Andrea Satta

Use of Testosterone To Prevent Cyclophosphamide-Induced Azoospermia

Several cytotoxic agents used to treat neoplastic or immunologic diseases may alter endocrine function in humans [1-5]. In particular, gonadal failure with azoospermia, amenorrhea, or anovulatory cycles has been reported in patients treated for various diseases [1-3]. Cyclophosphamide, an alkylating drug, is widely used as an antineoplastic or immunosuppressive agent. Severe gonadal failure with transient or permanent azoospermia is found in 50% to 90% of men treated with cyclophosphamide [6, 7]. Prepubertal patients who receive large doses of cyclophosphamide seem to recover gonadal function better than adults do; normal pubertal development and normal spermatogenesis have been reported in many of these patients [3, 8-10]. These findings may indicate that active germinal cells are more sensitive to cyclophosphamide because of their elevated mitotic activity. In designing our study, we assumed that the use of testosterone to inhibit germinal cell activity might reduce the sensitivity of these cells to the effects of cyclophosphamide. We administered cyclophosphamide as an immunosuppressive agent to manage glomerulonephritis, either orally or in an intravenous bolus [11, 12]. We therefore could evaluate the effects of the two methods of administration on germinal cell function. Methods We studied 15 men who had the nephrotic syndrome, (age range, 23 to 35 years). Each patient was told of the possible risks of treatment and gave informed consent. Patients were randomly divided into three groups. The five patients in group A (three with membranous nephritis, one with mesangial nephritis, and one with lupus nephritis) were given oral cyclophosphamide at an average dosage of 150 mg daily for 6 to 8 months; the total dose was 27 to 36 g. The five patients in group B (two with membranous nephritis, two with mesangial nephritis, and one with focal glomerulosclerosis) and the five patients in group C (two with membranous nephritis, one with mesangial nephritis, and two with lupus nephritis) received cyclophosphamide as a monthly intravenous bolus, 15 mg/kg of body weight, according to the protocol reported elsewhere [11, 12]. The average duration of treatment was 8 months; the total dose was 10.4 g. Patients in group C also received testosterone, 100 mg intramuscularly every 15 days, in addition to cyclophosphamide. Patients began receiving testosterone (a commercial blend of enanthate and propionate esthers of testosterone) 30 days before starting cyclophosphamide therapy, and they continued to receive it during the course of immunosuppressive therapy. Because ours was a pilot study, the testosterone dose that we used was lower than that currently used to treat hypogonadism; however, it proved to be effective in inhibiting gonadotropin secretion and spermatogenesis [13]. To ensure the possibility of future paternity, sperm samples from each patient were collected before the start of therapy and stored in liquid nitrogen. Additional sperm samples were collected from each patient before therapy, during the third and sixth months of cyclophosphamide therapy, and 3 and 6 months after the end of the therapy. All sperm samples were analyzed in the same laboratory by the same technician. Serum samples for luteinizing hormone and follicle-stimulating hormone assays were collected at the same intervals as the sperm samples. Hormone levels were measured in duplicate by specific immunoradiometric methods using commercial kits (Serono Diagnostic, Milan Italy). Both of the preceding assays are sensitive to about 0.15 IU/L. Levels are reported in IU/L in accordance with the First International Reference Preparation 68/40 and Second International Reference Preparation 78/549 guidelines for reporting luteinizing hormone levels and follicle-stimulating hormone levels, respectively. Statistical analysis was done using the Student t-test. All results are given as the mean SE. Results Under control conditions, all patients had normal sperm counts; the means were 43.70 6.56 106/mL in group A, 45.52 7.04 106/mL in group B, and 42.91 5.27 106/mL in group C. During immunosuppressive therapy, sperm counts abruptly decreased in all patients in groups A and B (Table 1), and all patients were azoospermic 6 months after starting immunosuppressive therapy. Three and 6 months after completing therapy, all patients in group A and four of the five patients in group B were azoospermic. During the third and sixth months of immunosuppressive therapy, three patients in group C had azoospermia or severe oligospermia; sperm counts were lower than 1 106/mL. After therapy was discontinued, sperm counts increased progressively in group C; mean counts were 28.64 3.47 106/mL 3 months after therapy ended and 45.78 3.89 106/mL 6 months after therapy ended. In these patients, mean percentages of sperm