Increase In Protein Excretion Research Articles

Nitric oxide/cytochrome P450 interactions in cyclosporin A-induced effects in the rat

The present study evaluated the contribution of 20-hydroxyeicosatetraenoic acid (20-HETE) and its interaction with nitric oxide (NO) in cyclosporin A-induced nephrotoxicity and hypertension. The treatment of rats with cyclosporin A (25 mg/kg) for 7 days increased the renal microsomal conversion of arachidonic acid (AA) to 20-HETE (93 +/- 6%, P < 0.05), increased systolic blood pressure (SBP), reduced the urinary excretion of nitrite (53 +/- 8%, P < 0.05), induced renal damage as indicated by a marked increase in protein excretion (163 +/- 14%, P < 0.05), increased renal vasoconstrictor responses to AA (82 +/- 5%, P < 0.05) but not endothelin-1 or phenylephrine, and decreased vasodilator responses to bradykinin (42 +/- 10%, P < 0.05) and sodium nitroprusside (SNP; 56 +/- 13%, P < 0.05) in the renal preglomerular vessel treated with indomethacin and NO synthase inhibitor. The pretreatment of rats with HET0016 (10 mg/kg) or 1-aminobenzotriazole (50 mg/kg), inhibitors of cytochrome P450 (CYP450) activity, attenuated or prevented cyclosporin A-induced increases in 20-HETE production, SBP, and protein excretion, as did L-arginine (4 g/l), a substrate for NO synthase. L-Arginine but not HET0016 or 1-aminobenzotriazole blunted the cyclosporin A-induced decrease in nitrite excretion. Similarly, L-arginine blunted the enhanced vasoconstriction by AA as did HET0016 or 1-aminobenzotriazole. However, cyclosporin A-blunted dilator responses to bradykinin and SNP were not affected by L-arginine, HET0016, or 1-aminobenzotriazole. These data suggest that cyclosporin A-induced nephrotoxicity can be accounted for by reduced NO production and a consequent increase in 20-HETE. The cyclosporin A-induced nephrotoxicity is thus an ideal model for evaluating NO/CYP450 interactions.

Viral hepatitis and proteinuria in an area endemic for hepatitis B and C infections: another chain of link?

Virus hepatitis may lead to nephropathy as one of its multiple extrahepatic manifestations. Proteinuria by dipstick, a simple test in practice, is a useful and cardinal sign of underlying renal abnormalities. The aim of this study was to elucidate the impact of hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections on the occurrence of proteinuria amongst adults. A prospective, cross-sectional, community-based study was conducted in an HBV/HCV endemic area of southern Taiwan. Eligible subjects aged 40-65 years (n=9934) underwent testing of hepatitis B surface antigen (HBsAg), HCV antibody (anti-HCV) and other related biochemical profiles. Urinalysis with repeated dipstick for proteinuria detection was performed. Anti-HCV-positive rate amongst proteinuria subjects was significantly higher than nonproteinuria subjects (9.6% vs. 6.2%, P<0.001). By contrast, HBsAg-positive rate did not differ between subjects with and without proteinuria (13.0% vs. 13.8%, P=0.57). Prevalence of proteinuria amongst anti-HCV-positive subjects (10.2%) was significantly higher than that in HBsAg-positive subjects (6.4%, P=0.004) and in HBsAg-negative or anti-HCV-negative subjects (7.0%, P=0.004). The difference persisted even after excluding diabetics. Multivariate logistic regression analyses showed that diabetes was the most important significant factor associated with proteinuria, followed by hypertension, anti-HCV seropositivity, body mass index, age and triglyceride levels. We demonstrated the significant association between proteinuria and HCV, but not HBV, infection in this HBV/HCV-endemic area.

