Massimino Senatore

Effect of fluvastatin on proteinuria in patients with immunoglobulin A nephropathy

3‐Hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors are established drugs for the treatment of hypercholesterolemia, but several studies have shown that benefits obtained with these drugs are not causally related only to regression of cholesterol lowering. Moreover, in experimental models of progressive renal disease, statins have reduced the extent of glomerulosclerosis. This study evaluated the antiproteinuric effect of a daily dose of 40 mg fluvastatin for 6 months in moderately proteinuric patients with immunoglobulin A nephropathy, stable renal function, and no indicators of poor long‐term prognosis. The effects of therapy were evaluated on the basis of 24‐hour proteinuria (total proteinuria and albuminuria), albuminemia, creatinine clearance, cholesterol, and triglyceride values. Renal function remained stable in all patients. A significant decrease in proteinuria was observed after 6 months of therapy and persisted for all the observations. An increase in serum albumin was observed after 6 months of therapy. This study suggests that there is an antiproteinuric effect of HMG‐CoA reductase inhibitors in moderately proteinuric patients with immunoglobulin A nephropathy. (Clin Pharmacol Ther 2000;67:427‐31.)

Pro-apoptotic effect of fluvastatin on human smooth muscle cells.

The antiatherosclerotic effect of statins has been attributed to their hypocholesterolemic action. We therefore evaluated the effect, in vitro, of the addition of the serum of patients taking fluvastatin on human smooth muscle cells in order to ascertain the effect of the drug on cell proliferation and apoptosis. We found that the addition of serum from patients treated with fluvastatin for 6 days caused a significant reduction in cell proliferation, increased cell apoptosis and reduced the B cell leukemia-2 (bcl-2) concentration. It is concluded that the induction of apoptosis by statins could be a supplementary mechanism in the prevention of atherosclerotic lesions in humans.

Effects of homocysteine on proliferation, necrosis, and apoptosis of vascular smooth muscle cells in culture and influence of folic acid.

BACKGROUND It is known that hyperhomocysteinemia is associated with an increased risk of vascular disease, yet little is known about the pathogenic mechanisms underlying the action of homocysteine (Hcy) itself. METHODS We evaluated the effects of Hcy on cell proliferation, apoptosis, and necrosis in smooth muscle cells (SMCs) cultured for 24 h with different amounts of Hcy. The percentage of apoptotic and necrotic cells from the culture was evaluated using two different techniques: annexin V-FITC and propidium iodide (PI) fluorescence and apoptosis TUNEL assay. RESULTS The addition of 10 microM/l of Hcy to the medium was followed by a significant increase in cell proliferation and death, through apoptosis and necrosis, respectively. Notwithstanding this apparent balance, a significant increase was found in the total number of cells present in Hcy-treated culture, thus demonstrating a positive dose-dependent correlation with Hcy concentrations in the culture medium. The addition of folic acid to the culture medium significantly reduced both Hcy concentrations in media and the effects of Hcy on the proliferation/apoptosis/necrosis balance of cells in culture. The percentages for apoptotic cells and for cells with a necrotic morphology continued to increase as Hcy concentrations increased, although the absolute values were lower in the culture treated than in that not treated with folic acid. CONCLUSIONS In the presence of folic acid, at increasing concentrations of Hcy, the total number of cells in culture showed increases far less relevant with respect to the control. Also the percentage of apoptotic cells to that of cells with a necrotic morphology, although conserving the tendency to increase to growth of the concentrations of Hcy, have shown absolute values that were lower in the folic acid-treated cultures.

