A study in vivo of peritubular oncotic pressure and proximal tubular reabsorption in the rat

1. Peritubular capillary microperfusion was used to examine the effects of protein-free and hyperoncotic homologous plasma on fluid reabsorption by proximal convoluted tubules in the hydropenic rat. 3H-labelled p-aminohippurate was added to perfusates for the purpose of estimating the extent to which tubules under study were bathed by the perfusates. [14C]Mannitol was added to perfusates in order to detect contamination of collected tubular fluid by perfusates. 2. Hydrostatic pressures were monitored in the peritubular microvasculature and adjacent proximal tubules during perfusion. Evidence for secretion of p-aminohippurate from perfusate into tubules under study was determined by collecting tubular fluid from both early and late puncture site. Fractional and absolute reabsorption were not affected by either the protein-free or the hyperoncotic plasma. 3. When acetazolamide was added to the perfusate both fractional and absolute reabsorptive rates decreased by an average of 36%, indicating that the techniques were capable of detecting a decrease in proximal tubular reabsorption. 4. It is concluded that under the conditions of this study changes in peritubular capillary protein concentrations have no detectable effect on the rate of proximal convoluted tubule fluid reabsorption.     

Mechanism of NaCl and water reabsorption in the proximal convoluted tubule of rat kidney

The role of chloride concentration gradients in proximal NaCl and water reabsorption was examined in superficial proximal tubules of the rat by using perfusion and collection techniques. Reabsorptive rates (Jv), chloride concentrations, and transtubular potential difference were measured during perfusion with solutions (A) simulating an ultrafiltrate of plasma; (B) similar to (A) except that 20 meq/liter bicarbonate was replaced with acetate; (C) resembling late proximal fluid (glucose, amino acid, acetate-free, low bicarbonate, and high chloride); and (D) in which glucose and amino acids were replaced with raffinose and bicarbonate was partially replaced by poorly reabsorbable anions (cyclamate,sulfate, and methyl sulfate). In tubules perfused with solutions A and B, Jv were 2.17 and 2.7 nl mm-1 min-1 and chloride concentrations were 131.5 +/- 3.1 and 135 +/- 395 meq/liter, respectively, indicating that reabsorption is qualitatively similar to free-flow conditions and that acetate adequately replaces bicarbonate. With solution C, Jv was 2.10 nl mm-1 min-1 and potential difference was +1.5 +/- 0.2 mV, indicating that the combined presence of glucose, alanine, acetate, and bicarbonate per se is not an absolute requirement. Fluid reabsorption was virtually abolished when tubules were perfused with D solutions; Jv was not significantly different from zero despite sodium and chloride concentrations similar to plasma; chloride concentration was 110.8 +/- 0.2 meq/liter and potential difference was -0.98 mV indicating that chloride was close to electrochemical equilibrium. These results suggest the importance of the chloride gradient to proximal tubule reabsorption in regions where actively reabsorbable solutes (glucose, alanine, acetate, and bicarbonate) are lacking and provide further evidence for a passive model of NaCl and water transport.     

Renal cell tumours in mice: electron microscopy versus immunohistochemistry

15 renal cell tumours induced in CBA male mice by 1,2-dimethylhydrazine were studied electronmicroscopically (EM). All these tumours earlier were investigated histochemically and immunohistochemically with the use of gamma-glutamyltranspeptidase (GGT) as a marker of the proximal tubules and antigen A6 as a marker of distal tubules and/or collecting ducts. One of the tumours was GGT-positive and antigen A6-negative and ultrastructurally well developed brush border was found. This correlation between immunohistochemistry and EM data allowed to conclude that the site of origin of this tumour were the cells of the proximal tubules. All other tumours were GGT-negative and antigen A6-positive, i.e. the development of these tumours from the distal tubules (and not from the proximal tubules) could be suspected. However in 5 of these neoplasms reduced brush border was found. Microvilli were of a smaller size than in normal proximal tubules and were frequently located not on the apical cell surface but in the narrow spaces between two cells. Invaginations and apical vesicles typical for the normal proximal tubules were also found in some tumour cells. EM and immunohistochemical data combined allow to suggest the origin of these tumours from the common precursor cell capable of differentiation, in the course of tumour progression, into the cells with properties of proximal and/or distal tubules.     