motility (88%; range, 63% to 92%) and sperm that showed no structural abnormalities (80%; range, 73% to 89%) were in the normal range. Table 1. Sperm Counts under Control Conditions and during and after Cyclophosphamide Therapy* In groups A and B, serum luteinizing hormone levels did not change throughout the observation period (Table 2) but follicle-stimulating hormone levels progressively increased. The following are the mean follicle-stimulating hormone levels: group A, 8.54 0.69 IU/L and 14.82 2.15 IU/L during the third and sixth months of cyclophosphamide therapy and 18.40 1.79 IU/L and 19.20 1.28 IU/L 3 and 6 months after discontinuation of therapy; group B, 9.30 0.87 IU/L and 18.48 2.81 IU/L during the third and sixth months of cyclophosphamide therapy and 16.80 2.15 IU/L and 16.04 2.22 IU/L 3 and 6 months after discontinuation of therapy. All of these values were significantly higher than those seen under control conditions (P = 0.002). Luteinizing hormone and follicle-stimulating hormone levels decreased in group C during cyclophosphamide therapy because of the inhibitory effect of testosterone (Table 2). In particular, luteinizing hormone levels during the third and sixth months of immunosuppressive therapy were 1.62 0.24 IU/L and 1.06 0.19 IU/L, respectively; follicle-stimulating hormone levels at the same time points were 2.12 0.38 IU/L and 1.56 0.33 IU/L, respectively. The differences with respect to baseline values were statistically significant (P < 0.005). In these patients, luteinizing hormone levels measured 3 and 6 months after discontinuation of therapy were 6.44 0.87 IU/L and 4.80 0.40 IU/L, respectively; follicle-stimulating hormone levels at the same time points were 6.60 0.86 IU/L and 5.08 0.56 IU/L, respectively. Differences between the follicle-stimulating hormone levels in group C and those in groups A and B were statistically significant (P < 0.002). All 15 patients in the study had clinical remission of their underlying diseases. No side effects related to testosterone administration, such as liver enlargement or gynecomastia, were seen in group C. Table 2. Serum Follicle-Stimulating Hormone and Luteinizing Hormone Levels under Control Conditions and during and after Cyclophosphamide Therapy* Discussion Our findings confirm that cyclophosphamide therapy can affect germinal cell function in humans [1-36, 7]. All patients who received daily oral cyclophosphamide and four of five who received a monthly intravenous bolus of cyclophosphamide were azoospermic for as long as 6 months after the end of immunosuppressive therapy. Serum follicle-stimulating hormone levels were elevated in all patients who had become azoospermic, a finding that reflects a severe alteration of the germinal epithelium. In contrast, spermatogenesis returned to normal in the five patients who received testosterone before and during immunosuppressive therapy; follicle-stimulating hormone levels for all patients were within the normal range. This finding indicates that the use of testosterone to inhibit germinal cell activity may protect against the effects of cyclophosphamide. Leydig cell function was not affected in any study patient, as shown by normal levels of luteinizing hormone; previous reports have shown that Leydig cells are more resistant than germinal cells to the action of alkylating agents [6, 10]. Testosterone has been used as a contraceptive in men; it induces azoospermia or severe oligospermia by reducing levels of gonadotropins and intragonadal testosterone [13, 14]. In accordance with this observation, our data show a substantial reduction in gonadotropin levels during testosterone administration. Moreover, other studies [13, 14] have shown that testosterone can be used safely at high doses and for long periods. No data are available on the effects of testosterone in nephrotic patients receiving cyclophosphamide, and previous reports on gonadal protection during therapy with cytotoxic drugs are conflicting. Experimental studies in rats have shown that the use of triptorelin to suppress gonadal function may protect the testes from the adverse effects of cyclophosphamide therapy [15]. In contrast, nafarelin given to dogs was shown to potentiate the damaging effects of the alkylating agents [16]. Oral contraceptives and luteinizing hormone-releasing hormone agonists have also been used in humans in attempts to preserve gonadal function during chemotherapy. However, results of previous studies are conflicting and inconclusive [3, 17-20]. Our data were obtained from a small number of patients observed for only 6 months after the discontinuation of immunosuppressive therapy. However, our findings may indicate a safe way to preserve gonadal function in nephrotic patients who are treated with cyclophosphamide. Dr. Bartoli: Medicina Interna, University of Udine, Piazzale S. Maria della Misericordia 1, 33100 Udine, Italy.