Permselective Dysfunction of Podocyte-Podocyte Contact upon Angiotensin II Unravels the Molecular Target for Renoprotective Intervention

Ameliorating the function of the glomerular barrier to circulating proteins by blocking angiotensin II (Ang II) translates into less risk of progression toward end-stage renal failure in diabetic and nondiabetic nephropathies. However, the mechanisms underlying this barrier protection are not clear. Specialized contacts between adjacent podocytes are major candidate targets, and the actin cytoskeleton is emerging as a regulatory element. Here, we present data demonstrating that Ang II induced reorganization of F-actin fibers and redistribution of zonula occludens-1 (ZO-1) that is physically associated with actin in murine podocytes. These effects were paralleled by increased albumin permeability across podocyte monolayers. The F-actin stabilizer jasplakinolide prevented both ZO-1 redistribution and albumin leakage, suggesting that actin cytoskeleton rearrangement is instrumental to podocyte permselective dysfunction induced by Ang II. Changes in both F-actin and ZO-1 patterns were confirmed in glomeruli of rat isolated perfused kidneys on short infusion of Ang II, leading to increased protein excretion. Podocyte dysfunction was mediated by Ang II type 1 receptor and was partly dependent on Src kinase-phospholipase C activation. These data demonstrate that strategies aimed at stabilizing podocyte-podocyte contacts and targeting the relevant intracellular signal transduction are crucial to renoprotection.

Contributions of Sodium and Chloride to NaCl-Induced Hypertension

The recommendation to avoid high dietary “salt” intake for the prevention and treatment of hypertension is often expressed in terms of dietary sodium. However, a consistent body of evidence suggests that the chloride component of salt is an important contributor to NaCl-induced elevations of blood pressure.1 In several rat models of salt-sensitive hypertension (Dahl salt-sensitive [S] rat, DOCA-salt hypertension, stroke-prone SHR [SHRSP], angiotensin II-induced salt-sensitive hypertension), selective dietary sodium loading, in the absence of chloride, fails to produce hypertension. In various feeding protocols, anions provided with sodium included various combinations of bicarbonate, phosphate, aspartate, glutamate, and glycinate. Overall, the failure of selective dietary sodium loading to produce hypertension in these studies was not related to group differences of body weight, net sodium balance, blood pH, or serum concentrations of sodium, potassium, or chloride. Similarly, a limited number of clinical observations also indicate that blood pressure is not increased in humans by high dietary sodium intakes in the absence of chloride. In 1929, Berghoff et al reported that blood pressure increased in 7 hypertensive individuals on a high NaCl intake, but not on a high sodium bicarbonate intake.2 This observation was subsequently confirmed.3 Similarly, other investigators have also observed that in contrast to the increase of blood pressure induced by a high NaCl intake in hypertensive patients, blood pressure is not increased by a high sodium intake provided as sodium phosphate or sodium citrate.1 Further suggesting a modulating effect of dietary chloride on blood pressure, in hypertensive and normotensive subjects, substitution of dietary NaCl with equimolar sodium bicarbonate leads to a reduction of blood pressure.4,5 Additionally, in hypertensive humans, the reduction of blood pressure by dietary potassium is attenuated by potassium chloride compared with that of potassium citrate.6 Because the blood pressure increment in response …

Protein-to-creatinine ratio in spot urine samples as a predictor of quantitation of proteinuria

Background Normal individuals usually excrete very small amounts of protein in the urine. Persistently increased protein excretion is usually a marker of kidney damage. Quantifying protein in urine is commonly used in the diagnosis of kidney diseases, detection of treatment effects and evaluation of prognosis. We evaluated the use of the total protein-to-creatinine ratio (P/C) in spot urine specimens as a predictor of urine protein excretion in 24-h collections. Methods The correlation between P/C in first morning and random urine specimens and urinary protein excretion in 24-h collections were analyzed. The cutoff value of P/C in first morning urine specimens for screening urinary protein excretion of 1 and 3 g in 24-h collections was determined by receiver operating characteristics (ROC) curve. Results For patients with Ccr≤10 ml/min, correlation between the urine protein excretion in 24-h collections and the P/C in first morning urine specimens was not significant. For patients with Ccr>10 ml/min, the correlation was highly significant. Similar results were obtained for random urine specimens. By ROC curve analysis, the P/C of 0.94 and 2.84 g/gcr in first morning urine specimens represent the best threshold to detect urine protein excretion of 1 and 3 g in 24-h collections, respectively. There is a good correlation between P/C in first morning urine specimens and random urine specimens from inpatients and outpatients. But the P/C in random specimens is significantly higher than that in first morning specimens in outpatients. Conclusion The P/C in spot urine samples could be used as an alternative to urine protein excretion in 24-h collections in patients with Ccr>10 ml/min. The P/C in first morning urine samples is better than that in random specimens, especially for outpatients.