Increased oxidative stress in renal proximal tubules of the spontaneously hypertensive rat: a mechanism for defective dopamine D1A receptor/G-protein coupling

AIM: Defective dopamine D1A dopamine receptor/G-protein coupling has been demonstrated in renal proximal tubules of the spontaneously hypertensive rat (SHR). In the present study, we aimed to analyze the underlying mechanisms through which such defects are introduced into the D1A receptor protein of SHR. MATERIALS AND METHODS: The oxidative state of SHR proximal tubules was analyzed by measuring lipid peroxidation. D1A receptor/G-protein coupling was measured following the induction of oxidative stress in normotensive Wistar-Kyoto (WKY) rats. RESULTS: For the first time, an increased state of oxidative stress was demonstrated in SHR proximal tubules compared with those of normotensive controls, WKY and Sprague-Dawley rats. Lipid peroxidation levels in SHR were significantly higher by 66 and 79%, relative to WKY or Sprague-Dawley rats, respectively. Hydrogen peroxide treatment of proximal tubules from SHR, WKY and Sprague-Dawley rats induced an additional increase in lipid peroxidation in a dose-dependent manner, although the percentage induction was lower in SHR than in WKY and Sprague-Dawley rats. This induction of lipid peroxidation in WKY rats resulted in a loss of D1A/G-protein coupling, with no decrease in receptor protein. Treatment of WKY rat proximal tubules with an antioxidant, ascorbic acid, or a reducing agent, dithiothreitol, induced D1A receptor/G-protein coupling. CONCLUSIONS: These data indicate that D1A receptor/G-protein coupling is modulated by changes in redox states. Therefore, the D1A receptor/G-protein coupling in SHR may have been damaged by reactive oxygen species released as a result of the elevated oxidative stress seen in the proximal tubules.     

Cytotoxic effect of Shiga toxin-1 on human proximal tubule cells

BACKGROUND: Cytolytic Shiga toxins (Stx) are believed to be largely responsible for renal damage in post-diarrheal hemolytic-uremic syndrome (D + HUS). Despite the general belief that endothelial cells are the primary target of Stx, there is evidence that proximal tubules may be a site of toxin action. We hypothesized that cultured proximal tubular cells are sensitive to the cytotoxic effects of Stx. METHODS: Cultured human proximal tubular cells were exposed to Stx-1 in the presence and absence of a variety of inflammatory factors likely to be elevated in the kidney or serum of patients with D + HUS. Cell survival, protein synthesis, total cell levels and synthesis of Stx receptors (GB3), and Stx binding were measured. RESULTS: Proximal tubules were extremely sensitive to the cytotoxic effect of Stx-1 with an LD50 at least equal to, if not less than, that seen with Vero cells. Interleukin-1 (IL-1), lipopolysaccharide (LPS), and butyrate (but not tumor necrosis factor or interleukin-6) up-regulated proximal tubule sensitivity to Stx-1. IL-1 increased Stx-1 binding, but did not alter total cell levels or synthesis of GB3, the glycosphingolipid receptor for Stx-1. In contrast, LPS and butyrate, despite increasing Stx-1 sensitivity, had no effect on Stx-1 binding. CONCLUSIONS: These studies indicate that proximal tubules are exquisitely sensitive to Stx-1 cytotoxicity and that inflammatory factors can increase toxin responsiveness through a variety of mechanisms. It is suggested that proximal tubules may be an important early target of Stx-1 action in D + HUS.     