Effects of Add-on Fluvastatin Therapy in Patients with Chronic Proteinuric Nephropathy on Dual Renin-Angiotensin System Blockade: The ESPLANADE Trial

BACKGROUND AND OBJECTIVES This open, prospective, randomized trial aimed to assess the effects of statins in chronic kidney disease patients on optimized antiproteinuric treatment with combined angiotensin-converting enzyme inhibition and angiotensin receptor blockade. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS After 1-month benazepril therapy followed by 1-month benazepril-valsartan combined therapy (run-in), 186 consenting patients with residual proteinuria >0.5 g/24 h were randomized to 6-month benazepril-valsartan therapy alone or combined with fluvastatin. Between-groups changes in proteinuria (primary outcome), serum lipids, and GFR were compared by ANCOVA. Analyses were blinded and by intention to treat. RESULTS During the run-in, proteinuria decreased more on benazepril-valsartan than on benazepril alone. Proteinuria reduction correlated with concomitant reduction in total, LDL, and HDL cholesterol, and apolipoprotein B and apolipoprotein A levels. After randomization, median proteinuria similarly decreased from 1.2 (0.6 to 2.2) to 1.1 (0.5 to 1.7) g/24 h on fluvastatin and from 1.5 (0.8 to 2.7) to 1.0 (0.5 to 2.4) g/24 h on benazapril-valsartan therapy alone. Fluvastatin further reduced total and LDL cholesterol and apolipoprotein B versus benazepril-valsartan alone, but did not affect serum triglycerides and GFR. Treatment was well tolerated. CONCLUSIONS In chronic kidney disease patients with residual proteinuria despite combined angiotensin-converting enzyme inhibitor and angiotensin receptor blockade therapy, add-on fluvastatin does not affect urinary proteins, but further reduces serum lipids and is safe. Whether combined angiotensin-converting enzyme inhibitor, angiotensin receptor blockade, and statin therapy may improve cardiovascular outcomes in this high-risk population is worth investigating.

Immunosuppressive Treatment of Membranoproliferative Glomerulonephritis

The treatment of membranoproliferative glomerulonephritis (MPGN) is considered by most authors as unrewarding, and the disease progresses to end-stage renal disease (ESRD). We studied the effectiveness of a new immunosuppressive (IS) regimen by analyzing the rates of remission, relapse and progression to ESRD in 19 patients with MPGN. The treatment consisted of 4 phases: (1) induction with intravenous boluses of methylprednisolone plus cyclophosphamide (CPM) orally; (2) maintenance with oral prednisone (PDN) in an alternate-day regimen and CPM in a daily oral dose; (3) tapering during which PDN alone was slowly decreased; (4) discontinuation when CPM was omitted and PDN slowly withdrawn according to the steroid withdrawal schedule. At the end of the treatment that lasted on average 10 +/- 1 months, 15 patients remitted, 3 improved and 1 progressed. There were 8 relapses in 6 patients: 4 in 3 patients were treated with repeat cycles and remitted completely. Four patients who had relapsed after 4, 8, 11 and 13 years of remission refused retreatment and progressed rapidly to ESRD. All patients treated and retreated after relapsing had remissions, while renal failure and disease progression occurred in 1 patient only. Plasma creatinine averaged, in the whole group, 165 +/- 26 before, 156 +/- 30 after treatment and 224 +/- 57 microM/l at the end of 7.4 +/- 0.8 years of follow-up. An intensive IS regimen combining steroids and alkylating agents in high doses and for a prolonged time is effective in inducing remission and halting progression to ESRD in patients with MPGN.

Blunting of Furosemide Diuresis by Aspirin in Man

Experiments were performed on humans to study the blunting on the diuretic action of furosemide by prostaglandin synthetase inhibitors. Maximal water diuresis was instituted. At the peak of urine flow, clearance periods were performed during baseline conditions and repeated after the injection of aspirin and, subsequently, of furosemide. Control subjects did not receive aspirin. Urine flow rate (V), Cosm, and Na excretion (UNa) . V were significantly lower when the administration of the diuretic had been preceded by that of aspirin. In the absence of furosemide, however, aspirin did not influence renal hemodynamics nor Na and water reabsorption. Therefore, the same experimental protocol was repeated in paired experiments where each normal subject served as his own control, being studied twice, in the presence and absence of aspirin, respectively. The average changes in water and Na excretion induced by furosemide were not different when the patients were pretreated with aspirin as compared with those measured in the absence of prostaglandin inhibition. Changes occurring in individual experiments were significantly correlated (r = 0.95, P less than 0.01) with those in calculated furosemide clearance. Since aspirin, indomethacin, and meclophenamate are secreted by the organic acid transport system of the proximal tubule, competition for a common secretory mechanism, rather than prostaglandin inhibition, could mediate the blunting of furosemide diuresis.