Glomerular charge selectivity is impaired in hypertensive nephropathy.

In hypertensive nephropathy the morphological changes and albuminuria seem to start in the inner cortex but the mechanism of proteinuria is unknown. We tested the hypothesis of reduced glomerular charge selectivity in the juxtamedullary cortex of old spontaneously hypertensive rats (SHR) as a cause of proteinuria in rats with hypertensive nephropathy. The glomerular charge barrier was evaluated in 80-week-old SHR and age-matched normotensive Wistar-Kyoto rats (WKY) by measuring the glomerular clearance of radiolabelled cationic and anionic chymotrypsinogen (Chym and aChym, MW 25,000) accumulated by the proximal tubular cells in the outer, middle and inner cortex following intravenous injection. The glomerular filtration rates (GFR) in the cortical zones were obtained by aprotinin (MW 6500) and the sieving coefficient (theta) of the protein tracers calculated as their glomerular clearance/GFR. The theta aChym was similar in SHR and WKY except in the inner cortex where it was 35% higher in SHR (0.65+/-0.05) than in WKY (0.48+/-0.03) (P = 0.01). The ratio of theta aChym to theta Chym was increased from 0.55+/-0.06 to 0.77+/-0.05 (P<0.02) in the inner cortex of SHR, whereas theta Chym remained the same in all cortical zones. Finally, the percentage of glomeruli with adsorption droplets in podocytes quantified by light microscopy was higher in the inner than the outer cortex of SHR (P<0.05). The study supports the theory of a functioning glomerular charge barrier. An increased relative clearance of aChym in the inner cortex of SHR indicates impairment of the charge barrier, which, at least in part, could explain the increased protein excretion in SHR with hypertensive nephropathy.

Increased Urinary Protein Excretion in Children from Families with Balkan Endemic Nephropathy

Background: Balkan endemic nephropathy (BEN) has not been described in children, however some previous studies of children from families with BEN have revealed abnormalities of the urinary tract including an increased urinary protein excretion. Methods: In the present study, urinary excretion of total protein was studied in 703 healthy children, aged 9–13, from endemic and non-endemic settlements around the South Morava River. Since BEN is an environmentally induced disease, with possible seasonal variation of toxin(s), children were studied three times a year: in spring, autumn and winter, during a 3-year period. After a water load of 15 ml/kg body weight, a 3-hour urine sample was collected, from 7 to 10 a.m. Results: Protein excretion in urine was highest in children from families with BEN compared with the excretion in children from the city, non-endemic villages, and those from non-endemic families living in the same settlements. This was the case during all three periods investigated in the second year of the study. In the autumn term of all three years of the study, protein excretion was significantly higher in children from families with BEN than in children from the city and from non- endemic families living in the same settlements. If the upper limit of protein excretion is set at 34 mg/mmol creatinine, then increased protein excretion in autumn was found in the first year of study in 9/229 children from endemic settlements and in only 4/474 children from non-endemic areas (p = 0.004); in the second year in 5/229 and 3/474 children (p = 0.069), and in the third year in 10/229 and 4/474 children (p = 0.002), respectively. Conclusions: We have presented evidence that children from families with BEN and endemic villages consistently excreted significantly more protein in the autumn, and in three seasons (spring, autumn, winter) in 1 year of our study. These results are consistent with seasonal variations in exposure to an environmental toxin(s).

Tannins — a dietary problem for hand-reared grey partridge Perdix perdix after release?