Segmental distribution of the endocytosis receptor gp330 in renal proximal tubules

The subcellular distribution and segmental variations in location of gp330, a scavenger receptor for filtered proteins in renal proximal tubules, was analyzed. Kidney tissue from rats (4 different strains), rabbits and humans were analyzed by light- and electron microscope immunocytochemistry, using cryosections or Lowicryl sections from cryosubstituted tissue. Gp330 was located mainly in apical coated pits, small and large endocytic vacuoles and in dense apical tubules in the proximal tubule cells. The labeling density was markedly higher in segments 1 and 2 as compared to segment 3 of the proximal tubule. In addition to the location in the early part of the endocytic pathway, gp330 was also present in lysosomes, especially in segments 1 and 2. The lysosomal labeling was not restricted to the membrane, but was also seen in the matrix. Localization of gp330 in lysosomes was confirmed on sections from purified lysosomal fractions from rat renal cortex. The brush border localization of gp330 in proximal tubules exhibited a characteristic segmental variation. In the initial part of segment 1, there was virtually no brush border labeling. In the remaining part of segment 1 and in segment 2, there was a distinct but sometimes patchy labeling of the brush border. In segment 3, groups of microvilli of approximately 10 as seen in sections were intensively labeled from bottom to tip and there were often more than one of these groups on a single cell, the remaining microvilli were unlabeled. No differences in the cellular and subcellular localization of gp330 were observed between species or rat strains. In conclusion, the present study demonstrates that in addition to its location in the early endocytic and recycling pathway, gp330 is also present in microvilli and the protein and degradation products thereof is present in lysosomes, consistent with its role as a protein scavenger receptor.     

Use of digitalis glycosides to identify the mechanisms of amantadine transport by renal tubules

The mechanism(s) for uptake of organic cations by renal cortical tubules was (were) examined further. Renal cortical tubules were purified from rat kidneys by a Percoll gradient centrifugation technique. Bicarbonate buffer (Krebs-Henseleit, KHS) conditions were altered, and chemical modulators were used which affect the activity of the basolateral Na+/K+-ATPase. Renal tubule uptake of the achiral organic cation amantadine was determined. The cardiac glycosides digoxin and acetylstrophanthidin and ouabagenin did not alter amantadine uptake by either proximal or distal tubule fragments in KHS. However, ouabain inhibited proximal tubule amantadine uptake in a dose-dependent manner with lower potency than distal tubule amantadine uptake in KHS. Ouabain did not inhibit amantadine tubule uptake in phosphate buffer. However, inhibition of amantadine uptake by ouabain returned in a time-dependent manner upon addition of bicarbonate to the phosphate buffer. Low extracellular sodium or potassium did not alter amantadine uptake by proximal tubules. Hypokalemic and hypokalemic/ hyponatremic conditions decreased the inhibitory potency of ouabain for amantadine uptake by proximal tubules. For distal tubules, both hyponatremic and hypokalemic conditions, alone and together, decreased the inhibitory potency of ouabain, but did not affect amantadine uptake in the absence of ouabain. Hypochloremic conditions decreased affinity for amantadine uptake by distal, but not proximal tubules. No change in maximal transport capacity for amantadine uptake was observed under hypochloremic conditions for either tubule fragment. These studies challenge the widely accepted concept of Na+/ K+-adenosine triphosphatase activity and maintenance of the basolateral membrane potential as rate-limiting steps for the energy-dependent renal tubule uptake of organic cations. Furthermore, these studies suggest a mechanism for ouabain inhibition of organic cation renal tubule uptake that may not involve the Na+/K+-adenosine triphosphatase and may be possibly bicarbonate-dependent.     

Estrogen effects on the renal handling of calcium in the ovariectomized perfused rat

Estrogen deficiency is a major cause of bone loss in women but the mechanism is unclear. The ovariectomized (OVX) rat is a well recognized model for post-menopausal osteoporosis. In this study we have examined the effects of OVX and estrogen replacement in the OVX rat on the renal handling of calcium in response to alterations in the calcium load in the perfused rat. The interaction of estrogen administration and parathyroid hormone (PTH) was also examined in the OVX, parathyroidectomized (PTX) rat. Calcium or EDTA was infused into sham or OVX rats to obtain a range of filtered calcium loads. The excretion of calcium, was compared to the filtered load for the data from both perfusions indicating a lower calcium (P = 0.006) and sodium (P = 0.009) excretion in the OVX rat. A similar result was seen in the OVX rat replaced with 20 micrograms of estrogen valerate 48 and 24 hours prior to perfusion with calcium excretion being greater with estrogen administration (P = 0.005) compared to vehicle alone. This was not observed in the parathyroidectomized rat. Correlations between sodium and water reabsorption and calcium and sodium reabsorption during perfusion indicate that the results of OVX were due primarily to proximal tubule effects. Prior to the perfusion experiment PTH (sham vs. OVX pmol/liter, mean +/- SD; 20 +/- 6 vs. 18 +/- 4) and calcitriol (128 +/- 85 vs. 97 +/- 74) were similar in both groups, indicating that the results were not dependent on calcitropic hormone effects. It is concluded that, in the perfused rat, OVX results in decreased excretion of calcium and sodium as a result of estrogen effects on the renal proximal tubule, an effect dependent on PTH. This effect is opposite to that found in postmenopausal women, perhaps due to the high filtered load of calcium used in the experimental design and species differences in the relative importance of proximal versus distal calcium handling.     