Interleukin 1, Interleukin 6, Interleukin 10, and Tumor Necrosis Factor α in Active and Quiescent Systemic Lupus Erythematosus

Objectives Several studies have investigated the cytokine profile of patients with systemic lupus erythematosus (SLE); however, their role is still controversial, mostly because SLE has a heterogeneous disease manifestation. We measured 4 of the most important cytokines in patients with SLE after dividing them in uniform groups according to disease activity and organ involvement. Materials and Methods Eighty-two adult female patients with SLE were divided into 3 groups according to disease activity and organ involvement: Group A (SLE activity index [SLEDAI] score, 7 ± 0.4) included subjects with newly diagnosed, active SLE, investigated before starting therapy. Group B (SLEDAI score, < 6) included patients without renal involvement, treated with prednisone and azathioprine or hydroxychloroquine. Group C (SLEDAI score, < 6) included patients with lupus nephritis, treated with methylprednisolone and cyclophosphamide, reaching complete remission. Fourteen healthy females served as controls. Results Interleukin-1 levels were 1.0, 0.8, 0.7, and 0.25 pg/mL in groups A, B, C, and D, respectively. Interleukin-6 levels were 3.2, 3.6, 4.0, and 1.4 pg/mL in groups A, B, C, and D, respectively; Il-10 levels, 3.05, 1.1, 1.5, and 1.65; tumor necrosis factor-α levels, 8.75, 5.8, 5.4, and 3.6. Interleukin 1, IL-6, and tumor necrosis factor-α were significantly higher in the patients with SLE than in the healthy controls; IL-1 was significantly higher in group A than in group C. Interleukin 10 showed positive correlation with C-reactive protein, whereas it showed negative correlation with C3. Conclusions Data from our cohort, one of the largest so far reported, add to the evidence that proinflammatory cytokines such as Interleukin-1, Interleukin-6, Interleukin-10 and tumor necrosis factor-α are important in SLE pathogenesis.

Hormonal Strategies for Fertility Preservation in Patients Receiving Cyclophosphamide to Treat Glomerulonephritis: A Nonrandomized Trial and Review of the Literature

BACKGROUND Prepubertal patients receiving chemotherapy are relatively resistant to cyclophosphamide-induced germinal cell alterations. We studied the possible protective effect of testosterone and triptorelin to inhibit gonadal activity in men and women receiving cyclophosphamide, respectively. STUDY DESIGN Nonrandomized trial. SETTING & PARTICIPANTS 28 consecutive patients, 11 men and 17 women, from a university medical center with various forms of glomerulonephritis, treated with cyclophosphamide. INTERVENTION Men received cyclophosphamide plus testosterone; women were divided into 2 groups: 13 patients (group A) received cyclophosphamide plus triptorelin; 4 (group B) received only cyclophosphamide. OUTCOMES & MEASUREMENTS Serum follicle-stimulating hormone (FSH) and serum luteinizing hormone levels and, in addition, sperm counts and testosterone levels in men and estradiol levels in women were measured before and after treatment with cyclophosphamide. RESULTS All 10 men became azoospermic or severely oligospermic during treatment; after 12 months, all except 1 had a normal sperm count and FSH levels were normal. In women during cyclophosphamide therapy, amenorrhea occurred in all patients. After cessation of therapy, all women in group A started to menstruate regularly, and at the end of follow-up, ovulatory cycles were demonstrated in all women. Hormone levels showed no significant changes throughout the observation period. Six women conceived, and the pregnancies were brought to term successfully without complications. In group B, all 4 women developed sustained amenorrhea; serum FSH and luteinizing hormone levels at the end of therapy and follow-up were significantly higher with respect to baseline; estradiol levels at the end of follow-up were significantly lower compared with baseline and corresponding values in group A. LIMITATIONS The substudy in men is uncontrolled, the substudy in women is nonrandomized. CONCLUSIONS The study suggests a protective effect of testosterone and triptorelin against cyclophosphamide-induced gonadal damage in men and women with various forms of kidney disease, respectively.

Nephrotic syndrome and angiotropic lymphoma report of a case.

A case of angiotropic lymphoma involving renal glomeruli and interstitial vessels associated with nephrotic syndrome and with minor lesions in the glomerular basal membrane is reported. A 56-year-old woman had fever, weakness and clinical findings of a nephrotic syndrome with normal renal function. Renal biopsy revealed that the glomeruli were infiltrated by neoplastic lymphoid cells positive for CD20 and CD45; the glomerular basement membranes showed a pattern of minimal change disease. This case and our review of the literature suggest that the rare association of intravascular lymphoma and glomerular disease is more than coincidental.

