Jorge E. Toblli

Iron management in chronic kidney disease: conclusions from a “Kidney Disease: Improving Global Outcomes” (KDIGO) Controversies Conference

Before the introduction of erythropoiesis-stimulating agents (ESAs) in 1989, repeated transfusions given to patients with end-stage renal disease caused iron overload, and the need for supplemental iron was rare. However, with the widespread introduction of ESAs, it was recognized that supplemental iron was necessary to optimize hemoglobin response and allow reduction of the ESA dose for economic reasons and recent concerns about ESA safety. Iron supplementation was also found to be more efficacious via intravenous compared to oral administration, and the use of intravenous iron has escalated in recent years. The safety of various iron compounds has been of theoretical concern due to their potential to induce iron overload, oxidative stress, hypersensitivity reactions, and a permissive environment for infectious processes. Therefore, an expert group was convened to assess the benefits and risks of parenteral iron, and to provide strategies for its optimal use while mitigating the risk for acute reactions and other adverse effects.

Morphological changes in cavernous tissue in spontaneously hypertensive rats

Erectile dysfunction has an increased prevalence in hypertensive patients and is associated with cardiovascular diseases. For many years the discussion has been polarized on whether in hypertensive patients, it is the arterial hypertension or the antihypertensive therapy that is the cause of male erectile dysfunction. The aim of our study was to determine the morphologic changes in cavernous tissue (CT) in an animal model of arterial hypertension. Male spontaneously hypertensive rats (SHR) (n = 15) and normotensive Wistar-Kyoto (WKY) rats (n = 15) were studied for 8 months. Animals were allowed to drink tap water and fed a standard rat chow ad libitum. Systolic blood pressure (SBP) was measured monthly by the tail/cuff method. At the end of the experiment all the animals were sacrificed for microscopic studies. Cavernous tissue was processed by hematoxylin and eosin, Massons trichrome, and monoclonal anti-alpha smooth muscle actin. Cavernous smooth muscle (CSM) and vascular smooth muscle (VSM) proliferation and CT fibrosis were evaluated by a semiquantitative score. SHR showed a higher proliferative score in CSM (2.7 +/- 0.28 v 1.1 +/- 0.07; P < .001), as well as in VSM (2.7 +/- 0.25 v 1 +/- 0.05; P < .001), and higher CT fibrosis score (2.8 +/- 0.28 v 0.1 +/- 0.07; P < .001), when compared to WKY rats. Furthermore, SHR showed a positive correlation between SBP and CSM proliferative score (r2 = 0.9277), SBP and VSM proliferative score (r2 = 0.8828), and SBP and CT fibrosis score (r2 = 0.7775). In addition, an increase in the surrounding connective tissue at the perineurium and endoneurium of the amielinic nerves in CT was observed in the SHR group. According to these results we conclude that SHR present morphologic changes in vessels as well as in cavernous spaces of the erectile tissue that have a high positive correlation with high blood pressure. Moreover, the increase in extracellular matrix expansion seems to affect not only the interstitium but also the neural structures of the penis.

ACE Inhibition and AT1 Receptor Blockade Prevent Fatty Liver and Fibrosis in Obese Zucker Rats

Objective: Non‐alcoholic steatohepatitis (NASH), which is a common liver disease in industrialized countries, is associated with obesity, hypertension, and type‐2 diabetes (metabolic syndrome). Since angiotensin II (ANG II) has been suggested to play an important role in liver inflammation and fibrosis, the purpose of this study was to investigate whether therapy against renin‐angiotensin system (RAS) may provide some beneficial effect in liver of an animal model of metabolic syndrome.

Effects of angiotensin II subtype 1 receptor blockade by losartan on tubulointerstitial lesions caused by hyperoxaluria.

