. These findings are suggestive of a carrier-mediated transtubular influx of urate in rat proximal tubule. Absence of competition with PAH may suggest differences in the secretory mechanisms for organic acids.     

The early phase of experimental acute renal failure

Summary Experiments were designed to determine whether leakage of substances across the tubular epithelium, which are impermeant in the normal kidney, falsifies the measurement of glomerular filtration rate in acute renal failure. Permeability to those substances most commonly used for filtration rate determination, polyfructosan, inulin and ferrocyanide, was estimated by measuring their recoveries following perfusion through various nephron segments in haeme pigment, ischaemic and nephrotoxic models of actue renal failure. Late proximal recovery of¹⁴C ferrocyanide was only marginally decreased compared to controls, by a maximum of 6%. Distal recovery of polyfructosan,¹⁴C and³H inulin were depressed somewhat more, by a maximum of 11%. Urinary recovery of¹⁴C inulin was reduced by only 15% in kidneys showing severely restricted renal function. It is concluded that tubular leakage is not a feature of significance in the early phase of moderate acute renal failure, that ferrocyanide and inulin are reliable markers for the determination of nephron filtration rate and water reabsorption, and that the reduction in whole kidney inulin or polyfructosan clearance reflects primarily a reduction in glomerular filtration rate.     

Comparison between the renal handling of14C-Na ferrocyanide and3H-methoxy inulin in the rat

Summary ¹⁴C-sodium ferrocyanide and³H inulin were simultaneously microinjected into surface convolutions of proximal tubules or cortical peritubular capillaries of the left kidney. Ureteral urine was fractionally collected from both kidneys after each injection in order to demonstrate the subsequent excretory pattern of each substance. The results of 25 intratubular injections revealed no significant difference between the excretory pattern of either indicator. The mean total recovery of³H inulin was not significantly different from that of¹⁴C-Na ferrocyanide (P for mean difference 0.5 pairedt test). However a comparison between the time of 50% excretion of each substance revealed a slight but significant delay in the excretion of ferrocyanide [0.086±0.03 min (SE)].Each of the five cortical peritubular capillary microinjections gave rise to a similar excretory pattern for both indicators from each kidney; in every case the kinetic excretory pattern of¹⁴C ferrocyanide closely followed that of³H inulin. The mean ratio amount of ferrocyanide excreted/amount of inulin excreted, obtained for the experimental (left) kidney, did not differ significantly from that obtained for the control (right) kidney. (P for mean difference 0.2, pairedt test.)It was concluded that after simultaneous microinjection into proximal tubules or cortical peritubular capillaries no significant difference exists between the renal handling of¹⁴C-Na ferrocyanide and³H-inulin, also no loss of ferrocyanide occured either into or through the cells bordering the tubular lumen.     

Influence of uranyl nitrate upon tubular reabsorption and glomerular filtration in blood perfused isolated dog kidneys

The early changes in tubular reabsorption, glomerular filtration, blood flow and sodium excretion brought about by uranyl nitrate were investigated in isolated, bloodperfused dog kidneys during water diuresis. No significant changes in urine volume were observed; the decrease in fluid reabsorption was counterbalanced quantitatively by a reduction in glomerular filtration rate; only a small diminution of renal blood flow was found. The balance between reabsorption and filtration was observed as well when angiotensin action or prostaglandin synthesis were inhibited. The intrarenal venous pressure rose, suggesting that an increase in proximal intratubular hydrostatic pressure caused the decrease in filtration. Tubular back-leak of fluid, or backdiffusion, induced by the toxin, were excluded. The presence of natriuretic compounds in the urine was confirmed.     

Effect of hemoglobin loads on the function and morphology of ischemic kidneys in the rat

Male Wistar rats were used to investigate the effects of stromafree hemoglobin (200 mg Hb/100 g body weights, i.v. as a 16.4% solution) on kidney function and morphology. Ischemia of the kidney was induced by bilateral clamping of the renal pedicle. The most severe disturbances of kidney function occurred in kidneys damaged by ischemia during the peak of Hb excretion; endogenous creatinine clearance decreased to 5% of control values, serum creatinine concentration rose 6 times as high as control values, and Hb excretion in the urine was reduced. Kidney damage after Hb loading and ischemia was more severe than damage caused by ischemia alone. These results demonstrate that the vulnerability of kidneys to damage by ischemia is increased by Hb loading.     