Induction of rabbit renal mixed-function oxidases by phenobarbital: cell specific ultrastructural changes in the proximal tubule

Administration of phenobarbital (60 mg/kg) daily for 4 days to male rabbits resulted in induction of renal cytochrome P-450 (3.5-fold) and a corresponding increase in ethoxycoumarin-O-deethylase and benzphetamine-N-demethylase activity (17- and 4-fold, respectively). Kidney weight to body weight ratio and renal ethoxyresorufin-O-deethylase were not affected by phenobarbital pretreatment. Numerous focal areas of proliferation of smooth endoplasmic reticulum (SER) were evident in proximal tubule cells from phenobarbital treated rabbits while proximal tubular cells from control rabbits had only small and sparcely located aggregates of SER. Phenobarbital-induced SER proliferation was specifically localized to the S3 segment of the proximal tubule. Proliferation was not observed in S2 cells of the proximal tubule, cells of Henle's loop, distal tubules, or collecting tubules in rabbits pretreated with phenobarbital. These data demonstrate the biochemical heterogeneity of cell types within the proximal tubules of rabbits. Furthermore, induction of mixed-function oxidases specifically in S3 cells of the proximal tubule may be of toxicological significance in the metabolic activation of certain nephrotoxicants.     

Anatomy and ultrastructure of the excretory system of the lizard, Sceloporus cyanogenys

The anatomy and ultrastructure of the lizard kidney (Sceloporus cyanogenys) have been studied by light and electron microscopy. The number of glomeruli was counted in serial sections and estimated to be 2,000 (in the two kidneys). Beginning with the glomerulus and Bowman's capsule the nephron segments are sequentially: (a) proximal tubule; (b) intermediate ciliated segment consisting of a proximal and distal part; (c) distal tubule, which can be divided into two segments, followed by (d) connecting tubule and (e) initial collecting duct. The initial collecting ducts from several nephrons open into the collecting duct. Tubular epithelium in this lizard has similarities to that of other reptiles. The lateral borders do not overlap like in mammals, but interdigitate by fingerlike projections. The length of the nephron segments was measured in disected tubules and the diameter was measured on light and electron micrographs. From these measurements estimates of inner tubular surface area were made. Together with data from physiological studies (Stolte et al., '76; Schmidt-Nielsen, '76) the estimated surface area was used to calculate transport rates per unit area across the epithelium. Comparisons of structure and transport rates per unit area across the epithelium. Comparisons of structure and transport rates were made between S. cyanogenys and other reptiles and mammals.     

Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were colocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Genetics of idiopathic scoliosis

