Tacrolimus Therapeutic Drug Monitoring in Stable Kidney Transplantation and Individuation of CYP3A5 Genotype.

The posology of tacrolimus (TAC) is usually guided by its therapeutic drug monitoring. Some patients reach target concentrations (CTs) quickly, others more slowly. In a retrospective study, 20 kidney transplant recipients were included (mean age, 50.7 ± 14.1 years; weight 64.0 ± 14.2 kg; patients clinically stable for over a year). We studied cytochrome CYP3A5 genotype, in particular CYP3A5 6986A>G, the most important polymorphism related to the metabolism of TAC (wild genotype CYP3A5 *1 genotype, and CYP3A5 *3 variants). One year after transplantation, the CTs were 5.0 to 8.0 ng/mL. The patients were divided into group A (TAC doses < 6.0 mg/d) and group B (TAC doses > 6.0 mg/d). All were tested for the CYP3A5 gene sequence to characterize their polymorphism. Patients with CYP3A5 *1/*1 and *1/*3 were extensive metabolizers, and those with CYP3A5 *3/*3 were poor metabolizers. In group A and group B, the average TAC doses at the time of therapeutic drug monitoring were 3.0 ± 1.4 ng/mL (0.05 ± 0.03 mg/kg) and 12.8 ± 3.7 ng/mL (0.2 ± 0.1 mg/kg), respectively (P < .001). Group A was the poor metabolizers genotype, while in group B, the extensive metabolizers genotype was present. Patients with the CYP3A5 *1/*1 or *1/*3 genotype required 1.5 to 2 times higher doses than patients *3/*3 to reach CT. This genetic test allows clinicians to know, before the kidney transplant, the patient's TAC metabolism pattern and then to optimize the drug exposure.

Racemic synthesis and solid phase peptide synthesis application of the chimeric valine/leucine derivative 2-amino-3,3,4-trimethyl-pentanoic acid.

The synthesis of non natural amino acid 2-amino-3,3,4-trimethyl-pentanoic acid (Ipv) ready for solid phase peptide synthesis has been developed. Copper (I) chloride Michael addition, followed by a Curtius rearrangement are the key steps for the lpv synthesis. The racemic valine/leucine chimeric amino acid was then successfully inserted in position 5 of neuropeptide S (NPS) and the diastereomeric mixture separated by reverse phase HPLC. The two diastereomeric NPS derivatives were tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPS receptor where they behaved as partial agonist and pure antagonist.

Chronic periaortitis associated with membranous nephropathy: clues to common pathogenetic mechanisms.

Chronic periaortitis (CP) is a rare disease hallmarked by the presence of a periaortic retroperitoneal fibro-inflammatory tissue which can often cause obstructive uropathy. CP is isolated in most cases but it may also be associated with other sclerosing inflammatory and immune-mediated diseases. We here present the case of a patient who was initially diagnosed as having CP and subsequently developed membranous nephropathy and chronic sclerosing sialoadenitis of the right parotid gland. As these conditions were all characterized by either pronounced infiltration of IgG4-positive plasma cells or marked IgG4 tissue deposition, we hypothesize that they are part of the same disease spectrum, and discuss the immune-mediated pathogenetic mechanisms potentially shared by these conditions. In particular, we consider the role of Th2-mediated immune reactions and of immunogenetic factors such as HLA genotype as common determinants of these disorders.

Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

ANCA-positive periaortic vasculitis: does it fall within the spectrum of vasculitis?

Retroperitoneal fibrosis (RPF) is a disease of unknown aetiology that has sometimes been reported in association with connective tissue disorders and systemic vasculitis. We report here two cases of antineutrophil cytoplasmic antibody (ANCA)-positive RPF showing clinical evidence of rapidly progressive glomerulonephritis. Although treatment with prednisone and cyclophosphamide led to a remission of RPF in both cases, renal function was restored in only one patient and the other progressed to chronic renal failure. The paper reviews the literature concerning ANCA-positive RPF and discusses the relationship between ANCA-positive vasculitis and RPF.

Interferon-alpha in combination with ribavirin as initial treatment for hepatitis C virus-associated cryoglobulinemic membranoproliferative glomerulonephritis.