Studies of the antigens involved in an immunologic renal tubular lesion in rabbits.

Rabbits injected with nonglomerular components of rabbit kidney incorporated in Freund's complete adjuvant develop a lesion characterized a) extensive interstitial fibrosis, tubular degenerative changes, and sparse focal lymphocytic infiltrates; b) the deposition of IgG and C3 in a granular pattern along the basement membranes of proximal convoluted tubules; and c) functional tubular defects if the lesions are severe. The antibodies were eluted from kidneys with such lesions and labeled with fluorescein isothiocyanate. It was shown that these fluorescein-labeled eluates reacted with the corresponding antigens in the tubular deposits and also with the antigens present in the brush border and/or cytoplasm of the proximal tubules. The antigens are found in proximal tubules of the kidney but not in brain, lung, heart, liver, spleen, bowel, muscles, or urine. They appear to be soluble but may also be present in the plasma membrane.     

Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis.

The present work was designed to elucidate the in vivo role of complement in the proteinuria-associated tubulointerstitial injury. Rats were intravenously injected with puromycin aminonucleoside, and massive proteinuria was observed within 5 days. Prominent tubulointerstitial injury characterized by proximal tubular degeneration, tubular dilatation, and leukocyte infiltration were observed 7 days after injection. C3 and C5b-9 were observed in the luminal side of proximal tubular cells. Renal function, assessed by inulin and para-aminohippurate clearance, was significantly decreased. To-assess the role of complement in this model, rats were injected with either cobra venom factor or soluble recombinant human complement receptor type 1 starting at day 3. These manipulations significantly improved tubulointerstitial pathology and para-aminohippurate clearance without affecting the degree of proteinuria. Deposition of C3 and C5b-9 was not detected in the kidney of rats depleted of complement by cobra venom factor. In rats treated with soluble complement receptor, C3 was still detected in the tubules, but deposition of C5b-9 was not observed. Soluble complement receptor was detected at the site of C3 deposition and in the urine. These data strongly suggest that complement plays a pivotal role in proteinuria-associated tubulointerstitial injury and that systemic complement depletion or inhibition of complement in the tubular lumen may diminish the tubulointerstitial damage.     

Age-related cell proliferation and apoptosis in the kidney of male Fischer 344 rats with observations on a spontaneous tubular cell adenoma

Cell proliferation rate and apoptosis were examined in archival kidneys from young, middle-aged, and old male F344 rats. Immunohistochemical expression of proliferating cell nuclear antigen (PCNA) and apoptosis were quantified in the same cell populations of the proximal tubule epithelium. A total of 79 kidneys from 40 rats were examined. There was a progressive increase in cell proliferation rates in rats from 4 and 6-10 months of age. In 23-month-old rats, proliferative activity appeared to be reduced. No age-related variations in apoptotic indices were found. One of the 16 rats aged 23 months had a tubular cell adenoma. In the tumor-affected kidney, cell proliferation rate was dramatically higher than in the contralateral kidney as well as in all the other kidneys examined. This high proliferative activity was not balanced by variation in cell death.     

