Denise Maria Avancini Costa Malheiros

Intrarenal Renin-Angiotensin System Is Upregulated in Experimental Model of Progressive Renal Disease Induced by Chronic Inhibition of Nitric Oxide Synthesis

Locally generated angiotensin II (AngII) may be involved in the pathogenic mechanisms of chronic renal diseases. Renal expression of AngII and other components of the renin-angiotensin system (RAS) were analyzed by immunohistochemistry and Western blot in a model of chronic progressive nephropathy induced by inhibition of nitric oxide synthesis. Renal injury was evaluated by histology and albumin excretion. Systemic RAS status was evaluated through plasma renin activity (PRA) and plasma AngII concentration. In addition, the effects of enalapril, losartan, and mycophenolate mofetil (MMF) on AngII expression in animals with chronic renal disease was also analyzed. Plasma renin activity and plasma AngII were not different between rats with nephropathy and controls (2.08 +/- 0.7 versus 2.03 +/- 0.5 ng/ml/h and 94.3 +/- 18 versus 78.9 +/- 16 fmol/ml, respectively). However, rats with chronic progressive nephropathy showed augmented renal content of angiotensinogen protein (13.5 +/- 3.5 versus 2.2 +/- 0.4 pixels in control rats; P < 0.05), enhanced expression of cathepsin D-a renin-like enzyme-in cortical collecting tubules (103.5 +/- 27.0 versus 66.2 +/- 3.6 cells/mm2 in controls; P < 0.01), and increased expression of AT1 receptor in interstitium (54.7 +/- 7.8 versus 1.3 +/- 0.4 cells/mm2 in controls; P < 0.001). Kidney angiotensin-converting enzyme content did not differ among the groups. Notably, an increased number of interstitial cells expressing AngII was detected in the renal interstitium (9.5 +/- 1.6 versus 1.7 +/- 0.6 cells/mm2 in controls; P < 0.05). Rats treated with Nomega-nitro-L-arginine-methyl-esther and losartan presented a decreased local AngII formation, in contrast to its known effect on plasma AngII. Moreover, mycophenolate mofetil lowered interstitial AngII expression, suggesting that inflammatory signaling may be involved in interstitial AngII generation. This study demonstrates the upregulation of local RAS in the kidney in a model of chronic progressive nephropathy.

Effect of Salt Intake and Inhibitor Dose on Arterial Hypertension and Renal Injury Induced by Chronic Nitric Oxide Blockade

Long-term nitric oxide blockade by N omega -nitro-L-arginine methyl ester (L-NAME) leads to severe and progressive hypertension. The role of salt intake in this model is unclear. To verify whether salt dependence in this model is related to the extent of nitric oxide inhibition, we gave adult male Munich-Wistar rats a low salt, standard salt, or high salt diet and oral L-NAME treatment at either 3 or 25 mg/kg per day. At 10 to 15 days of treatment, the slope of the pressure-natriuresis line was decreased in rats receiving low-dose L-NAME compared with untreated controls. In rats treated with the higher dose, the line was shifted to the right but remained parallel to that obtained in untreated controls. Renal vascular resistance was moderately increased in rats receiving low-dose L-NAME, whereas high-dose L-NAME induced a marked vasoconstriction that was aggravated by salt overload. Low-dose L-NAME treatment induced hypertension only when associated with sodium overload. In rats receiving high-dose L-NAME, hypertension was aggravated by sodium excess but was not ameliorated by sodium restriction. Long-term (6 weeks) L-NAME treatment was associated with progressive hypertension, which was aggravated by salt overload, and with the development of albuminuria, focal glomerular collapse, glomerulosclerosis, and renal interstitial expansion. These abnormalities were worsened by salt overload and largely prevented by salt restriction. In the model of chronic nitric oxide blockade, salt dependence is a function of the inhibitor dose, and renal injury varies directly with the level of salt intake.

