Alport syndrome: impact of digenic inheritance in patients management.

Alport syndrome (ATS) is a genetically heterogeneous nephropathy with considerable phenotypic variability and different transmission patterns, including monogenic (X-linked/autosomal) and digenic inheritance (DI). Here we present a new series of families with DI and we discuss the consequences for genetic counseling and risk assessment. Out of five families harboring variants in more than one COL4 gene detected by next generation sequencing (NGS), minigene-splicing assay allowed us to identify four as true digenic. Two families showed COL4A3/A4 mutations in cis, mimicking an autosomal dominant inheritance with a more severe phenotype and one showed COL4A3/A4 mutations in trans, mimicking an autosomal recessive inheritance with a less severe phenotype. In a fourth family, a de novo mutation (COL4A5) combined with an inherited mutation (COL4A3) triggered a more severe phenotype. A fifth family, predicted digenic on the basis of silico tools, rather showed monogenic X-linked inheritance due to a hypomorphic mutation, in accordance with a milder phenotype. In conclusion, this study highlights the impact of DI in ATS and explains the associated atypical presentations. More complex inheritance should be therefore considered when reviewing prognosis and recurrence risks. On the other side, these findings emphasize the importance to accompany NGS with splicing assays in order to avoid erroneous identification of at risk members.

Excellent long-term results in de novo renal transplant recipients treated with proliferation signal inhibitors and reduced calcineurin inhibitors exposure.

BACKGROUND: A new class of immunosuppressants, proliferation signal inhibitors (PSI)--sirolimus and everolimus--has the potential to prevent chronic allograft nephropathy (CAN). This retrospective analysis reports a 6-year practice using PSI at a single center, comparing a regimen based on reduced-dose calcineurin inhibitors (CNI) and PSI versus full-dose CNI and mycophenolic acid (MPA). METHODS: The study population included 70 patients (group A) who received de novo PSI therapy in combination with reduced dose of CNI, standard steroids, and basiliximab induction, and 216 patients (group B) with full-dose CNI, MPA, steroids, and basiliximab induction. RESULTS: No statistically significant differences were recorded in the baseline donor and recipient characteristics. A difference was observed in cold ischemia time, which could represent a bias for the analysis. No differences were recorded in actuarial patient survival, delayed graft function, biopsy-proven acute rejection rates, and renal function analysis. A significant difference was recorded in the actuarial graft survival rate at years 2, 3, and 4 (P< .01), as well as overall graft survival rates (P= .025). DISCUSSION: The reduction of cold preservation time seemed to be an important factor to improve both short- and long-term renal function. This regimen revealed a long-term trend toward better renal function and graft survival. The use of PSI with reduced doses of CNI seems to be indicated for suboptimal grafts, especially when a reduced quality of the kidney is associated with prolonged cold ischemia time.

State of the art on autologous mesothelial transplant in animals and humans.

Sixteen years ago rabbit and human mesothelial cells were successfully cultured and autoimplanted. The aim of the study was merely to demonstrate that mesothelial implant was possible and interesting not only in peritoneal dialysis, but also in the vaster field of medicine and surgery concerning all the mesothelial districts of the body. The aim of this paper is to recollect the steps which have led to autologous mesothelial transplantation and verify if the technique has been validated and adopted by others. Review of the literature published in the last 15 years shows that intraperitoneal transplantation of mesothelial cells has been effective in reducing the formation of peritoneal adhesions, and in remodeling the area of mesothelial denudation. New studies on the mesothelial cell opened the way to construction of transplantable tissue-engineered artificial peritoneum, to the utilization of mesothelial progenitor cells and to find simple methods to collect autologous mesothelial cells. Finally mesothelial transplantation may represent a new neovascular therapy in the prevention and treatment of ischemic coronary heart disease.

Possible role of milky spots in mesothelial transplantation.

Milky spots are very small omental organs, in contact with peritoneal membrane, devoid of capsule and consisting of macrophages, lymphocytes and a few plasma cells supported by blood and lymphatic vessels. The exact role of these particular organs is still not clear, but they are similar to lymphatic structures and it is clear that they play a role in peritoneal infection and abdominal tumors. Peritoneal dialysis seems to activate the milky spots changing their morphology. The authors try to formulate some hypotheses on the role played by these little omental organs during autologous mesothelial transplant.