A 4-week feeding trial on 22 grey partridges Perdix perdix was conducted in this study. Seven birds were fed commercial poultry food, seven natural food and eight commercial poultry food containing 6% of quebracho–tannin. Our results suggest that 6% dietary tannin, when added to a commercial food with high protein content, effects the grey partridge only slightly. No difference was seen in food consumption and body mass remained stable. However, birds fed tannin had longer small intestines, which most probably indicate gastrointestinal detoxication. They also excreted a high amount of tannin in their faeces. In addition, no between-group variation was seen in cytochrome P450 enzymes. Birds fed natural food had high concentration of nitrogen in intestinal excreta and high plasma alanine concentrations. They also suffered a rapid decrease in body mass after the change in diet and their body mass remained low. This may indicate increased protein excretion and/or catabolism of endogenous nutrient reserves. Potential short-term effects of the change in diet were seen in plasma. These findings coincide with the high mortality period of birds released into the wild.

Increased serum angiotensin-converting enzyme activity and plasma angiotensin II levels during pregnancy and postpartum in the diabetic rat.

The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, electrolyte balance and renal function in normal human pregnancy. The present study was designed to assess various components of the RAS and renal function during pregnancy and immediately after pregnancy in the streptozotocin (STZ)-diabetic rat. Pregnant Wistar rats were allocated to three groups: I-control, non-diabetic rats (n=24), II-STZ-diabetic rats (STZ 55 mg/kg body weight, i.v. on day 10 of pregnancy, n=24), III-diabetic rats, as above, treated with insulin (4 units/day, s.c. n=21). On days 17-18 of pregnancy, or within 24 hours after delivery, the rats were sacrificed and the various components of the RAS were determined. Urinary protein excretion (UP) and creatinine clearance(CCr) were greater in group II, four days after STZ, than in group I (UP: I-7.6+/-2.8, II-18.6+/-6.3 mg/24-hour, p<0.001, CCr: I-1.04+/-0.33, II-2.38+/-0.7 ml/minute, p<0.001). Mean (+/-SD) serum angiotensin-converting enzyme (ACE) activity and plasma angiotensin II(Ang II) levels at days 17-18 of pregnancy were greater in the untreated diabetic rats than in control pregnant rats (ACE: 163+/-18 vs. 111+/-21 nmol/ml/minute, p<0.001, Ang II: 115+/-45 vs. 43+/-10 pg/ml, p<0.005). Postpartum serum ACE activity and plasma Ang II levels were greater in group II (ACE: I-123+/-14, II-142+/-24, III-108+/-21 nmol/ml/minute, p<0.01, Ang II: I-56+/-38, II-148+/-62, III-38+/-17 pg/mI, p<0.001). ACE activity in the lung was greater, whereas the activity in the renal cortex was less, in group II than in group I. Kidney weight in untreated diabetic rats was greater than in the other two groups. Increased serum ACE activity during pregnancy and postpartum in the untreated diabetic rat is associated with enhanced serum Ang II levels, which may contribute to increased protein excretion and renal hypertrophy.

Nephrotoxicity of ifosfamide, carboplatin and etoposide (ICE) alone or combined with extracorporeal or radiant-heat-induced whole-body hyperthermia.

Although whole-body hyperthermia combined with specific genotoxic chemotherapy can be shown to enhance neoplastic cell killing without a concomitant rise in bone marrow toxicity, nephrotoxicity can become treatment-limiting. This study compares the kidney toxicity to the kidney of ifosfamide, carboplatin and etoposide (ICE) chemotherapy alone, and ICE chemotherapy combined with either extracorporeal (e-WBH) or radiant-heat-induced hyperthermia (r-WBH) in 43 patients with refractory sarcoma. Within 3 days of ICE chemotherapy treatment there was a significant increase in urinary protein excretion and a reduction of the glomerular filtration rate. These effects were more pronounced if WBH was added. The use of immunoluminometric assays revealed a predominance of low-molecular-mass proteins. This increase in protein excretion persisted in the e-WBH-treated group, whereas it vanished within 3 weeks in both the group treated with ICE alone and that treated with r-WBH. Our findings suggest that ICE chemotherapy causes transient tubular and glomerular damage, which is enhanced by WBH. In terms of long-term nephrotoxicity e-WBH was more nephrotoxic than r-WBH. This finding is consistent with our clinical observations.

