Increased intrarenal renin-angiotensin system activity contributes to diabetic nephropathy. ANG II generation in mesangial cells (MC) is increased by high-glucose (HG) exposure. This study assessed the mechanisms involved in the glucose-induced ANG II generation in rat MC. Under basal conditions, MC mainly secreted prorenin. HG decreased prorenin secretion and induced a striking 30-fold increase in intracellular renin activity. After 72 h of HG exposure, only the mRNA levels for angiotensinogen and angiotensin-converting enzyme (ACE) were significantly elevated. However, after shorter periods of 24 h of HG stimulation the mRNA levels of the enzymes prorenin and cathepsin B, besides that for ACE, were significantly increased. The results suggest that the HG-induced increase in ANG II generation in MC results from an increase in intracellular renin activity mediated by at least three factors: a time-dependent stimulation of (pro)renin gene transcription, a reduction in prorenin enzyme secretion, and an increased rate of conversion of prorenin to active renin, probably mediated by cathepsin B. The increase in angiotensinogen mRNA in parallel to increased renin activity indicates that HG also increased the availability of the renin substrate. The consistent upregulation of ACE mRNA suggests that, besides renin, ACE is directly involved in the increased mesangial ANG II generation induced by HG.
The activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. The ratios found for ACE1, ACE2, ACEInt1 and ACEInt2 in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation.
The mechanism of cyclosporine A (CyA) nephrotoxicity is unclear. In order to evaluate renal microcirculation seven euvolemic Munich-Wistar (MW) rats were studied after acute CyA treatment (50 mg/kg, i.v.). Both total glomerular filtration rate (GFR, 0.96 +/- 0.04 vs. 0.47 +/- 0.07 ml/min) and single nephron GFR (27.90 +/- 3.39 vs. 14.02 +/- 3.49 nl/min) declined significantly (P less than 0.001). It was observed an increase in afferent (RA, increases 188%) and efferent (RE, increases 360%) arteriolar resistances that caused a decrease on glomerular plasma flow rate (QA) from 100.99 +/- 17.09 to 44.37 +/- 13.37 nl/min (P less than 0.001). Mean glomerular capillary hydraulic pressure (PGC) increased from 45 +/- 1 to 55 +/- 4 mm Hg (P less than 0.05) and the glomerular ultrafiltration coefficient (Kf) decreased by 70% (0.096 +/- 0.030 to 0.031 +/- 0.010 nl/sec X mm Hg, P less than 0.05). Additionally, in order to study hormonal participation in this nephrotoxicity, other three groups of MW rats were previously treated with captopril (2 mg/kg, i.v.), verapamil (20 micrograms/kg/min, i.v.) or indomethacin (2 mg/kg, i.v.). Both captopril and verapamil minimized the renal effects of CyA, with a decline of approximately 25% instead of approximately 50% on GFR and RPF. Moreover, two groups of Brattleboro rats were studied. Acute CyA administration in homozygote Brattleboro rats produced a decline of only approximately 22% and approximately 31%, respectively, in GFR and renal plasma flow (RPF), when compared with MW rats (P less than 0.05). Similar results were observed in heterozygote Brattleboro rats when compared with MW rats, disclosing differences due to a different strain of rats.(ABSTRACT TRUNCATED AT 250 WORDS)
The angiotensin-converting enzyme (ACE) profile in urine of hypertensive patients and spontaneously hypertensive rats (SHR; 90- and 65-kDa N-domain ACEs) is different from that of healthy subjects and Wistar rats (190 and 65 kDa). In addition, four ACE isoforms were purified from mesangial cells (MC) of Wistar rats in the intracellular compartment (130 and 68 kDa) and as secreted forms (130 and 60 kDa). We decided to characterize ACE forms from SHR MC in culture. Analysis of the ACE gene showed that SHR MC are able to express ACE mRNA. The concentrated medium and cell homogenate were separately purified by gel filtration and then subjected to lisinopril-Sepharose chromatography. The molecular masses of purified enzymes, 90 kDa for ACEm1A and 65 kDa for ACEm2A (secreted enzymes) and 90 kDa for ACEInth1A and 65 kDa for ACEInth2A (intracellular), were different from those of Wistar MC. The purified enzymes are Cl- dependent, inhibited by enalaprilat and captopril, and able to hydrolyze AcSDKP. Immunofluorescence and cell fractionation followed by Western blotting showed predominant immunoreaction of the 9B9 antiserum for N-domain ACE in the nuclei. The N-domain ACE was localized in the glomerulus from Wistar rats and SHR. ANG II and ANG-(1-7) were localized in the cell cytoplasm and nuclei. The 90-kDa N-domain ACE, described recently as a possible genetic marker of hypertension, was found inside the cell nuclei of SHR MC colocalized with ANG II and ANG-(1-7). The presence of ANG II in the cell nuclei could suggest an important role for this peptide in the transcription of new genes.