Cell Proliferation/Cell Death Balance in Renal Cell Cultures after Exposure to a Static Magnetic Field

The effect of a static magnetic field (MF) of 0.5 mT of intensity on the cell proliferation/cell death balance was investigated in renal cells (VERO) and cortical astrocyte cultures from rats. Magnetic stimulation was delivered by magnetic disks at known intensities. The percentage of apoptotic and necrotic cells was evaluated using flow cytometry and morphological analysis following Hoechst chromatin staining. An index of cell proliferation was determined using sulfonated tetrazolium (WST-1). Control cultures were prepared without exposure to MFs. After 2, 4 and 6 days of exposure to a MF, we observed a gradual decrease in apoptosis and proliferation and a gradual increase in cells with a necrotic morphology with respect to the control group. In astrocyte cultures, over a 6-day exposure period. A gradual increase was observed in apoptotic, proliferating, and necrotic cells. Our findings suggest that the effect of exposure to MFs varies, depending on the cell type; MFs may also have a nephropathogenic effect.

Water Immersion Increases Urinary Excretion of Aquaporin-2 in Healthy Humans

Many previous studies have shown that aquaporin-2 (AQP2), the vasopressin-regulated water channel, is excreted in the urine and that the excretion increases in response to vasopressin. Moreover, recently a close correlation between AQP2 excretion in urine and kidney AQP2 expression has been demonstrated, showing that urinary excretion of AQP2 is a reliable indicator for AQP-2 function. As head-out water immersion causes an expansion in the central vascular volume equal to that induced by 2 liters of saline, without modifying plasma composition, we used immersion in water to evaluate if the response to acute expansion of the central vascular volume could involve vasporessin (AVP) and AQP2. In healthy subjects, concentrations of plasma atrial natriuretic factor (ANF) and AVP, and urinary AQP2 were measured during a 2-hour immersion period. In all subjects, immersion caused a prompt and marked increase in immunoreactive ANF (23.0 ± 2.12 pg/ml at second hour vs. 2.17 ± 0.42 pg/ml at baseline) and in urinary excretion of AQP2 (23.9 ± 2.69 pmol/mg creatinine at second hour vs. 4.42 ± 0.14 pmol/mg creatinine at baseline), while a significant decrease was found in plasma AVP. Recovery was associated with a prompt return to pre-study levels. These findings demonstrate that heat-out water immersion stimulates urinary excretion of AQP2 in absence of an increase in plasma AVP.

Are There Potential Non-Lipid-Lowering Uses of Statins in the Kidney?

Numerous recent clinical and experimental studies have suggested that hyperlipidemia and atherogenic lipoproteins are involved in the onset and progression of atherosclerotic disease. These lipidic and lipoproteic abnormalities are involved in the physiopathologic mechanisms that accelerate the progression of renal failure [1]. With this observation as a starting point, in many experimental models of progressive renal damage, the inhibitors of the 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase have been found to reduce glomerulosclerosis [2]. HMG-CoA reductase, the main limiting enzyme in the biosynthesis of cholesterol [3], converts HMG-CoA into mevalonate, which is an early precursor of cholesterol, and the precursor of nonsteroidal complexes essential to cellular activity. In 1976, an antibiotic isolated from Phytium ultimum seemed to inhibit cholesterol synthesis [4]. Mevastatin and compactin were isolated from Penicillium citrium [5] and Penicillium brevicompactum, respectively [6]. A series of cholesterol-lowering drugs that are all competitive inhibitors of HMG-CoA reductase has appeared since this original discovery. Lovastatin and simvastatin are lactones and are enzymatically hydrolyzed in the liver into the active form, an open-ring hydroxy acid [5, 7]. Pravastatin and fluvastatin are already in the active open-ring configuration. Fluvastatin is the first fully synthetic compound and is different from other compounds in its lipophilic moiety [8]. Recently the family of lipophilic statins has further grown with the arrival of two new molecules: cerivastatina and atorvastatina. Although the exact mechanism by which statins improve experimental renal damage is unknown, recent studies suggest that HMG-CoA reductase inhibitors may have a direct effect on the mechanisms underlying progressive renal failure [9, 10]. Possible ‘key features’ underlying the protective activity of statins, independent of their hypocholesterolizing activity, are: the cellular proliferation/apoptosis balance; the production of inflammatory cytokines, and the pathways of transduction of intracellular signals.