Sodium excretion in goldblatt hypertension

Summary Separate kidney function with special attention to sodium excretion was studied during 18 days in hypertensive rats with unilaterally constricted renal artery. For urine collection the bladder was divided into two separate chambers and drained by a new simple technique. Urinary flow rate of the untouched kidney was always elevated as compared to the clamped kidney. However the clearance of PAH and inulin of the untouched kidney was higher than that of the clamped kidney only during the first postoperative days and during saline intake. The osmolality and the inulin and potassium concentration were usually higher in the urine from the clamped kidney, whereas sodium concentration was higher in the urine from the contralateral kidney. Sodium excretion of the untouched kidney was not only greater than that of the clamped kidney but even greater than that of a normotensive control kidney. On the other hand sodium excretion of the clamped kidney was less than that of a normal kidney. Thus, total renal sodium output of the hypertensive and the normotensive rats did not differ. The reduced sodium excretion of the clamped kidney is interpreted as a consequence of the exaggerated sodium excretion of the untouched kidney, which is exposed to the high blood pressure.Preliminary results of this study have been presented at the Seventh Symposion of the Nephrological Society, Tübingen 1970.Supported by the Deutsche Forschungsgemeinschaft im Rahmen des SFB 89 Kardiologie Göttingen.     

Experimental avian nephropathy. Changes of renal function and structure induced by ochratoxin A-contaminated feed.

One-day-old chickens were fed ochratoxin A-contaminated diets at 2 levels: 0.3 and 1 mg ochratoxin A per kg feed, for 341 days. The only observable lesion to develop was a kidney damage comparable with the naturally occurring avian nephropathy. The changes in renal function were characterized by impairment of glomerular and tubular function, indicated by a decreased inulin clearance, TmPAH and decreased urine concentrating capacity. The changes of renal structure included degeneration of the tubular epithelium accompanied by regeneration. At slaughter, the kidneys, liver and muscular tissue of the birds contained residues of ochratoxin A (up to 50 mug per kg). As all the birds would have passed the meat inspection because no macroscopical lesions were present, this represents a possible health problem.     

骨髓单个核细胞移植对肾缺血再灌注后肾小管坏死及细胞凋亡的影响

目的：观察和分析外源性骨髓单个核细胞（BMMNCs）对肾缺血再灌注诱发的肾小管坏死及凋亡的影响。方法：密度梯度离心法分离获取BMMNCs并行DAPI标记。制作SD大鼠肾缺血再灌注损伤模型，通过下腔静脉进行BMMNCs移植。分别于缺血再灌注后不同时相获取肾脏标本，荧光显微镜下观察DAPI的标记情况，HE染色做肾组织学检测，TUNEL法检测肾组织凋亡细胞，免疫组织化学染色观察增殖细胞核抗原（PCNA）的表达。结果：BMMNCs移植组大鼠肾脏组织中可见DAPI荧光细胞，部分存在于肾小管上皮组织中，未见明显细胞坏死及变性征象，其凋亡细胞计数显著减少，而PCNA阳性细胞数增多。结论：外源性BMMNCs移植可减少缺血再灌注诱发的肾小管上皮细胞的变性、坏死和凋亡，促进缺血再灌注损伤后肾小管上皮细胞的增殖，从而提示BMMNCs移植有助于缺血再灌注损伤后肾小管的修复及其结构完整性的维持。     

Accelerated cellular recovery after an ischemic renal injury.

To determine the mode of action of the beneficial effect of adenosine triphosphate (ATP)-MgCl2, recovery of microinjected inulin, proximal tubular pressure (PTP), and cellular damage as quantitated by histomorphometric analysis of necrosis and swelling were evaluated at 2, 6, and 24 hours after 45 minutes of renal ischemia in rats treated with either normal saline or ATP-MgCl2. At 2 hours both groups of rats demonstrated increased permeability to inulin, elevated PTP, and severe ischemic damage and necrosis. By 6 hours ATP-MgCl2 rats had less tubular back leak of inulin, PTP was modestly reduced, and ultrastructural studies demonstrated improved cellular morphologic features with evidence of early regenerative changes. The saline rats had progressive ischemic cellular damage. At 24 hours ATP-MgCl2 rats had reestablished tubular integrity, PtP had fallen, and ischemic alterations were improved, with only focal evidence of necrosis. Saline-treated rats still had a back leak of inulin, elevated PTP, and progressive ischemic injury. This study demonstrates that 1) cellular damage continues to occur for 6 hours after renal ischemia; 2) ATP-MgCl2 enhances recovery of tubular integrity and cellular morphologic features. The salutary effect of ATP-MgCl2 appears related to the preservation of sublethally injured cells and acceleration of the process of restoration and repair of damaged cells.     