Mixed cryoglobulinemia (MC) and glomerulonephritis are the most important extrahepatic manifestations of chronic hepatitis C virus (HCV) infection. In HCV-infected patients with MC, renal involvement worsens the overall prognosis because of a high incidence of infection or cardiovascular disease. The relationship between MC and HCV infection has prompted the use of antiviral therapy. Two patients with chronic HCV infection, type-II MC and membranoproliferative glomerulonephritis (MPGN), presenting as nephrotic syndrome were treated with interferon (IFN)-alpha (3 MU 3 times per week) and ribavirin (15 mg/kg daily) for 6 months. Laboratory tests included measurement of anti-HCV antibodies, HCV RNA, and HCV genotyping, and characterization of circulating cryoglobulins. A pretreatment renal biopsy was performed, and the histopathologic lesions were scored according to the index of disease activity. Viremia and cryoglobulinemia were suppressed in both patients. However, a complete remission of proteinuria was observed in 1 patient only. The evaluation of the renal biopsy specimens revealed a mild MPGN (activity score: 5/24) in the patient with remission of proteinuria and a severe MPGN (activity score: 15/24) in the patient who maintained a nephrotic-range proteinuria. Although a fully satisfactory treatment is not yet available, we feel that a reasonable therapeutic strategy for HCV-infected patients with MC nephritis could be as follows: (1) antiviral treatment alone for patients with a low-grade kidney involvement, and (2) a short-term course of steroids and cytotoxic drugs followed by antiviral therapy for acute exacerbations and/or rapidly progressive GN.

Fluorescent in situ hybridisation on tissue sections: a quantitative approach with confocal laser scanning microscopy.

The use of fluorescent detection methods in association with digital microscopy technologies is an innovative approach for tissue localisation of messenger RNA. The success of such methods relies on the tissue preservation, local availability of the probe and on the existence of high resolution tridimensional analysis systems. Cryostatic sections, mild denaturation, short oligonucleotide probes (20mer) and confocal laser scanning microscopy allow the fulfillment of all these conditions avoiding photobleaching and tissue autofluorescence. In this paper, we describe in detail a method for in situ hybridisation set up with digoxigenin-coupled oligonucleotide complementary to beta-actin mRNA as a probe and an anti-hapten fluorescent antibody as second step for detecting specific hybridisation. Fluorescence was analysed by means of a confocal laser scanning microscope (CLSM) that provides images with low out-of-focus blurring also with relatively low numerical aperture (NA) objectives. We propose also an easy method to perform semi-quantitative thresholding analysis which allows to discriminate between background and specific signal.

Hypertension as an etiopathological factor in the development of cerebral atrophy in hemodialyzed patients.

Twenty-five patients on long-term regular hemodialysis treatment (RDT) at our dialysis unit who underwent diagnostic cerebral computed tomography (CCT) participated in a study aimed at clarifying the pathogenesis of cerebral atrophy occasionally found at their original scan. The upper age limit was 55 years to exclude the physiological involutive brain changes occurring with age. Cerebral atrophy (CA), as defined morphologically (enlargement of cerebral sulci or an increased Evan's Index), was detected in all cases. Seventeen patients underwent magnetic resonance imaging (MRI) to define possible white matter changes more accurately. No significant correlation was found between the degree of atrophy and the following uremia-altered hematoseric parameters: creatinine, hematocrit, cholesterol, triglyceridemia, albumin, PTH, calcium, inorganic phosphate. There was no correlation between degree of atrophy and number of months the patients had been on RDT or time that passed between the finding of a creatinine clearance <30 ml/min and the start of RDT. Very high correlations were found between the degree of CA and predialytic blood pressure values, and between CA and the duration of hypertension (n = 13, r = 0.66, p < 0.013). Thus, hypertension seems to be an early cause of cerebral parenchymal damage in RDT patients, and should be promptly corrected.

[Physiopathology, clinical aspects and prevention of renal insufficiency caused by contrast media]. / Fisiopatologia, clinica e prevenzione dell'insufficienza renale da mezzi di contrasto.

Contrast-media associated nephropathy (CMAN) consists in a sudden impairment of glomerular filtration rate following exposure to radiographic contrast materials. Damage may be limited to an asymptomatic mild increase of blood creatinine, or reach the highest levels of nitrogen retention compatible with acute renal failure. Some preexisting clinical conditions or pathologies may lead to CMAN: not only renal insufficiency, diabetes mellitus, multiple myeloma, congestive heart failure and severe hypertension, but also simple dehydration and a growing series of immunologic diseases are recognized as predisposing condition. The exact mechanism responsible for renal injury is still doubtful but recently animal models have shown substantial ischemic changes that may be added to the traditional presumed pathogenesis of direct tubular toxicity and intra-tubular obstruction. As renal ischemia stimulates both endogenous vasoconstrictor and vasodilator substances, it is now supposed that CMAN acts similarly to non-steroidal anti-inflammatory agents, selectively inhibiting the vasodilatory prostaglandin phase and therefore causing a derangement of the physiologic vasoconstriction/vasodilatation balance of renal circulation. The role of oxygen free radicals to contribute to renal dysfunction is considered. Low osmolality non ionic contrast media when compared to conventional high osmolality ionic contrast media have reduced but not eliminated CMAN. Simple but effective lines of prevention include the previous selection of patients predisposed to CMAN for concomitant pathology, suspension of FANS or any other recognized nephrotoxic substance, the least amount of contrast media compatible with radiologic visualization of the patient's problem, careful hydration of the patient before contrast injection and sustained diuresis afterwards. The usefulness of pre-treatment with Ca-channel blockers or atrial natriuretic factors remains sub judice.