Endothelin-1 and CYP450 arachidonate metabolites interact to promote tissue injury in DOCA-salt hypertension.

Inhibition of cytochrome P-450 (CYP450) enzymes with cobalt chloride (CoCl2) prevented hypertension, organ hypertrophy, and renal injury induced by DOCA and salt (1% NaCl) in uninephrectomized (UNx) rats. Systolic blood pressure (SBP) rose to 193 +/- 6 mmHg by day 21 from control levels of 150 +/- 7 mmHg in response to DOCA-salt treatment, a rise that was prevented by CoCl2 (24 mg. kg-1. 24 h-1). The effects of DOCA-salt treatment, which increased protein excretion to 88.3 +/- 6.9 mg/24 h on day 21 from 9.0 +/- 1.1 mg/24 h on day 3, were prevented by CoCl2. CoCl2 also attenuated the renal and left ventricular hypertrophy and the increase in media-to-lumen ratio in hypertensive rats. DOCA-salt treatment increased excretion of endothelin (ET)-1 from 81 +/- 17 to 277 +/- 104 pg. 100 g body wt-1. 24 h-1 associated with a fourfold increase in 20-hydroxyeicosatetraenoic acid (20-HETE) excretion from 3.0 +/- 1.1 to 12.2 +/- 1.9 ng. 100 g body wt-1. 24 h-1 (days 3 vs. 21). CoCl2 blunted these increases by 58 and 72%, respectively. In aortic rings pulsed with [3H]thymidine, ET-1 increased its incorporation. Dibromododec-11-enoic acid, an inhibitor of 20-HETE synthesis, attenuated ET-1-induced increases in [3H]thymidine incorporation. We distinguished effects of CoCl2 acting via CO generation vs. suppression of CYP450-arachidonic acid metabolism by treating UNx-salt-DOCA rats with 1-aminobenzotriazole (ABT), which suppresses CYP450 enzyme activity, and compared these results to those produced by CoCl2. ABT reduced hypertension, as did CoCl2. Unlike CoCl2, ABT did not prevent organ hypertrophy and proteinuria, suggesting that these effects were partially related to CO formation. Blockade of the ETA receptor with BMS-182874 reduced SBP, organ hypertrophy, and proteinuria, indicating the importance of ET-initiated abnormalities to the progression of lesions in UNx-salt-DOCA.

Trimethylphosphate: a 30-month chronic toxicity/carcinogenicity study in Wistar rats with administration in drinking water.

Trimethylphosphate (TMPO) was administered to 50 male and 50 female Wistar rats through their drinking water at doses of 0, 1, 10, or 100 mg/kg body weight up to 30 months. The dosage of 100 mg/kg was reduced to 50 mg/kg in week 54 for reasons of tolerance, and the animals were euthanized in week 100. Additional 10 animals per dose and sex were treated for 12 months and then euthanized for interim analysis. Weakness of the hind limbs, increased incidences of sunken flanks, distended abdomen, and poor general condition were observed in both sexes of the 100/50 mg/kg group beginning with week 46. Food intake was reduced in high dose males. At 10 mg/kg body weights were up to 10% (males) and at 100/50 mg/kg up to 20% (males) or 15% (females) lower than in controls. Mortality was not affected in animals receiving up to 10 mg/kg. At 100/50 mg/kg it was markedly increased, reaching about 70% at week 100. Relatively slight hematologic changes (reduced hemoglobin, hematocrit, erythrocyte counts, increased reticulocyte numbers, and thrombocyte counts as well as a shift in the differential blood count) at 100/50 mg/kg are interpreted as changes most probably secondary to the other toxic effects. Increased cholesterol concentrations in plasma, shifts in the serum protein electrophoresis (males), increased organ weights (females), and an increased incidence of necroses and lymphocytic infiltrations point to a treatment-related effect on the liver at 100/50 mg/kg. Slightly increased protein excretion, increased relative kidney weights, and an increased incidence of chronic progressive nephropathy are considered treatment-related but rather secondary effects at 100/50 mg/kg. At 100/50 mg/kg an increased incidence and severity of bilateral tubular atrophy in the testes was diagnosed. The most important toxic effect was neurotoxicity, consisting of degeneration and loss of nerve fibers in the peripheral nerves and the spinal cord, associated with myopathic changes, and occurring at 100/50 mg/kg. The no-observed-adverse-effect-level, based on the suppression of body weight gain, is 1 mg/kg in males and 10 mg/kg in females. The incidence, time of occurrence, spectrum of types, and localizations of tumors provided no indication of a tumorigenic/carcinogenic effect of the test substance. TMPO is therefore considered not to be carcinogenic in Wistar rats.