The Number and Dimensions of Small Airways in Emphysematous Lungs

Effects of a single dose of lead (0.04 mg lead g body weight) on the proliferation of proximal tubular epithelium in rat kidneys were investigated by autoradiography over a period of 72 hours, using ³H-thymidine as a label. The results demonstrate that cell proliferation was greatly stimulated within 2 days after lead was injected. The increase in DNA synthesis began about 20 hours after intraperitoneal injection of lead, reached a sharp peak at 30 hours, and declined rapidly thereafter. At the peak, the mean labeling activity was 40 times that observed in control rats. Cumulatively, an average of 14.5% of the proximal tubular epithelial cells were labeled 72 hours after lead was injected. When uninephrectomy was followed immediately by injection of lead, the stimulation of DNA synthesis in the remaining kidney was, on the average, greater than the sum of the separate effects of the two treatments. This indicates that the stimulatory effects of uninephrectomy and injection of lead on renal cell proliferation were additive.     

High-resolution optical coherence tomography imaging of the living kidney

Optical coherence tomography (OCT) is a rapidly emerging imaging modality that can provide non-invasive, cross-sectional, high-resolution images of tissue morphology in situ and in real-time. In the present series of studies, we used a high-speed OCT imaging system equipped with a frequency-swept laser light source (1.3 mum wavelength) to study living kidneys in situ. Adult, male Munich-Wistar rats were anesthetized, a laparotomy was performed and the living kidneys were exposed for in situ observation. We observed the kidneys prior to, during and following exposure to renal ischemia induced by clamping the renal artery. The effects of intravenous mannitol infusion (1.0 ml of 25%) prior to and during renal ischemia were also studied. Finally, living kidneys were flushed with a renal preservation solution, excised and observed while being stored at 0-4 degrees C. Three-dimensional OCT data sets enabled visualization of the morphology of the uriniferous tubules and the renal corpuscles. When renal ischemia was induced, OCT revealed dramatic shrinkage of tubular lumens due to swelling of the lining epithelium. Three-dimensional visualization and volumetric rendering software provided an accurate evaluation of volumetric changes in tubular lumens in response to renal ischemia. Observations of kidneys flushed with a renal preservation solution and stored at 0-4 degrees C also revealed progressive and significant loss of tubular integrity over time. Intravenous infusion of mannitol solution resulted in thinning of the tubular walls and an increase in the tubular lumen diameters. Mannitol infusion also prevented the cell swelling that otherwise resulted in shrinkage of proximal tubule lumens during ischemia. We conclude that OCT represents an exciting new approach to visualize, in real-time, pathological changes in the living kidney in a non-invasive fashion. Possible clinical applications are discussed.     

The adaptation of renal urea excretion after unilateral nephrectomy and after overloading with urea

Summary Urea and inulin clearances were measured in unanesthetized rats 5 and 50 h, and 2–3 weeks after unilateral nephrectomy. At identical i-v infusion rates, urine flow in the uninephrectomized animals was similar to that of sham-operated controls. In the low range of urine flow rates, fractional urea excretion was higher in recently uninephrectomized animals than in controls. The increase appeared to be the consequence of the increased fractional excretion of water. At higher rates of urine flow, and after large loads of urea, the single kidney excreted urea in a manner similar to that of control kidneys. Urea clearances were, in 148/150 clearance determinations, lower than the simultaneously measured inulin clearances. A large increase of GFR was observed in rats chronically loaded and acutely infused with urea.Supported by Fonds National Suisse de la Recherche Scientifique, Grant No. 3440.70.     