Reduced bcl-2 concentrations in hypertensive patients after lisinopril or nifedipine administration

In 30 patients with essential hypertension and 30 healthy control subjects, we evaluated blood concentrations of B cell leukemia-2 (bcl-2), a protooncogene that can reduce apoptosis. Bcl-2 concentrations were higher in hypertensive than in normotensive subjects. The increase in pressure due to a cold pressor test caused a further increase in blood bcl-2 concentrations, in both hypertensive and normotensive subjects. Treatment of hypertensive patients with hypotensive drugs caused a reduction in bcl-2 concentrations, which was more marked after administration of lisinopril than of nifedipine. The results suggest that concentrations of bcl-2 are increased in patients with hypertension, which could be an important factor in cell proliferation underlying posthypertensive vascular remodeling. Moreover, lisinopril and nifedipine appear to be capable of reducing bcl-2 concentrations, with potentially beneficial effects on vascular modifications in patients with hypertension.

HMG-CoA reductase inhibitors and apoptosis.

Accessible online at: http://BioMedNet.com/karger Dear Sir, Recent studies have postulated that glomerulosclerosis is induced by hyperlipidemia and this is in common with atherosclerosis. Monocyte infiltration, mesangial cell proliferation and mesangial matrix expansion have been shown to be early events not only in the process of glomerulosclerosis but also in all mesangial proliferation-characterized processes; the recent opinion is that these processes can be lessened by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibition. HMG-CoA reductase is a major rate-limiting enzyme in cholesterol biosynthesis. It converts HMG-CoA to mevalonate, an early precursor of cholesterol. Mevalonate represents the precursor of essential nonsteroidal complexes for main cellular activity. Latest experimental data suggest that the inhibitors of HMG-CoA reductase may have glomerular and mesangial protective effects independent of a reduction in circulating lipids. We now know that lovastatin in vitro downregulates mesangial production of the monocyte chemoattractant protein-1, macrophage colony-stimulating factor, macrophage inhibitory factor, platelet-derived growth factor, endothelin, and angiotensin II [1–3]. But does HMG-CoA reductase inhibitor have a pro-apoptotic effect? We conducted a study on 12 patients (6 males, 6 females; mean age 25 B 4) who were not hypercholesterolemic and were administered 40 mg fluvastatin a day for 6 days. Blood samples were collected before beginning the protocol and 0, 0.5, 1 and 6 h after the administration of the last dose of fluvastatin. We thus evaluated the effects of the addition of wholeblood serum on both the proliferation and the appearance of apoptosis in VERO monkey kidney cell culture. The VERO cells were cultured in RPMI with 10% FCS, 5 mM Lglutamine, 100 U/ml penicillin and 100 Ìg/ ml streptomycin incubated at 5% CO2. The study was made on glass plates from 24 wells with inoculation of 3 ! 10 cells in 1 ml of culture medium. Burker’s counting cell was used to evaluate cellular proliferation, while the acridine Orange technique was used to study the appearance of apoptosis. Apoptosis and cell proliferation were evaluated 24, 48 and 72 h after the addition of serum from whole blood at 1.5 and 15%. In the liquid contained in the wells, we also measured the concentrations of B-cell leukemia-2 (Bcl-2) (Bcl-2 ELISA Oncogene Research Products, Cambridge, Mass., USA), a protooncogene that can reduce apoptosis. Each experiment was repeated at least 3 times. Results are expressed as mean B SEM. Statistical analysis was performed by one-way ANOVA followed by Turkey’s test (fig. 1). Fig. 1. Effect of sera from fluvastatin-treated people on proliferation and apoptosis of VERO monkey kidney cell culture and Bcl-2 concentrations. Target cells were incubated for 72 h in a medium containing 15% whole blood sera from fluvastatin-treated patients. Abscissa is time of sera collection after last dose. Means and SEMs of 12 experiments each run in triplicate, with different sera. The mean value of control (100%) for cell number, after subtracting t = 0, was 238 ! 105 cells plate and 3.5% for apoptosis. * p ! 0.05 vs. t 0. 0 0 30 ] ] ] ] ] ]

Is the Bioreactivity of Vitamin-E-Modified Dialyzer an Expression of Increased Plasmatic Vitamin E Concentration?