PURPOSE Hyperoxaluria is a recognized cause of tubulointerstitial lesions and this circumstance could contribute to cause chronic renal disease. The renin-angiotensin system has a critical role in the development of interstitial fibrosis, mostly by angiotensin II type 1 receptor stimulation of pro-fibrotic mechanisms. We evaluated whether angiotensin II type 1 receptor blockade prevents oxalate renal lesions. MATERIALS AND METHODS We divided 2-month-old male Sprague-Dawley rats into 4 groups, namely group 1-control, group 2-hyperoxaluria, group 3-hyperoxaluria plus losartan and group 4-losartan. For 4 weeks groups 2 and 3 received 1% ethylene glycol (precursor for oxalates) in drinking water. Losartan (40 mg./kg. body weight) was administered in groups 3 and 4 daily. At the end of the study renal lesions were evaluated using anti-alpha-smooth muscle actin, anti-collagen type III, anti-monocytes/macrophages and anti-transforming growth factor-beta1 antibodies. To evaluate oxidative stress in renal tissue total glutathione and thiobarbituric acid reactive substances in kidney homogenates were determined. Regarding renal functional parameters, creatinine clearance and urinary albumin excretion were also studied. RESULTS Despite similar urinary oxalate levels compared with group 2 group 3 rats showed fewer tubulointerstitial lesions, consisting of significant lower scores for tubular atrophy, unspecific inflammatory cell infiltrate, ED1 mouse anti-rat monoclonal antibody (Serotec, Ltd., Oxford, United Kingdom) (monocytes/macrophages), crystal deposits, interstitial fibrosis, alpha-smooth muscle actin, collagen type III and tubulointerstitial transforming growth factor-beta1. Moreover, urinary albumin excretion and creatinine clearance were significantly improved in group 3 (p <0.01). Higher total glutathione and lower thiobarbituric acid reactive substances were also observed in this group (p <0.01). Thiobarbituric acid reactive substances were the most important and significant independent variable correlating with interstitial fibrosis (t ratio 4.867, p <0.04). CONCLUSIONS We believe that the renal-angiotensin system interaction by losartan produces a beneficial effect against renal lesions caused by hyperoxaluria through a number of actions, including a reduction in crystal formation in the tubular fluid, inflammatory reaction control and interaction with oxidative stress. These factors lead concurrently to preserve tubular epithelial cell and renal interstitium integrity. In addition, these results suggest that the principal mechanism of action should be mediated by angiotensin II type 1 receptors.

Angiotensin-(1–7) reduces proteinuria and diminishes structural damage in renal tissue of stroke-prone spontaneously hypertensive rats

Angiotensin (ANG)-(1-7) constitutes an important functional end-product of the renin-angiotensin-aldosterone system that acts to balance the physiological actions of ANG II. In the kidney, ANG-(1-7) exerts beneficial effects by inhibiting growth-promoting pathways and reducing proteinuria. We examined whether a 2-wk treatment with a daily dose of ANG-(1-7) (0.6 mg·kg(-1)·day(-1)) exerts renoprotective effects in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). Body weight, glycemia, triglyceridemia, cholesterolemia, as well as plasma levels of Na+ and K+ were determined both at the beginning and at the end of the treatment. Also, the weekly evolution of arterial blood pressure, proteinuria, and creatinine clearance was evaluated. Renal fibrosis was determined by Massons trichrome staining. Interleukin (IL)-6, tumor necrosis factor (TNF)-α, and nuclear factor-κB (NF-κB) levels were determined by immunohistochemistry and confirmed by Western blotting analysis. The levels of glomerular nephrin were assessed by immunofluorescence. Chronic administration of ANG-(1-7) normalized arterial pressure, reduced glycemia and triglyceridemia, improved proteinuria, and ameliorated structural alterations in the kidney of SHRSP as shown by a restoration of glomerular nephrin levels as detected by immunofluorescence. These results were accompanied with a decrease in both the immunostaining and abundance of IL-6, TNF-α, and NF-κB. In this context, the current study provides strong evidence for a protective role of ANG-(1-7) in the kidney.