Impact of cholesterol lowering treatment on plasma kynurenine and tryptophan concentrations in chronic kidney disease: Relationship with oxidative stress improvement

BACKGROUND AND AIM Tryptophan (Trp) degradation via indoleamine (2,3)-dioxygenase (IDO), with consequent increased in kynurenine (Kyn) concentrations, has been proposed as marker of immune system activation. Oxidative stress (OS) might contribute to the pro-inflammatory state in chronic kidney disease (CKD) through the activation of NF-kB, with consequent activation and recruitment of immune cells. METHODS AND RESULTS Serum concentrations of Trp and Kyn, oxidative stress indices malondialdehyde (MDA) and allantoin/uric acid (All/UA) ratio and anti-oxidant amino acid taurine were measured in 30 CKD patients randomized to 40 mg/day simvastatin (group 1), ezetimibe/simvastatin 10/20 mg/day (group 2) or ezetimibe/simvastatin 10/40 mg/day (group 3) and treated for 12 months. Baseline Kyn and Kyn/Trp ratio were higher in CKD patients vs. healthy controls (1.67 ± 0.62 μmol/L vs 1.25 ± 0.40 μmol/L, p < 0.01 and 0.036 ± 0.016 vs 0.023 ± 0.010, p < 0.001 respectively). Both Kyn and Kyn/Trp ratio significantly decreased after cholesterol lowering treatment, to values comparable with healthy controls after one year treatment (1.67 ± 0.62 μmol/L vs 1.31 ± 0.51 μmol/L, p < 0.0001 and 0.036 ± 0.016 vs 0.028 ± 0.012 p < 0.0001, respectively). This was paralleled by a significant decrease in MDA (218 ± 143 nmol/L vs 176 ± 123 nmol/L, p < 0.01) and All/UA ratio (1.47 ± 0.72 vs 1.19 ± 0.51, p < 0.01) in CKD patients. CONCLUSIONS Amelioration of both oxidative and inflammation status after cholesterol lowering treatment in CKD might be mediated by restoration of antioxidant taurine concentrations during therapy (from 51.1 ± 13.3 μmol/L at baseline to 63.1 ± 16.4 μmol/L, p < 0.001 by ANOVA), suggesting that improvement of both oxidative and inflammation status in CKD patients could be explained, at least partly, by the cholesterol lowering effects.

Quali-quantitative analysis of urinary glycosaminoglycans for monitoring glomerular inflammatory activity.

Objective. A 2-year follow-up study was carried out in patients with IgA nephropathy (IgAN) in order to verify the possible use of quali-quantitative analysis of urinary glycosaminoglycans (GAGs) as a prognostic index of disease and for drug treatment monitoring. Material and methods. Ten patients with IgAN were evaluated at four time points: baseline, and 6, 9 and 24 months later. GAGs were isolated from 24-h urine using ion-exchange chromatography on diethylaminoethyl–Sephacel, and concentrations were expressed as milligrams of hexuronate per gram of creatinine. GAG composition was determined by cellulose acetate electrophoresis and expressed as relative percentages by means of densitometric scanning of Alcian Blue-stained strips. Results. The relative content of total low-sulphated chondroitin sulphate species decreased significantly during the study period compared to baseline, whereas the relative percentages of heparan sulphate and chondroitin sulphate increased significantly. Moreover, a significant correlation was noted between the relative contents of urinary GAGs, renal function and inflammation indexes. Conclusions. It is likely that the excretion of various types of GAGs may be related to different glomerular pathophysiological conditions. Therefore, the determination of urinary GAG composition may represent a reliable indicator of disease activity.

Increased Low-Density Lipoprotein S-Homocysteinylation in Chronic Kidney Disease

Background: Since low-density lipoprotein (LDL) S-homocysteinylation has been recently reported to enhance atherogenicity of lipoprotein, we have investigated the levels of homocysteine (Hcy) linked to LDL in chronic proteinuric patients in which lipid abnormalities highly contribute to the excess of morbidity and mortality. Methods: We used capillary electrophoresis to measure LDL-bound thiol Hcy, cysteine (Cys), cysteinylglycine (Cys-Gly), glutathione (GSH), and glutamylcysteine (Glu-Cys) in 30 chronic kidney disease (CKD) individuals and 60 healthy volunteers. Results: We found more elevated levels of total plasma Hcy, Cys, GSH and Glu-Cys in patients than in controls and also found that Hcy and Cys bound to LDL were significantly increased in nephropathic subjects. By multiple linear regression, we found that in healthy people, total Hcy was the most important determinant of LDL-bound Hcy and Cys-Gly was negatively associated with apoB-Hcy concentrations. In CKD the most important determinant of homocysteinylation was creatinine while total plasma Hcy is weakly associated with apoB-Hcy. Conclusions: The increased levels in Hcy-LDL observed in CKD patients might account, at least in part, for the excess of cardiovascular risk; thus LDL S-homocysteinylation can be considered a key marker of risk for cardiovascular disease in these individuals.