To define the luminal agent(s) responsible for the reduction of nephron filtration rate following increases of loop of Henle flow rate early proximal flow rate (EPFR) during loop perfusion with 17 different salt solutions were compared to the non-perfused tubules. During orthograde microperfusions a reduction of EPFR as indication of a feedback response was noted with a number of monovalent Cl- and Br- salts (LiCl, KCl, NaCl, RbCl, CsCl, NH4Cl, choline Cl, NaBr, KBr), with Na+ salts except Na acetate (NaHCO3, NaNO3, NaF, NaI, NaSCN), and with CaCl2 and MgCl2. These latter 2 solutions where used in a concentration of 70 mM while all other solutions had a concentration of 140 mM. During retrograde perfusion from the distal to the proximal end of the loop of Henle EPFR fell significantly with Cl- and Br- salts with percentage changes of EPFR ranging from -8.0 to -44.3%. In contrast, Cl- free salts and Cl- salts of divalent cations were associated with percentage changes of EPFR ranging from +7.1 to -6.2%, significance being reached only during perfusion with NaSCN. When furosemide (5 x 10(-4) M) was added to NaBr or KBr a feedback response was not observed. During orthograde perfusion with NaNO3 distal Cl- concentrations were 44.2 +/- 5.08, mM (mean +/- S.E.) at a perfusion rate of 10 nl/min and 59.1 +/- 3.93 mM at a rate of 40 nl/min. CaCl2 perfusion induced a marked elevation of distal Cl- concentrations to levels higher than 140 mM. Loop chloride handling was normal during RbCl perfusion. The magnitude of the feedback response during retrograde perfusion was not changed by lowering NaCl concentration from 140 to 60 mM, but fell when NaCl concentration was further reduced. In contrast to orthograde perfusions it was insensitive to changes in flow rate. Our results are compatible with the thesis that feedback responses depend critically upon the rate of Cl- transport probably across the macula densa cells. Br- ions can replace Cl- because they appear to share a common transport pathway which can be inhibited with furosemide. Unspecificity of feedback responses during orthograde microperfusions is due to presence of Cl- ions in the macula densa region even when solutions are initially Cl- free. Cl- salts of divalent cations do not elicit a feedback response because Cl- transport is severely curtailed.     

The differentiation of proximal and distal tubules in the male rat kidney: the appearance of aldolase isozymes, aminopeptidase and alkaline phosphatase during ontogeny

1. The specific activities of aminopeptidase, alkaline phosphatase and aldolase isozymes were measured in homogenates of kidneys taken at different stages of ontogeny. The cellular localization of these enzymes was studied in cryostat tissue sections using substrate linked assays for aminopeptidase and alkaline phosphatase and the mixed aggregation immuno-cytochemical technique for aldolase isozymes; local enzyme concentrations were estimated photometrically. 2. The presence of both aldolase-A and aldolase-B was demonstrated in all metanephrogenic cells (and at still higher concentrations in collecting tubule cells) of the rat fetus 16 days after conception and in the undifferentiated cells of the neogenetic zone of kidney up to 8 days after birth; no aminopeptidase or alkaline phosphatase could be found in these cells. 3. Measurements made on stained tissue sections show that the shift towards aldolase-B, seen in homogenate analyses, is due to a change in the relative amounts of proximal tubules. No evidence was seen for repression in the synthesis of aldolase-A or aldolase-B monomers in the different kidney cells during ontogeny. 4. Two transitions in the mode of nephron differentiation were observed: one was shortly after birth, the other followed weaning. Before the first transition the concentrations of the enzymes increased to different degrees, such that the enzymes reached concentrations comparable with those as in the cells of adult rats by 2 to 4 days post partum. After the second transition proximal tubule size and specific activity of brush border membrane enzymes increased 3 fold. In contrast, the distal tubules did not increase significantly in size, but their aldolase-A concentration increased 3 fold. 5. Evidence based on enzyme quantification and morphometry in kidney sections is presented to demonstrate that the proximal tubule cells show functional adaptation by two independent mechanisms: specific amplification of gene expression and hypertrophy. In contrast, the distal tubule shows functional adaptation only by specific amplification of gene expression.     

Histogenetic stereological reconstruction of rat basophilic, clear, and oncocytic neoplastic renal cell lesions using carbonic anhydrase type II-PAS double-stained sections