Cyclosporine-induced interstitial fibrosis and arteriolar TGF-β expression with preserved renal blood flow

BACKGROUND Cyclosporine A (CsA)-induced chronic nephrotoxicity is characterized by interstitial fibrosis and afferent arteriole hyalinosis. CsA lesion has been linked to maintained renal vasoconstriction and narrowing of the afferent arteriole lumen diameter, leading to preglomerular ischemia. We investigated the role of renal hemodynamics in CsA-induced transforming growth factor (TGF-beta) expression and interstitial fibrosis. METHODS Groups of rats fed a low salt diet were given CsA 5 mg/kg/day (CsA) or the vehicle (olive oil, [VH]) s.c. and had the renal blood flow (RBF), glomerular filtration rate (GFR), mean arterial pressure, renal vascular resistance, renal histologic changes, and immunohistochemical features for macrophages and TGF-beta evaluated after 1, 2, and 8 weeks of treatment. RESULTS At week 1, despite normal renal hemodynamics and MAP, there was a significant macrophage interstitial influx in CsA-treated rats (70+/-16 vs. 29+/-4 cells+/0.5 mm2, in CsA vs. VH, P=0.02) that was progressive with treatment (80+/-13 vs. 32+/-8 cells+/0.5 mm2, P=0.016 and 197+/-36 vs. 23+/-3 cells+/0.5 mm2, P=0.0002, CsA vs. VH at 2 and 8 weeks, respectively). After 2 weeks of treatment, CsA animals developed a significant interstitial fibrosis, with preserved RBF, even when it was assessed 2 hr after CsA injection. There was a significant increase in the immunostaining for TGF-beta in the juxtaglomerular arterioles in CsA-treated rats (48.6+/-3.8 vs. 35.1+/-1.1%, CsA vs. VH at 2 weeks, P<0.05 and 59.0+/-3.2 vs. 37.0+/-2.1%, CsA vs. VH at 8 weeks, P=0.0001). A significant and progressive GFR decrease followed the renal structural injury of CsA treatment. Arteriolar and glomerular anatomic injury were not found throughout the study. CONCLUSIONS Low CsA doses might generate interstitial fibrosis without any decrease in RBF or structural arteriolar lesion evidence, possibly through early macrophage influx and increased TGF-beta expression. It clearly seems that CsA-induced ischemia and tubulointerstitial injury may occur independently, suggesting that chronic CsA nephrotoxicity may be very hard to prevent or even not be preventable at all.

Nitroflurbiprofen, a new nonsteroidal anti-inflammatory, ameliorates structural injury in the remnant kidney

Cyclooxygenase derivatives and nitric oxide (NO) may influence the pathogenesis of progressive nephropathies. We investigated the effect of nitroflurbiprofen (NOF), a NO-releasing nonsteroidal anti-inflammatory drug (NSAID) without gastrointestinal toxicity, in rats with 5/6 ablation (NX). The following four groups were studied: Sham, sham-operated rats; Sham + NOF, Sham receiving oral NOF two times daily; NX, rats subjected to NX; and NX + NOF, NX receiving NOF. NOF was barely detected in plasma but released the parent compound flurbiprofen. At 30 days, glomerular hydraulic pressure (PGC) was 76 ± 3 mmHg in NX (52 ± 1 in Sham, P < 0.05). NOF slightly reduced PGC to 69 ± 2 mmHg in NX + NOF ( P > 0.05 vs. NX). Glomerular volumes behaved similarly. At 60 days, tail cuff pressure was 152 ± 6 mmHg, glomerulosclerosis index was 22.1 ± 9.5, and interstitial fractional area was 9.9 ± 1.2% in NX. NOF reduced these parameters to 137 ± 4 mmHg, 3.5 ± 0.7, and 6.4 ± 0.8%, respectively ( P < 0.05), without causing growth stunting or anemia. These beneficial effects could not be ascribed to NO donation and may reflect cyclooxygenase inhibition. This is the first evidence that chronic NSAID treatment may ameliorate progressive nephropathies.

Adipose tissue-derived stem cell treatment prevents renal disease progression.

Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle in mice for 1 h; after that, the kidney was reperfused immediately. Four hours after the surgery, 2 × 105 ASCs were intraperitoneally administered, and mice were followed for 24 h posttreatment and then at some other time interval for the next 6 weeks. Also, animals were treated with 2 × 105 ASCs at 6 weeks after reperfusion and sacrificed 4 weeks later to study their effect when interstitial fibrosis is already present. At 24 h after reperfusion, ASC-treated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4, IL-10, and HO-1 expression increased despite a lack of ASCs in the kidneys as determined by SRY analysis. As expected, untreated kidneys shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen deposition, and decreased staining for FSP-1, type I collagen, and Hypoxyprobe. The renal protection seen in ASC-treated animals was followed by reduced serum levels of TNF-α, KC, RANTES, and IL-1α. Surprisingly, treatment with ASCs at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia, thereby reducing the epithelial–mesenchymal transition.

Mycophenolate Mofetil Reduces Renal Injury in the Chronic Nitric Oxide Synthase Inhibition Model

We and others have recently shown that mycophenolate mofetil (MMF) reduces renal inflammation and glomerular and interstitial injury in the 5/6 renal ablation model. In the present study, we investigated whether MMF limits renal injury in a model of chronic nitric oxide (NO) inhibition associated with a high-salt diet and characterized by progressive systemic hypertension, albuminuria, glomerular sclerosis and ischemia, interstitial expansion, and progressive macrophage infiltration. Adult male Münich-Wistar rats were distributed among 3 groups: HS, rats receiving a high-salt diet (3.2% Na); HS+N, HS rats orally treated with the NO inhibitor N&ohgr;-nitro-l-arginine methyl ester (L-NAME), 25 mg · kg−1 · d−1; and HS+N+MMF, HS+N rats orally treated with MMF, 10 mg · kg−1 · d−1. Renal hemodynamics were studied after 15 days of treatment; histological and immunohistochemical studies were conducted after 30 days of treatment. MMF treatment did not reverse the hemodynamic alterations characteristic of this model. Renal injury in the HS+N group was associated with macrophage and lymphocyte infiltration. Treatment with MMF reduced glomerular and interstitial injury and limited macrophage and lymphocyte infiltration. These results suggest that renal inflammation is a strong independent factor in the pathogenesis of the nephropathy associated with the HS+N model.

Antifibrotic Effect of Tamoxifen in a Model of Progressive Renal Disease

Tamoxifen, a selective estrogen receptor modulator, has antifibrotic properties; however, whether it can attenuate renal fibrosis is unknown. In this study, we tested the effects of tamoxifen in a model of hypertensive nephrosclerosis (chronic inhibition of nitric oxide synthesis with L-NAME). After 30 days, treated rats had significantly lower levels of albuminuria as well as lower histologic scores for glomerulosclerosis and interstitial fibrosis than untreated controls. Tamoxifen was renoprotective despite having no effect on the sustained, severe hypertension induced by L-NAME. Tamoxifen prevented the accumulation of extracellular matrix by decreasing the expression of collagen I, collagen III, and fibronectin mRNA and protein. These renoprotective effects associated with inhibition of TGF-β1 and plasminogen activator inhibitor-1, and with a significant reduction in α-smooth muscle actin-positive cells in the renal interstitium. Furthermore, tamoxifen abrogated IL-1β- and angiotensin-II-induced proliferation of fibroblasts from both kidney explants and from the NRK-49F cell line. Tamoxifen also inhibited the expression of extracellular matrix components and the production and release of TGF-β1 into the supernatant of these cells. In summary, tamoxifen exhibits antifibrotic effects in the L-NAME model of hypertensive nephrosclerosis, likely through the inhibition of TGF-β1, suggesting that it may have therapeutic use in CKD treatment.

Regression of glomerular injury by losartan in experimental diabetic nephropathy.