Evaluation of exercise testing for subjects with isolated proteinuria and/or hematuria of unclear significance.

BACKGROUND: A frequent problem that family doctors face is the meaning of small quantities of blood or protein in urine samples. Patients with this problem are often either neglected or referred to specialists for complex, expensive, and often invasive diagnostic procedures. Exercise testing has never been considered in nephrology, except for some attempts in diabetic patients. METHODS: We report on a study conducted over 12 years with patients referred for slight hematuria and/or proteinuria to determine whether exercise testing could be a diagnostic aid in some or all of them. We performed exercise testing using a treadmill preceded and followed by urine analysis, with a kidney biopsy within 10 days. Of the 94 patients enrolled in the study, only those with a positive exercise test turned out to have parenchymal nephropathy. At the end of the study, we simplified the quantification of exertion, dispensing with the treadmill and drastically reducing the number of urinary parameters considered. RESULTS: In patients with histological evidence of kidney damage, most of the variables increased significantly after the test. Statistical analysis also showed that determination of proteinuria and hematuria alone guaranteed maximum predictability. We found that it is also possible to simplify the quantification of effort/exertion and to drastically reduce the number of urinary parameters and still obtain significant results. CONCLUSIONS: Exercise testing provides useful information about the significance of microhematuria and proteinuria, reducing the number of cases that need to be referred to specialists. The method needs to be validated in other studies, but our results suggest that family doctors could use simple dipsticks to screen the many cases of microhematuria or proteinuria observed in daily practice. The method seems useful in eliminating doubts and unnecessary diagnostic costs.

High doses of water increase the purifying capacity of the kidneys.

BACKGROUND: In previous studies we were successful in demonstrating that the administration of water over a short period of time increases the transport capacity in the excretory tract of rabbit ureters by increasing urinary volume in the ureter from 0.3 ml/min to 10 ml/min. This phenomenon may explain the effect of water therapy performed in thermal spas, where the administration of 1-2 liters of mineral water is performed in 30-60 minutes. OBJECTIVES: The aim of the present study is to investigate if this increased transport capacity can act also in the renal tubular apparatus to modify the excretion of some endogenous substances. MATERIALS AND METHODS: We evaluated daily renal clearances in ten subjects under basal conditions during supplemental administration of 25 ml/kg of mineral water over a 24-hour period and during the administration of the same amount of water over a 30-minute period. RESULTS: Subjects who drank a water load of 25 ml/Kg over 30 minutes showed a higher diuresis than that observed in those who drank the same amount over a 24-hour period. Creatinine and urea clearance at 24 hours were significantly higher in subjects who drank the water load over 30 minutes. Serum magnesium levels and folic acid levels were also significantly higher in subjects who drank the water load over 30 minutes. CONCLUSIONS: Water administration over a short period of time seems to modify the daily excretion of some endogenous metabolites.

[From the Mailing List SIN: Therapy and prevention of peritoneal sclerosis]. / Dalla Mailing-List soci SIN (ML-SIN). La sclerosi peritoneale: terapia e prevenzione.

Recently, in the Mailing List of the Italian Society of Nephrology (ML-SIN), a message asking for opinions on the diagnosis and treatment of peritoneal sclerosis gave rise to an extensive debate on this interesting clinical topic. The discussion evidenced significant differences both in the reported onset of clinical manifestations, emphasizing the difficulty in obtaining a definite early diagnosis, and in therapy approaches. Occasionally, this is limited to medical treatment, but surgery, although burdened with elevated complexity and high mortality rates due to post-operative complications, is usually advocated for intestinal obstruction. This is the second issue reserved for the review of the ML-SIN concerning this topic, following that dedicated to definition, etiology, pathology and clinical characteristics. In this section, two expert colleagues complete the analysis of the different aspects of peritoneal sclerosis, discussing the therapy and the prevention of this serious complication of peritoneal dialysis.

[From the Mailing List SIN: peritoneal sclerosis]. / Dalla Mailing-List soci SIN (ML-SIN): la sclerosi peritoneale.