[Treatment of arterial hypertension in diabetic nephropathy. Certainties and hypotheses]. / Le traitement de l'hypertension artérielle dans la néphropathie diabétique. Certitudes et hypothèses.

Clinical observation has long emphasized the importance of arterial hypertension in the course of diabetic nephropathy and recent studies suggest that hypertension might play a decisive pathogenetic role in the course of the disease, hence the necessity of correcting the hypertension of diabetic patients has by now been universally accepted. There is, however, still some uncertainty concerning the usefulness of acting preventively on so-called microhypertension; in other words, whether early antihypertensive drug treatment can prevent diabetic nephropathy. This paper discusses the criteria to be followed in the choice of antihypertensive medication during diabetic nephropathy giving special attention to pathophysiological considerations. Moreover, it also discusses the effects of antihypertensive drugs currently regarded as first-choice agents, i.e. calcium antagonists and the angiotensin converting enzyme inhibitors, on intrarenal hemodynamics.

Renal effects of captopril, indomethacin and nifedipine in nephrotic patients after an oral protein load.

BACKGROUND: In this study we investigated whether the increase in proteinuria induced by an oral protein load may be prevented by the angiotensin-converting enzyme inhibitor (ACEI) captopril in patients with nephrotic syndrome, and whether the effects of captopril on renal haemodynamics and/or glomerular selectivity are comparable to those obtained with the nonsteroidal anti-inflammatory drug (NSAID) indomethacin and the calcium-channel blocker (CaCB) nifedipine. METHODS: Twelve subjects underwent the following treatments: (1) low-protein meal (0.2 g protein/kg body wt), (2) high-protein meal (1.3 g protein/kg body wt), (3) high-protein meal plus oral captopril (50 mg), (4) high-protein meal plus oral nifedipine (10 mg), (5) high-protein meal plus oral indomethacin (50 mg). Urine and blood samples were obtained after meals and tested for total protein, immunoglobulin G and albumin. GFR and renal plasma flow (RPF) were calculated from iothalamate and p-aminohippuric acid clearances respectively. RESULTS: Mean arterial pressure decreased significantly after both captopril (-4%, P = 0.001) and nifedipine (-5%, P = 0.0019). Compared with the low-protein meal, mean values of GFR and RPF increased significantly after the high-protein meal alone (+21%, P = 0.0002; +10%, P = 0.0491 respectively), and after captopril (+18%, P = 0.0025; +24%, P = 0.0034 respectively) or nifedipine administration (+30%, P = 0.0001; +21%, P = 0.0036 respectively), whereas they remained unchanged after the high-protein meal plus indomethacin administration. FF did not change significantly under the five experimental conditions. The increase in urinary protein excretion induced by the meat load (total protein +18%, P = 0.0102; albumin +26%, P = 0.0316; IgG +28%, P = 0.0203) was entirely blocked by both captopril and indomethacin, whereas it was further increased by nifedipine administration. CONCLUSIONS: Both captopril and indomethacin, but not nifedipine, are able to prevent the increase in urinary protein excretion rate following a meat meal. The antiproteinuric effect of captopril is comparable to that of indomethacin, but the renal haemodynamic changes induced by these drugs differ considerably, because the filtration capacity and the renal functional reserve were preserved by captopril and decreased by indomethacin. The reduction in systemic blood pressure following administration of both captopril and nifedipine does not account for changes in proteinuria, since, with a similar degree of blood pressure lowering, urinary protein excretion is reduced by captopril and increased by nifedipine.

Effectiveness of dietary protein augmentation associated with angiotensin-converting enzyme inhibition in the management of the nephrotic syndrome.

To determine whether the increase in proteinuria resulting from high dietary protein intake could be prevented by angiotensin-converting enzyme inhibition (ACEI), we performed paired studies on 8 nephrotic patients with normal GFR. They were fed sequential diets with a protein content of 0.8 (LPD) and 1.6 g/kg BW (HPD) each for 8 weeks. Patients on HPD received enalapril (ENAL) 10 mg/day. Despite the significant difference in protein intake, urinary protein excretion, at the end of the two dietary periods, was not statistically different. However, total serum protein and serum albumin increased significantly with HPD + ENAL treatment. The capability of ACEI to prevent the increase in proteinuria induced by HPD may be due to changes in glomerular hemodynamics, possibly mediated by changes in the activity of angiotensin II. Our study indicates that protein metabolism in nephrotic patients is better maintained with HPD + ENAL than with LPD alone.