Angiotensin-(1–7) attenuates diabetic nephropathy in Zucker diabetic fatty rats

Angiotensin (ANG)-(1-7) is known to attenuate diabetic nephropathy; however, its role in the modulation of renal inflammation and oxidative stress in type 2 diabetes is poorly understood. Thus in the present study we evaluated the renal effects of a chronic ANG-(1-7) treatment in Zucker diabetic fatty rats (ZDF), an animal model of type 2 diabetes and nephropathy. Sixteen-week-old male ZDF and their respective controls [lean Zucker rats (LZR)] were used for this study. The protocol involved three groups: 1) LZR + saline, 2) ZDF + saline, and 3) ZDF + ANG-(1-7). For 2 wk, animals were implanted with subcutaneous osmotic pumps that delivered either saline or ANG-(1-7) (100 ng·kg(-1)·min(-1)) (n = 4). Renal fibrosis and tissue parameters of oxidative stress were determined. Also, renal levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), ED-1, hypoxia-inducible factor-1α (HIF-1α), and neutrophil gelatinase-associated lipocalin (NGAL) were determined by immunohistochemistry and immunoblotting. ANG-(1-7) induced a reduction in triglyceridemia, proteinuria, and systolic blood pressure (SBP) together with a restoration of creatinine clearance in ZDF. Additionally, ANG-(1-7) reduced renal fibrosis, decreased thiobarbituric acid-reactive substances, and restored the activity of both renal superoxide dismutase and catalase in ZDF. This attenuation of renal oxidative stress proceeded with decreased renal immunostaining of IL-6, TNF-α, ED-1, HIF-1α, and NGAL to values similar to those displayed by LZR. Angiotensin-converting enzyme type 2 (ACE2) and ANG II levels remained unchanged after treatment with ANG-(1-7). Chronic ANG-(1-7) treatment exerts a renoprotective effect in ZDF associated with a reduction of SBP, oxidative stress, and inflammatory markers. Thus ANG-(1-7) emerges as a novel target for treatment of diabetic nephropathy.

Comparison of the renal, cardiovascular and hepatic toxicity data of original intravenous iron compounds

BACKGROUND Intravenous (i.v.) iron is essential for managing haemoglobin levels in haemodialysis patients. However, i.v. iron may cause variable degrees of toxicity. This is mainly related to the pharmacological characteristics of any given i.v. iron compound. METHODS This blinded study examines the effects of five i.v. iron preparations on haemodynamic and functional parameters. Sixty Sprague-Dawley rats (n = 10/group) received high or low molecular weight (HMW/LMW) iron dextran, ferric gluconate (FG), ferric carboxymaltose (FCM), iron sucrose (ISC) or isotonic saline solution (control). Five i.v. doses of iron (40 mg iron/kg) or saline were administered over 4 weeks. RESULTS Systolic blood pressure was significantly reduced in the LMW dextran group, whereas serum iron and percentage transferrin saturation were significantly elevated in all treatment groups. Creatinine clearance was reduced and urinary protein excretion increased in the FG group only (P < 0.01). Liver enzyme levels in the blood were increased (P < 0.01) in the FG and two dextran groups compared with the FCM and ISC groups. Analysis of liver, heart and kidney homogenates showed a significant increase in catalase and malondialdehyde levels in the FG group, and an increase in CuZn-superoxide dismutase and glutathione (GSH) peroxidase activity accompanied with a decrease in the reduced-to-oxidized GSH ratio in the FG and two dextran groups (P < 0.01). Tumour necrosis factor alpha and interleukin-6 levels were significantly elevated in liver, heart and kidney samples from the FG and two dextran groups but not the FCM, ISC or control groups. CONCLUSIONS These findings indicate that FG and HMW/LMW iron dextran have less favourable safety profiles than FCM and ISC in normal rats.

Angiotensin-(1-7) improves cardiac remodeling and inhibits growth-promoting pathways in the heart of fructose-fed rats.

The present study examined whether chronic treatment with angiotensin (ANG)-(1-7) reduces cardiac remodeling and inhibits growth-promoting signaling pathways in the heart of fructose-fed rats (FFR), an animal model of insulin resistance. Sprague-Dawley rats were fed either normal rat chow (control) or the same diet plus 10% fructose in drinking water. For the last 2 wk of a 6-wk period of the corresponding diet, control and FFR were implanted with osmotic pumps that delivered ANG-(1-7) (100 ng.kg(-1).min(-1)). A subgroup of each group of animals (control or FFR) underwent a sham surgery. We determined heart weight, myocyte diameter, interstitial fibrosis, and perivascular collagen type III deposition as well as the phosphorylation degree of ERK1/2, JNK1/2, and p38MAPK. FFR showed a mild hypertension that was significantly reduced after ANG-(1-7) treatment. Also, FFR displayed higher ANG II circulating and local levels in the heart that remained unaltered after chronic ANG-(1-7) infusion. An increased heart-to-body weight ratio, myocyte diameter, as well as left ventricular fibrosis and perivascular collagen type III deposition were detected in the heart of FFR. Interestingly, significant improvements in these cardiac alterations were obtained after ANG-(1-7) treatment. Finally, FFR that received ANG-(1-7) chronically displayed significantly lower phosphorylation levels of ERK1/2, JNK1/2, and p38MAPK. The beneficial effects obtained by ANG-(1-7) were associated with normal values of Src-homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity in the heart. In conclusion, chronic ANG-(1-7) treatment ameliorated cardiac hypertrophy and fibrosis and attenuated the growth-promoting pathways in the heart. These findings show an important protective role of ANG-(1-7) in the heart of insulin-resistant rats.