Many features of chronic kidney disease may be reversed, but it is unclear whether advanced lesions, such as adhesions of sclerotic glomerular tufts to Bowmans capsule (synechiae), can resolve during treatment. We previously showed, using a renal ablation model, that the renoprotective effect of the AT-1 receptor blocker, losartan, is dose-dependent. Here we determined if moderate and advanced glomerular lesions, associated with streptozotocin-induced diabetes, regress with conventional or high-dose losartan treatment. Using daily insulin injection for 10 months, we maintained diabetic adult male Munich-Wistar rats in a state of moderate hyperglycemia. Following this period, some rats continued to receive insulin with or without conventional or high-dose losartan for an additional 2 months. Diabetic rats pretreated with insulin for 10 months and age-matched non-diabetic rats served as controls. Mesangial expansion was found in the control diabetic rats and was exacerbated in those rats maintained on only insulin for an additional 2 months. Conventional and high-dose losartan treatments reduced this mesangial expansion and the severity of synechiae lesions below that found prior to treatment; however, the frequency of the latter was unchanged. There was no dose-response effect of losartan. Our results show that regression of mesangial expansion and contraction of sclerotic lesions is feasible in the treatment of diabetes, but complete resolution of advanced glomerulosclerosis may be hard to achieve.

Deletion of bradykinin B1 receptor reduces renal fibrosis.

The Kallikrein-kinin system works through activation of two receptors. One constitutive, named B2 receptor (B2R) and another inducible, denominated B1 receptor (B1R). In renal fibrosis, B2R receptor activation appears to be protective, however B1R participation is unveiled. The aim of this study was to analyze how the deletion of the B1R would modify tissue responses after unilateral ureteral obstruction (UUO). For that, B1R knockout (B1KO) and wild-type mice (B1B2WT) were subjected to UUO and sacrificed at days 1, 5 and 14. Renal dysfunction was assayed by urine proteinuria/creatinine ratio and percentage of tubulointerstitial fibrosis. Kidneys were harvested at day 5 to analyze anti and pro-inflammatory molecules expression by real-time PCR. We demonstrated that at all time points, B1KO mice presented lower proteinuria/creatinine ratio from bladder urine. B1KO protection was reinforced by its lower tubular interstitial fibrosis percentage at day 14 (B1B2WT: 12.16+/-1.53% vs. B1KO: 6.73+/-1.07%, p<0.02). UUO was able to induce B1R expression and its highest transcription was achieved at day 5. At this day, B1KO had significant lower expression of pro-inflammatory molecules such as TGF-beta, MCP-1, OPN and IL-6 and higher anti-inflammatory components, as IL-10 and HO-1. Herein, we observed that B1R deletion may be an important component in renal fibrosis prevention.

Granulosa cell tumor of the adult testis: report of a case and review of the literature

A 59-year-old man, referred to us with a 2-year historyof a painless growing mass in the left testis without pasthistory of trauma, infection, or lower urinary tract symp-toms. Physical exams revealed a normal right epididymisand testis, and a 13-cm mass and hydrocele in the left tes-tis, without hernia. Ultrasonography showed a 15 x 11 x12 cm mass with solid and cystic components and a largehydrocele. Serum levels of alpha-fetoprotein, beta-hCG,and LDH were within normal ranges. He underwent in-guinal surgical exploration of the left testis in October2000, when a large testicular mass was found. A radicalorchiectomy was performed, and the hydrocele fluid wasanalyzed. No frozen section biopsy was performed. His-topathological examination showed a 9-cm yellow tumor,with a small degenerated cystic area. Final analysis revealeda granulosa cell tumor with oval nuclei having a longitu-dinal nuclear groove (coffee bean appearance – Figure 1),focal invasion of the tunica albuginea and rete testis, nomitotic figures, and no invasion of surgical margins, sper-matic cord, or epididymis. Cytological analysis of the hy-drocele fluid was negative for tumor cells. Immunohisto-chemical tests were positive for vimentin, actin HFN 35,desmin, and cytokeratin AE-1/AE-3, but negative for epi-thelial membrane antigen. These findings strongly suggestthe diagnosis of a granulosa cell tumor.Follow-up was carried out with determinations of se-rum levels of alpha-fetoprotein, beta-hCG, and LDH, aswell as computerized tomography of the abdomen and chestx-ray every 6 months for 4 years, with no clinical recur-rence or exam abnormalities.Described for the first time in 1952,