Platelet-activating factor mediates angiotensin II-induced proteinuria in isolated perfused rat kidney.

Isolated kidney preparations (IPK) from male Sprague Dawley rats perfused at constant pressure were used to evaluate the effect of angiotensin II (AII) and platelet-activating factor (PAF) on renal function and urinary protein excretion. Compared with basal, intrarenal infusion of AII at 8 ng/min caused a progressive increase in protein excretion (11 +/- 6 versus 73 +/- 21 micrograms/min) in parallel with a decline in renal perfusate flow (RPF) (29 +/- 3 versus 18 +/- 3 ml/min). Addition to the perfusate of PAF at 50 nM final concentration also induced proteinuria (9 +/- 4 versus 55 +/- 14 micrograms/min) but did not change RPF (29 +/- 3 versus 30 +/- 3 ml/min). Preexposure of isolated kidneys to the PAF receptor antagonist WEB 2086 prevented the increase in urinary protein excretion induced by AII infusion (basal: 13 +/- 6; post-AII: 12 +/- 7 micrograms/min) but failed to prevent the vasoactive effect of AII (RPF, basal: 30 +/- 2; post-AII: 21 +/- 3 ml/min). In additional experiments, dexamethasone reduced the proteinuric effect of PAF remarkably. These results indicate that in isolated kidney preparation: (1) AII infusion induced proteinuria and decreased RPF; and (2) the effect of AII in enhancing urinary protein excretion was completely prevented by a specific PAF receptor antagonist, which, however, did not influence the AII-induced fall in RPF. It is suggested that PAF plays a major role in AII-induced changes in the permselective function of the glomerular capillary barrier.

The importance of urinary protein excretion during conservative management of severe preeclampsia

OBJECTIVES: We determined the natural course of urinary protein excretion during conservative management of severe preeclampsia and investigated whether changes in urinary protein excretion can predict maternal or perinatal outcome. STUDY DESIGN: We reviewed the medical charts of 66 women with severe preeclampsia which was managed conservatively before 32 weeks of gestation and who had at least two 24-hour urinary protein determinations 4 or more days apart after admission. RESULTS: Fifty-nine (89%) of 66 women had an increase in proteinuria during conservative management of severe preeclampsia. The median increase in protein excretion after admission was 660 mg/24 hours (range -4580 to 18,960 mg/24 hours). Patients were divided into two groups. The first group ( n = 24) had an increase in 24-hour urinary protein excretion of ≥2 gm; the second group ( n = 42) had a 24-hour urinary protein excretion that decreased ( n = 7) or increased by <2 gm ( n = 35). There were no cases of eclampsia or stillbirth in either group. The rate of HELLP (hemolysis, elevated liver enzyme levels, low platelet counts) syndrome, abruptio placentae, cesarean delivery because of fetal distress, 5-minute Apgar scores ≤6, and the admission-to-delivery intervals were all similar in the two groups. CONCLUSIONS: Proteinuria increases in most women with severe preeclampsia managed conservatively. No differences in maternal or fetal outcomes were found between pregnancies with marked increases in proteinuria and those with modest or no increases. (Am J Obstet Gynecol 1996;175:1313-6.)

Examination of lesions in the urinary bladder and kidney of dogs induced by nefiracetam, a new nootropic agent.