The effects of angiotensin-converting-enzyme inhibitors on the fibrous envelope around mammary implants.

Background: Late capsular contraction around breast implants is one of the most difficult complications to prevent or resolve. The authors studied the mechanisms that control the fibrotic process in an animal model. Using angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonist, the authors previously described a significant reduction in fibrosis in different experimental models. Methods: Four groups of six rats each had a mini breast implant, 12 with a smooth surface and 12 with a textured surface. In two groups, the angiotensin-converting enzyme inhibitor enalapril was administered in drinking water, ad libitum, to determine its effect on both implant types. Two control groups were given plain drinking water. Three months postoperatively, all of the rats were killed and the capsule sections were cut and stained with hematoxylin and eosin and Masson’s trichrome. Immunolabeling of collagen III and transforming growth factor (TGF)-&bgr;1 was performed using monoclonal antibodies. Results: Significant differences were found between smooth and textured implants, with a uniformly low inflammatory response found on textured implants. For both surfaces, the enalapril-treated group had a significant reduction of the inflammatory process that was especially marked in the textured implants. Immunostaining for collagen III and TGF-&bgr;1 showed a consistent reduction in both fibrous tissue and cytokine mediator. Conclusions: Enalapril lowers the expression of fibrotic mediators, TGF-&bgr;1, inflammatory markers, anti-ED1, anti–collagen III monoclonals, and the periprosthetic fibrosis process. The reduction of TGF-&bgr;1 indicates that the probable main cytokine mediator of the fibrotic cascade is attenuated. This hypothesis may provide the basis for a safe and cheap therapeutic strategy with which to modify the capsular contracture that sometimes affects women with mammary implants.

Antifibrotic effects of pioglitazone on the kidney in a rat model of type 2 diabetes mellitus.

BACKGROUND Recent evidence suggests that treatment of type 2 diabetes with thiazolidinediones [peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists], ameliorates glomerulosclerosis and tubulointerstitial fibrosis in the rat kidney. In the current work, we have investigated whether these drugs, and specifically pioglitazone (PGT), act by preventing fibrosis and kidney dysfunction mainly through antioxidant and anti-inflammatory effects, independently of glycaemic control. METHODS Male 2- to 3-month-old obese Zucker fa/fa (OZR) and ZDF fa/fa rats (ZDFR), and their control the lean Zucker rat (LZR), were used. Diabetic rats were given either a low dose (0.6 mg/kg/day) or a high dose (12 mg/ kg/day) of PGT in the chow for 2 or 4-5 months. Glycaemia, blood pressure, creatinine clearance and proteinuria were determined, and the underlying histopathology was defined with markers of fibrosis, glomerular damage, oxidative stress and inflammation by immunohistochemistry/ quantitative image analysis in tissue sections, and western blots and ad hoc assays in fresh tissue. RESULTS PGT at low doses given for 4-5 months considerably reduced blood pressure, proteinuria and creatinine clearance. This was associated with amelioration of renal tissue damage and fibrosis, evidenced by the glomerulosclerosis, tubulointerstitial fibrosis, tubular atrophy and podocyte injury indexes, and of oxidative stress and inflammation, as shown by the decrease in the respective markers, although glycaemia remained high and obesity was not affected. CONCLUSIONS These results indicate that low doses of PGT ameliorate renal fibrosis and preserve renal function in this animal model of metabolic syndrome, independently of glycaemic control or effects on body weight.