Renal function was evaluated in normal and after 30 days of % renal mass reduction (CRF) in Munich-Wistar (MW) rats, spontaneously hypertensive rats with superficial glomeruli (EPM), and in Brattleboro rats with congenital diabetes insipidus (DI). Mean arterial pressure was higher in EPM-Control and EPM-CRF rats as compared with MW and DI rats. MW and EPM rats with CRF showed increases of 120% and 196%, respectively, in single nephron glomerular filtration rate as compared with their controls. However, DI rats with CRF did not show any increase in single nephron glomerular filtration rate as compared with the control group. Therefore, the data suggest that the presence of hypertension enhances the adaptive mechanisms on remnant kidney's function. Conversely, in the absence of antidiuretic hormone, adaptive mechanisms of remnant nephrons did not occur. In addition, it was observed that rats with CRF submitted to prostaglandin blockade with indomethacin showed for MW rats a 55% and 20% reduction in ultrafiltration coefficient and in single nephron glomerular filtration rate, respectively. Decreases of 60% and 30% in ultrafiltration coefficient and single nephron glomerular filtration rate, respectively, were observed for EPM rats. Antidiuretic hormone (ADH) has been proposed to have a kidney trophic action, 4 and homozygous Brattleboro rats (DI), which lack ADH, do not show hyperfiltration after being fed a hyperproteic diet, 3 as is observed in other strains of rats. 2 Thus, the objectives of the present study were to evaluate the remnant kidney's function in hypertensive states and the role of ADH and PGs in this model by using spontaneously hypertensive rats with superficial glomeruli (EPM rats), DI rats, and indomethacin administration. MethodsStudies were performed in Munich-Wistar (MW) rats (Simonsen Laboratories, Gilroy, California), EPM rats, and DI rats. EPM rats are the first generation of spontaneously hypertensive rats (SHR) (National Institutes of Health [NIH], Bethesda, Maryland) mated with MW rats and, thus, present spontaneous hypertension and superficial glomeruli.5 DI rats (NIH) have hereditary hypothalamic diabetes insipidus. All studies were performed in 3-4-month-old male rats, and they all were bred in the Animal House of Escola Paulista de Medicina. Each of the three strains of rats were divided into a control group and a group submitted to Y6 renal mass ablation (chronic renal failure [CRF]), which was studied 30 days after. The subtotal 5 /6 nephrectomy was performed by ligation of two or three branches of the left renal artery and right nephrectomy at the same time. All rats were allowed free access to regular rat chow and tap water until the morning of the study. Inactin (Byk, Gulden, Konstanz, Germany) anesthetic was used, and replacement of surgical volume losses was
There is evidence that fructose-1,6-diphosphate (FDP) provides protection from hepatic and cardiac toxic-induced damage and ischemic renal insult. To determine if FDP also protects against cyclosporine (CsA)-induced nephrotoxicity, two groups of adult male Wistar rats were studied for whole kidney clearance rates. After two initial control periods, group 1 received only CsA (CsA, n = 8). Group 2 received FDP 350 mg/kg, followed by CsA 50 mg/kg (FDP-CsA, n = 6). In both groups, after a 30-min equilibration period, two additional clearance rates were measured (Postl and Post2). A significant reduction in clearance rates was observed after drug infusion in both groups (approximately 58 and 64% in CsA and FDP-CsA groups, respectively, p < 0.05) with a recovery to control values in the Post2 period in the FDP-CsA group. These data suggest a protective effect of FDP on CsA-induced renal impairment.