The histogenesis of 3 types of rat renal cell tumors (basophilic cell, clear cell, and oncocytic) was stereologically analyzed, with particular attention paid to transitions from normal tubules. Early nitrosamine-induced preneoplastic lesions, including dysplastic tubules (altered tubules), epithelial hyperplasias, and small adenomas, were reconstructed using serially sectioned specimens processed for carbonic anhydrase type II (CA) and periodic acid-Schiff (PAS) (CA-PAS) double staining to allow easier distinction of the nephron segments: Proximal tubules had a PAS-positive brush border and were weakly positive for CA in the cytoplasm; distal tubules were PAS negative and weakly positive for CA; collecting ducts were PAS negative and strongly positive for CA. Similarly, cytochrome c oxidase (CytOx) and CytOx-PAS double staining was also applied to confirm the character of oncocytic lesions. All basophilic lesions (7 of 7) showed transition to proximal tubules. Clear cell lesions positive for CA, on the other hand, showed transition to distal tubules in 4 of 9 (44.4%) lesions and to collecting ducts in 4 of 9 (44.4%) lesions, but in only 1 of 9 (11%) to a proximal tubule. All oncocytic lesions (16 of 16), characterized by positivity for both CA and CytOx, showed transition to collecting ducts. The results indicate that the origins of renal cell neoplasia are proximal tubules for the basophilic cell lesions, either proximal or distal tubules for their clear cell counterparts, and collecting ducts for oncocytic lesions.     

Immunohistochemical localization of peroxisomal enzymes in developing rat kidney tissues

We studied the developmental changes in the localization of peroxisome-specific enzymes in rat kidney tissues from embryonic Day 16 to postnatal Week 10 by immunoblot analysis and immunohistochemistry, using antibodies for the peroxisomal enzymes catalase, d-amino acid oxidase, l-alpha-hydroxyacid oxidase (isozyme B), and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein. Peroxisomal enzymes were detected in the neonatal kidney by immunoblot analysis and their amount increased with kidney development. By light microscopic immunohistochemistry, they were first localized in a few proximal tubules in the juxtamedullary cortex of 18-day embryos. The distribution of proximal tubules positive for them expanded towards the superficial cortex with development. The full thickness of the cortex became positive for the staining by 14 days after birth. Peroxisomes could be detected by electron microscopy in structurally immature proximal tubules in 18-day embryos. Their size increased and the ultrastructure of subcompartments became clear with continuing development of proximal tubules. These results show that peroxisomal enzymes appear in the immature proximal tubules in the kidney of embryos and that the ultrastructure of the peroxisomes and localization of the peroxisomal enzymes develop along with the maturation of proximal tubules and kidney tissues.     

D-Glucose enhancement of water reabsorption in proximal tubule of the rat kidney

Water reabsorption in the proximal convoluted tubule of the rat kidney was examined by in vivo microperfusion techniques in order to examine the effect of D-glucose within the tubular lumen. When tubules were perfused with a balanced artificial solution containing Na, K, Cl, HCO3, urea, and D-glucose, absolute reabsorption averaged 4.01 +/- 0.24 nl/min per mm. Addition of D-glucose to the NaCl perfusate enhanced water reabsorption to values similar to those obtained with the balanced artificial perfusate. The enhanced water reabsorption consequent to the addition of D-glucose to the NaCl perfusion solution was completely inhibited by addition of phloridzin to the perfusate. The addition of an unabsorbed hexose, 2-deoxy-D-glucose, to the NaCl perfusate failed to enhance water reabsorption, whereas the addition of an incompletely reabsorbed sugar that is not metabolized, 3-O-methyl-D-glucose, resulted in partial enhancement of theabsolute rate of water reabsorption. These studies demonstrate that D-glucose has the specific effect of augmenting water reabsorption in the proximal tubule of the rat kidney.     

An evaluation of splenopancreatic disconnection as a modification of the distal splenorenal shunt, studied in nonalcoholic patients by sequential angiography

To evaluate the validity and complications of modifying the distal splenorenal shunt (DSRS) by performing splenopancreatic disconnection (SPD), hemodynamic changes in the portal system were assessed by visceral angiography in 93 patients with nonalcoholic portal hypertension during early postoperative follow-up after DSRS. There were 40 patients who underwent DSRS alone and 53 who underwent DSRS plus SPD. Early follow-up angiography showed that portal vein perfusion was well maintained, and that the diameter of the portal vein had decreased significantly by the same degree in both groups. Hepatofugal collaterals for the shunt had developed to a greater extent in the DSRS group, while they were almost completely absent in the DSRS with SPD group. Nevertheless, partial portal vein thrombosis was not detected in the DSRS group, although it was seen in seven (13.2%) of the patients who underwent DSRS plus SPD, in whom the left proximal splenic vein was not visible. The proximal splenic vein was seen in significantly less of the DSRS with SPD patients (47.2%) than the DSRS group patients (85%). In conclusion, SPD more effectively prevented the early postoperative development of collateral pathways for the shunt compared with standard DSRS; however, the possible stagnation of blood flow in the left proximal splenic vein may predispose to a risk of partial portal vein thrombosis developing during the early postoperative period after DSRS with SPD.     