The present study was designed to test the biocompatibility of a new vitamin E-modified multilayer membrane compared with highly biocompatible polysulphone dialyzer and acrylonitrile dialyzer. Thirty patients (mean age 53.2 ± 15.3 SD years; dialytic age 36 ± 5.6 months) were selected for the study. The study was divided into three periods of 6 months (phases A, B and C). In the first phase (from Jan. 1999 to June 1999) patients undergoing maintenance bicarbonate dialysis were randomly divided into three filter groups composed, respectively, of 10 patients: acrylonitrile group, polysulphone group and vitamin E-coated dialyzer group. In the phase B (from July 1999 to Dec. 1999) and in the phase C (from Jan. 2000 to June 2000), all three groups changed their own dialysis membranes. Vitamin E-coated dialyzer causes significant decreases in β2-microglobulin, ferritin and immunoglobulin G, a normalization of complement C3 and an increase of plasmatic vitamin E compared to other filters. In the VE group homocysteine decreases but not in a significant manner. In addition, this dialyzer seems not to influence lipid pattern and protein-energy malnutrition parameters. These results clearly show a positive effect of this new filter in influencing different biochemical parameters, perhaps saving vitamin E and reducing polymorphonuclear cell activation.

Immune dysfunction and cytokine production in hemodialysis. Could they be lessened by vitamin E-coated dialyzer membrane?

Most uremic patients in dialysis therapy develop an immunodeficiency characterized clinically by frequent infectious complications and impaired response to vaccinations [1]. This became evident in the early period of hemodialysis treatment when endemic outbreaks of hepatitis B threatened dialysis centers [2]. Vaccination results against hepatitis B, in dialysis patients, are disappointing: low results are also documented for most other types of vaccinations in these patients, such as influenza [3], tetanus [4] or diphteria [5]. There is an only exception in using vaccination containing polysaccharide antigens like pneumococcus [6]. Polysaccharide antigens are recognized by B cells on their own, but for recognition of other antigens, B cells need specific T-cell help: these observations indicate that the major defects appear to be in cellmediated immunity [7–9] and in particular correlated to T-cell rather than B-cell function [10]. In normal conditions, in response to antigens, naive CD4+ T cells differentiate into effector T helper (Th) cells. Based on their pattern of cytokine production and their functional responses, Th cells can be subdivided into those that participate in cell-mediated immune responses such as delayed type of hypersensitivity reactions and macrophage activation (Th1 subset) and those releasing cytokines that induce B cells to secrete antibodies (Th2 subset). Several factors are known to polarize the differentiation of Th cells into either Th1 or Th2 direction, including the co-stimulatory action of antigen presenting cells (APCs), the cytokine environment, altered peptide ligands and the antigen dose. In particular, Th1 are characterized by elevated secretion of interleukin (IL)-2, transforming growth factor-s (TGF-s) and interferon-A (IFN-A), thereby activating cytotoxic T lymphocytes and macrophages. Moreover, IFN-A suppresses Th2 which induces humoral immunity. The Th2 subset of CD4+ T cells preferentially secretes IL-4, IL-5, IL-6 and IL-10 [11– 13], stimulating the proliferation of mast cells and eosinophils as well as the production of immunoglobulins and IL-10 which may suppress the Th1-induced cell-mediated immunity [14, 15]. In general, Th1 cytokines promote Th1 and inhibit Th2 activities and vice versa. Th1 and Th2 responses should, therefore, be stable [16]. Chronic renal failure induces a clinical state of immunodeficiency in most patients: immune defects may be caused by uraemic state itself and by a direct consequence of dialytic therapy [17, 18]. Dialysis has been associated with acute changes in the complement activation status, granulocyte markers, macrophage function, T-cell activation and the release of various pro-inflammatory cytokines. Although the physiolog-