Progression of cerebral atrophy in patients on regular hemodialysis treatment: long-term follow-up with cerebral computed tomography.

Fifteen patients (10 males, 5 females) on regular hemodialysis treatment (average age 43.6 +/- 4.0 years, average time on dialysis 100.7 +/- 62.8 months) underwent cerebral computed tomography between 1981 and 1984. Ten patients showed mild cerebral atrophy (CA) on the basis of cortical sulci exceeding 3 mm in breadth and an Evans ratio exceeding 0.31, for a total of 14 degrees of CA (mean 0.9 +/- 1). The same 15 patients underwent a second cerebral computed tomography during 1991/92 (101 +/- 23.7 months later). At that time, the patients exhibited a degree of CA of 2.6 +/- 1.4, for a total of 39 degrees with an overall increase of 25 degrees. Since CA is not detected before the age of 55 years in the normal population, we conclude that the CA in this patient group can only be attributed to uremia-related pathology and that it tends to worsen as regular hemodialysis treatment continues. Nevertheless, no evident cognitive, affective, or behavioural changes were verified in these patients. To our knowledge, this is the first presentation of radiologically documented progression of CA in the same patient population over time.

Significance of albuminuria in the follow-up of acute poststreptococcal glomerulonephritis.

The present study was aimed at assessing the diagnostic value of urinary albumin (uA) excretion rate in the long-term follow-up of patients suffering from acute post-streptococcal glomerulonephritis (APSGN). 26 patients, who had presented primarily with nephritic syndrome and showing increased uA without a concomitant rise in total proteinuria (uTP) were followed-up for 131 months on average (range 36-288). At the last check, 14 patients did not show urinary abnormalities, 9 had a persistent increase in uA, 1 increased uTP and 2 renal insufficiency. Urinary and clinical signs of the disease were not seen during observation periods prolonged for 79 months on average (range 20-156) after normalization of uA. No pathological findings were remarked in biopsy specimens obtained in 3 patients when uA was normalized; in contrast, when both uTP and uA (12 cases) or when isolated uA (14 cases) were increased a pattern of diffuse mesangial proliferative glomerulonephritis was constantly observed. These results indicate that the abnormal uA excretion rate during long-term follow-up of APSGN allows to identify a subset of patients with persistent renal disease; conversely, the occurrence of normal uA seems to point to a good diagnostic and prognostic significance.

[The treatment of arterial hypertension in diabetes mellitus. Choices and problems]. / Trattamento dell'ipertensione arteriosa in corso di diabete mellito. Scelte e problemi.

Recent studies indicate that arterial hypertension in diabetes mellitus is a paramount pathogenetic step in the evolution and acceleration of diabetic macro- and microangiopathy and in particular in the development of nephropathy and uremia. This paper deals with the clinical problems of antihypertensive treatment in diabetic patients and discusses the antihypertensive repertory with the aim at determining the best drug choice in the individual case. In the light of our present pathophysiologic knowledges of the intrarenal effects of the various classes of antihypertensive drugs the possibility of preventing diabetic nephropathy is discussed.

A diffusion immunogold procedure for accurate ultrastructural investigation of renal cell membrane epitopes.

A role for renal antigenic targets has been supposed and sometimes convincingly demonstrated in the development of various types of experimental glomerulonephritides. In this report we describe a reliable protocol for accurate ultrastructural investigation of antigens on the renal cell surface by means of a pre-embedding technique associated with colloidal gold staining. Sprague-Dawley rats were injected with a monoclonal antibody specific for a 90-kD cell membrane glycoprotein and killed 12 or 48 h later; after prefixation, renal fragments were cryoprotected and snap-frozen. Cryostat sections were incubated with a 5-nm colloidal gold-goat antimouse antibody, postfixed in osmium tetroxide reduced with potassium ferrocyanide and embedded in Durcupan ACM. At the glomerular level, gold granules were localized on the endothelial cell surface. In the proximal tubules uniform labelling was noticed on the brush border microvilli, followed by later marking of the basolateral membranes. By this pre-embedding immunogold method we obtained suitable histological preservation and fine resolution of the cell membrane immunoreactive sites. This procedure represents a useful tool for ultrastructural studies on the interaction of circulating antibodies with renal cell surface antigens.