Identification of three new single nucleotide polymorphisms in the human tumor necrosis factor-alpha gene promoter

To obtain insight into the basic mechanisms controlling endocytosis, we tested the effects of different perfusates containing the cytological stain light green on endocytosis and ultrastructure of the vacuolar apparatus in renal proximal tubule cells. Rat proximal tubules were microinfused in vivo for 2 min in the presence or absence of light green with the following solutions: (A) perfusates containing inorganic salts and (B) perfusates with organic components or protein. In other experiments, the tubules were first microinfused for 2 min with 0.9% NaCl in the presence or absence of light green, then 15 min later further microinfused with or without light green using either group A or B solutions in order to either aggravate or reverse possible changes. All infused tubules were fixed after 5 min with 1% glutaraldehyde and examined by electron microscopy. In tubules microinfused without light green, the endocytic vacuolar apparatus in the apical cytoplasm showed a normal ultrastructure. However, microinfusion of solutions containing light green in either inorganic salts or a low concentration of protein caused significant changes in the apical endocytic apparatus. Large endocytic vacuoles were absent, and invaginations and small endocytic vacuoles were decreased in frequency. On the other hand, the amount of dense apical tubules was significantly increased, and in some cells dense apical tubules had transformed into a cisternalike network. These changes were aggravated in tubules which received a second microinfusion of NaCl and reversed in tubules that received a second infusion of protein. Furthermore, in the tubules microinfused with light green using perfusates containing organic components or protein, the apical cytoplasm of proximal tubule cells showed an essentially normal endocytic apparatus. The present study demonstrates that microinfusion of renal proximal tubules with light green disrupted normal endocytic membrane trafficking and recycling. These changes could be prevented or reversed by microinfusion of solutions containing protein or organic components.     

A new model of diabetic nephropathy with progressive renal impairment in the transgenic (mRen-2)27 rat (TGR)

BACKGROUND: The tissue renin-angiotensin system (RAS) may modulate the structural and functional changes that occur in the diabetic kidney. METHODS: Hypertensive transgenic (mREN-2)27 rat (TGR) that exhibit increased tissue renin expression were administered streptozotocin (STZ, diabetic) or citrate buffer (non-diabetic) at six weeks of age, and sacrificed 4 and 12 weeks later. Further groups were treated for 12 weeks post-STZ or vehicle with the angiotensin converting enzyme inhibitor, perindopril. Comparisons were made with 18-week-old non-diabetic and diabetic spontaneously hypertensive rats (SHR). RESULTS: In diabetic TGR, the most florid lesion was seen after 12 weeks of STZ, with kidneys exhibiting vacuolated tubules, hylanized arterioles, medullary fibrosis and necrosis and severe glomerulosclerosis. In contrast, only mild glomerulosclerosis was seen in non-diabetic TGR and diabetic SHR. Glomerular filtration rate was increased after four weeks of diabetes in TGR and 12 weeks of diabetes in SHR, but declined by greater than 50% after 12 weeks of diabetes in TGR. In both TGR and SHR, diabetes increased albuminuria but did not modify systolic blood pressure. Renal renin content increased progressively in diabetic TGR, and this was associated with increased renin immunolabeling in the juxtaglomerular apparatus (JGA) and the appearance of renin in proximal convoluted tubules. In contrast, renal renin content and JGA renin immunolabeling were unchanged in diabetic SHR. Perindopril attenuated renal pathology, improved renal function and abolished proximal tubular renin immunolabeling in diabetic TGR. CONCLUSIONS: This is the first report of a diabetic rodent model developing rapid onset renal impairment. Furthermore, this study suggests a role for an activated renal RAS in the acceleration of diabetic renal disease and confirms the benefit of drugs that inhibit this system.