Whole-kidney function and glomerular hemodynamics were evaluated after acute (50 mg/kg, iv, in bolus) and short-term chronic (50 mg mg/kg, ip, 5 days) acyclovir (ACV) and short-term chronic ganciclovir (Gan; 30 mg/kg, ip, 5 days) treatment in envolemic Munich-Wistar rats. The evaluation of whole-kidney function of the ACV groups showed a significant reduction in total GFR (0.96 +/- 0.10 to 0.28 +/- 0.02 mL/min in the acute group, P < 0.05, and 1.04 +/- 0.09 to 0.33 +/- 0.04 mL/min in the chronic group, P < 0.05) with a marked increase in total renal vascular resistance (TRVR) (33 +/- 5 to 122 +/- 26 mm Hg.min/mL in the acute group and 28 +/- 3 to 74 +/- 18 mm Hg.min/mL in the chronic group, P < 0.05) and a reduction in RPF (2.29 +/- 0.25 to 0.81 +/- 0.15 mL/min in the acute group and 2.57 +/- 0.36 to 1.30 +/- 0.40 mL/min in the chronic group, P < 0.05). Conversely, urinary flow (V') was unchanged (3.6 +/- 0.4 to 3.6 +/- 0.2 microL/min in the acute group) or elevated (3.7 +/- 0.6 to 6.6 +/- 1.4 microL/min in the chronic group, P < 0.05). The evaluation of glomerular hemodynamics after ACV treatment showed a reduction in single-nephron GFR (SNGFR) (46.4 +/- 5.3 to 26.2 +/- 3.4 nL/min in the acute group and 38.7 +/- 5.7 to 21.1 +/- 5.7 nL/min in the chronic group, P < 0.05), a significant elevation in total arteriolar resistance (RT) (2.90 +/- 0.44 to 4.94 +/- 0.77 x 10(10) dyn.s.cm-5 in the acute group and 3.72 +/- 0.45 to 9.00 +/- 2.40 x 10(10) dyn.s.cm-5 in the chronic group, P < 0.05) and a severe reduction in glomerular plasma flow rate (QA) (152.6 +/- 29.5 to 103.8 +/- 27.8 nL/min in the acute group and 149.1 +/- 29.8 to 68.5 +/- 10.0 nL/min in the chronic group, P < 0.05). However, the glomerular ultrafiltration coefficient, Kf, was changed only in the chronic group (0.1002 +/- 0.0165 to 0.0499 +/- 0.0090 nL/(s.mm Hg), P < 0.05). After Gan treatment, no changes were observed in GFR (1.04 +/- 0.09 to 0.96 +/- 0.08 mL/min, with the maintenance of RPF (2.57 +/- 0.36 to 2.66 +/- 0.34 mL/min) and a nonsignificant reduction in TRVR (28 +/- 3 to 20 +/- 3 mm Hg.min/mL. The short-term Gan treatment also showed a different pattern in glomerular hemodynamics by inducing an elevation in SNGFR (38.7 +/- 5.7 to 50.3 +/- 2.8 nL/min, P < 0.05) with no changes in QA (150 +/- 30 to 135 +/- 22 nL/min) and a mild vasodilation, RT (3.7 +/- 0.5 to 2.7 +/- 0.3 x 10(10) dyn.s.cm-5, P < 0.05) associated with an increment in Kf (0.1002 +/- 0.0165 to 0.2400 +/- 0.0700 nL/(s.mm Hg), P < 0.05). Thus, ACV induced acute renal failure by reducing GFR and SNGFR by an increase in TRVR and RT with a reduction in RPF and QA. Also, after short-term treatment with ACV, a reduction in Kf led to a reduction of SNGFR. On the other hand, Gan treatment did not induce acute renal failure by the adopted techniques.
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