Early segmental changes in ischemic acute tubular necrosis of the rat kidney

The background and mechanisms of ischemic acute tubular necrosis are still essentially unclarified. Therefore a quantitative morphological technique was applied for evaluation of the early structural changes in different fractions of the proximal convoluted tubule in the rat renal cortex. In male pentothal-anesthetized Wistar rats (body weight 200-250 g) ischemia of the right kidney was obtained by clamping (clamp diameter 0.15 mm) the ipsilateral renal artery for varying periods of time (10 min to 6 h) followed by removal and instant freezing of the kidney in isopentane at -165 degrees C and subsequent freeze-substitution in alcohol. The microscopic slides from the kidneys were silver methenamine-PAS stained. In the segments of the proximal convoluted tubules of the nephrons, presence of nuclear pyknosis, places of denuded basement membranes and presence of exfoliated tubular cells were counted. The results were statistically treated for comparison between the extent of damage in the initial postglomerular fraction and the later tubular loops. All three parameters showed a systematic, statistically significant increased number of lesions in the initial fraction of the proximal convoluted tubule versus the subsequent loops. The distribution of the structural lesions is in accordance with the previously reported presence of a tubulo-capillary counter-current flow in the proximal convoluted tubule and, when related to the highly variable oxygen tension in the normal renal cortex of the rat, indicates that the peculiar location of the early lesions might well be determined by these functional conditions.     

Fate of labeled angiotensin II microinfused into individual nephrons in the rat.

14C-labeled angiotensin II ([14C]AII) and tritiated inulin ([3H]In) were infused into individual nephrons in Inactin-anesthetized rats and urinary excretion was measured. Site of infusion was identified by neoprene injection and microdissection. In other experiments with higher doses of [14C]AII, microperfused at 10-4-10-5 M (concentrations 10-5-10-6 higher than contained in plasma), [14C]AII and its urinary metabolites were identified and quantified by two-dimensional peptide mapping. Recovery of 14C was 10.9% when proximal tubules were infused and 94.8% when distal tubules were infused. There was no correlation with tubular length in either case. For proximal tubules, two-thirds of the 11% recovered from urine appeared as peptide fragments of AII. With distal tubules almost all 14C activity appeared as intact AII. The principal metabolic product recovered from urine after proximal injection was the chymotryptic peptide, and its recovery was inversely related to tubular length. It is suggested that rapid removal of [14C]AII by proximal tubular cells occurs by enzymatic cleavage at the luminal surface with reabsorption of most of the products and excretion of the remainder     

Normal tubular regeneration and differentiation of the post-ischemic kidney in mice lacking vimentin.

Proliferation and dedifferentiation of tubular cells are the hallmark of early regeneration after renal ischemic injury. Vimentin, a class III intermediate filament expressed only in mesenchymal cells of mature mammals, was shown to be transiently expressed in post-ischemic renal tubular epithelial cells. Vimentin re-expression was interpreted as a marker of cellular dedifferentiation, but its role in tubular regeneration after renal ischemia has also been hypothesized. This role was evaluated in mice bearing a null mutation of the vimentin gene. Expression of vimentin, proliferating cell nuclear antigen (a marker of cellular proliferation), and villin (a marker of differentiated brush-border membranes) was studied in wild-type (Vim+/+), heterozygous (Vim+/-), and homozygous (Vim-/-) mice subjected to transient ischemia of the left kidney. As expected, vimentin was detected by immunohistochemistry at the basal pole of proximal tubular cells from post-ischemic kidney in Vim+/+ and Vim+/- mice from day 2 to day 28. The expression of the reporter gene beta-galactosidase in Vim+/- and Vim-/- mice confirmed the tubular origin of vimentin. No compensatory expression of keratin could be demonstrated in Vim-/- mice. The intensity of proliferating cell nuclear antigen labeling and the pattern of villin expression were comparable in Vim-/-, Vim+/- and Vim+/+ mice at any time of the study. After 60 days, the structure of post-ischemic kidneys in Vim-/- mice was indistinguishable from that of normal non-operated kidneys in Vim+/+ mice. In conclusion, 1) the pattern of post-ischemic proximal tubular cell proliferation, differentiation, and tubular organization was not impaired in mice lacking vimentin and 2) these results suggest that the transient tubular expression of vimentin is not instrumental in tubular regeneration after renal ischemic injury.     

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× 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 repidly 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 broders, and closure of the peritubular capillary spaces. Upon relases 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.