Toxic lesions induced by nefiracetam, a nootropic drug, in the urinary bladder and kidney were examined by repeated oral administration of 300 mg/kg/day for 1, 2, 3, 4, or 11 wk to male and female beagle dogs. Each dog was sacrificed after each treatment period, and urinalysis and serum biochemistry were performed for surviving dogs at several time points. One male and 2 females died during week 10 or 11. Degeneration and desquamation of epithelial cells and edema and hemorrhage in the lamina propria were observed in the urinary bladder after 1 wk of treatment. These changes became severe as time progressed and were reflected in the clinical abnormalities of hematuria and increased protein excretion in urine. However, epithelial regeneration and hyperplasia were seen thereafter, and almost no change was seen in the urinary bladder after treatment for 11 wk. Instead of recovery as in the urinary bladder, the kidney showed epithelial degeneration and hyperplasia in the papilla and collecting duct and interstitial congestion and hemorrhage after treatment for 11 wk. Extensive hemorrhage and papillary necrosis were seen in animals that died during week 10 or 11 of dosing. These kidney changes were associated with increased urinary volume and decreased osmotic pressure. The lesions are thought to have a common etiopathogenesis and to be initiated by the epithelial damage with a time lag between expression of injury in the urinary bladder and the kidney.

Characterization of the renal phenotype of transgenic rats expressing the human endothelin-2 gene.

We have previously established a transgenic rat model termed TGR(hET-2)37 overexpressing the human endothelin-2 (ET-2) gene with high renal transgene expression. This renal overexpression is of pathophysiological interest because a long-term activated paracrine renal endothelin system has been implicated in chronic renal failure due to progressive glomerular injury. Therefore, our aim in the present study was to analyze renal transgene expression in detail and address the question of whether transgene expression causes phenotypic and functional changes in the kidney. We used reverse transcription-polymerase chain reaction and in situ hybridization techniques for transgene expression analysis. Tissue ET-2 concentrations were measured with a specific radioimmunoassay. For histological evaluation of renal tissue, all samples were subjected to hematoxylin-eosin and periodic acid-Schiff staining. Renal tissue ET-2 concentrations were significantly increased in TGR(hET-2)37 rats. Using in situ hybridization, we found that the human ET-2 gene was almost exclusively expressed within the glomeruli. The glomerular transgene expression resulted in a significantly increased glomerular injury score and likewise in a significantly increased protein excretion, whereas glomerular filtration rate was not altered. Blood pressure was similar in TGR(hET-2)37 rats and age-matched controls, suggesting that the local changes in the kidney were correlated with paracrine endothelin actions. In conclusion, our study revealed that the major renal expression site of the human ET-2 transgene in TGR(hET-2)37 rats was within the glomeruli and caused the development of glomerulo-sclerosis with significantly increased protein excretion that is independent of blood pressure. We suggest that TGR(hET-2)37 rats are a new monogenetic animal model for study of the paracrine renal endothelin system and its involvement in renal pathophysiology.

Alterations of the renal function in the isolated perfused rat kidney system after in vivo and in vitro application of S-(1,2-dichlorovinyl)-L-cysteine and S-(2,2-dichlorovinyl)-L-cysteine.

The nephrotoxic effects of the two isomers S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCVC) and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC) were investigated comparatively in the isolated perfused rat kidney with two different treatment regimens. In the first approach, the kidneys were exposed to the test compounds dissolved in the perfusion media after removal from the animal. In the second approach the test compounds were administered to rats in vivo and the nephrotoxicity was assessed in the isolated perfused kidney 6 h and 18 h post-treatment. The vicinal isomer 1,2-DCVC produced concentration- and time-dependent nephrotoxicity with both treatment regimens, as indicated by the impairment of glucose reabsorption, the increase of protein excretion and of gamma-glutamyltransferase and alkaline phosphatase activities in urine. In contrast to the marked toxicity observed after in vivo and in vitro administration of 1,2-DCVC, the geminal isomer, 2,2-DCVC, was not nephrotoxic at all concentrations (0.5 and 2.5 mM in vitro, 40 and 70 mg/kg in vivo) investigated.

Age-dependent enzymuria, proteinuria and changes in renal blood flow and glomerular filtration rate in rats

The time course of urinary excretion of two enzymatic indicators of renal damage, N-acetyl-β-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) was measured in female Wistar rats at different ages. NAG and AAP are localized at different sites of the nephron and are released into the urine when kidney damage occurs. Total protein flow, urinary volume and creatinine flow were also determined. In a parallel experiment, the effect of aging on renal blood flow (RBF) and glomerular filtration rate (GFR) was examined in young (1.5-month) adult (3-month) and elderly (20-month) female rats. Clearance following a single injection of [ 131I] o-iodohippurate (hippuran, OIH) was used for the measurement of effective RBF and as an index of tubular cell function. [ 125I]Iothalamate (IOT) clearance was used to measure GFR. With advancing age, an increase in NAG and AAP urinary flow appeared. The increases in protein excretion were greater than and previous to those of enzyme excretion. It is shown that absolute RBF and GFR (ml/min) in old rats are greater than in young or adult animals. When absolute RBF or GFR was divided by kidney weight (ml/min/g) no clearance changes appeared in any age group studied; only when clearance was expressed in relation to body weight (ml/min/100 g), a decrease in RBF and GFR was evidenced. This indicates that the rate of increase of both RBF and GFR with age is similar to that of kidney weight and lower than that of body weight. The present findings indicate that urinary markers of renal injury increase with age, whereas GFR and RBF only decrease when expressed as clearance related to body weight.

Water and protein permeability is regulated by the glomerular epithelial slit diaphragm.

The glomerular barriers to water and macromolecular movement were examined 2 and 24 h after the administration of a monoclonal antibody (mAb) specific to an antigen located on the epithelial slit diaphragm and the external aspect of the glomerular basement membrane. By micropuncture techniques 2 h after mAb administration, single-nephron GFR (SNGFR) and plasma flow were unchanged but the glomerular capillary hydrostatic pressure gradient and glomerular capillary hydrostatic pressure were increased and the glomerular ultrafiltration coefficient (LpA) decreased to values that were 50% of the normal control. There was no increase in urinary protein excretion at 2 h. However, at 24 h after mAb, nephron plasma flow (SNPF) and SNGFR increased and the glomerular ultrafiltration coefficient returned to values indistinguishable from the normal control. At 24 h, there was a marked increase in protein excretion. The administration of meclofenamate decreased values for SNGFR and SNPF to normal. Immunoglobulin G was exclusively bound to glomerular capillary walls in a linear or continuous fashion at 2 h, but in a discontinuous, granular pattern at 24 h. These studies suggest that after mAb, important limiting glomerular barriers for hydraulic conductivity and protein excretion reside on the epithelial aspect of the glomerular capillary basement membrane, specifically at the level of the slit diaphragm. Studies also suggest that alterations in glomerular capillary hydraulic conductivity can be effectively separated from increases in macromolecular permeability.

Evaluation of methods indicating higher susceptibility of immature rats to renal ischemia.

After 45-min bilateral warm renal ischemia lethality amounted to 45% and 82% in 55- and 20-day-old rats, respectively (n = 176). Lethality rates were not significantly different after 20-min unilateral ischemia followed by contralateral nephrectomy after 24 hours (34 vs. 48% in young vs. adult rats; n = 168). The latter experimental approach was used to characterize age dependent differences in the susceptibility to ischemia. The degree of postischemic renal damage was the highest at the 1st and 2nd days after ischemia; at this time, lethality rates were similar in young and adult rats. However, urea concentration in serum was significantly more enhanced in young animals whereas that of creatinine was increased to the same extent in both age groups. The increase in protein excretion into urine was similar in young and adult rats, too. Furthermore, urine flow rates and GFR were significantly diminished after ischemia, independent of age. However, excretion of Na+ and K+ was distinctly more depressed in immature individuals. Finally, the glutathione content in kidney tissue of both age groups was reduced and lipid peroxidation was significantly higher after ligation of the renal arteries. The relative changes were similar in both age groups although the glutathione content was significantly lower in 20-day-old control rats. 4-5 days after ischemia, most parameters returned to baseline values. In 55-day-old rats, 45-min ischemia has more severe consequences on renal function compared to 20-min ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)