In the course of previous months, in the Mailing List of the Italian Society of Nephrology (ML-SIN), a message asking for opinions on the diagnosis and treatment of peritoneal sclerosis, gave rise to an extensive debate concerning this clinically interesting topic. The discussion evidenced significant differences both in the reported onset of clinical manifestations, emphasizing the difficulty in obtaining a definite early diagnosis, and in therapy approaches. This is sometimes limited to medical treatment, but surgery, although burdened with elevated complexity and a high mortality rate, mainly due to post-operative complications, is usually advocated for intestinal obstruction. In this issue of the review dedicated to the ML-SIN, two expert colleagues will analyze the different aspects of peritoneal sclerosis. The argument is developed in two sections: the first section is dedicated to the discussion of definition, etiology, pathology and clinical characteristics of this serious complication in peritoneal dialysis (PD).

Simple peritoneal sclerosis and sclerosing peritonitis: related or distinct entities?

AIM: The etiopathogenesis of sclerosing peritonitis is still debated, with some sustaining that it is a rare form of progression of simple peritoneal sclerosis and others that it is a primitive form. The aim of the present research was to clarify this question. MATERIAL AND METHODS: 438 peritoneal biopsies from 253 patients were re-examined. 174 were obtained prior to peritoneal dialysis and 224 after various periods of dialysis. Forty biopsies were from peritoneal dialysis patients who developed sclerosing peritonitis. Peritoneal morphology was studied for signs of transition from simple sclerosis to sclerosing peritonitis. RESULTS: Evidence was found sustaining the hypothesis that simple sclerosis to sclerosing peritonitis patients have distinct pathologies. CONCLUSIONS: The results confirm previous observations, excluding the existence of any type of relation between simple peritoneal sclerosis to sclerosing peritonitis.

Recent advances in peritoneal morphology: the milky spots in peritoneal dialysis.

Milky spots are submesothelial lymphoid structures, essential for the maturation of resident peritoneal macrophages, for peritoneal defense, and for all peritoneal inflammatory and immune processes. We evaluated the number and size of milky spots in omentum of rats subjected to dialysis for 15, 30, and 60 days and in omentum of non dialyzed control rats (5 rats per group). After 15 days of dialysis, the number (4.2 +/- 1.5/cm2) and mean size (0.13 +/- 0.04 mm2) of milky spots were significantly lower than in the control group (7.6 +/- 2.3/cm2, p < 0.03; 0.25 +/- 0.04 mm2, p < 0.01). After 30 days of dialysis, values returned to a level similar to that in controls (6.8 +/- 1.9/cm2 and 0.20 +/- 0.04 mm2). After 60 days of dialysis, values were significantly greater than in all other groups (11.8 +/- 2.2/cm2 and 0.41 +/- 0.07 mm2, p < 0.03). The early decrease in milky spots seems to be due to washing of the peritoneum and replacement of resident white cells at the expense of the white cell population in the milky spots. At 30 days, a process of adaptation seems to establish functional equilibrium. The increase in milky spots after 60 days of dialysis may be due to the chronic inflammatory stimulus of dialysis solutions with poor biocompatibility.

The rabbit model in evaluating the biocompatibility in peritoneal dialysis.

Rat and rabbit are the most common animal models for peritoneal dialysis. Rats are cheap and easy to keep. Rabbits allow dialysis to be performed for longer periods and in a way very similar to that used in human patients. Recent progress in histomorphometry enables accurate comparison of the biocompatibility of different peritoneal dialysis solutions. Preliminary data in the rabbit indicate that peritoneal dialysis is associated with a change in both the number and size of milky spots, which are peritoneal corpuscles involved in peritoneal defence.

Morphological aspects of peritoneal sclerosis.

The term peritoneal sclerosis simply means the presence of sclerotic tissue in the peritoneum. Qualitative and quantitative morphological aspects indicate that there are two nosological entities: simple sclerosis and sclerosing peritonitis. Simple sclerosis is a thin (<40-50 microm) layer of submesothelial sclerotic tissue often limited to certain peritoneal areas, with monotonous histology. It is a component of the slight anatomical alterations constantly detectable in peritoneal dialysis patients. Sclerosing peritonitis is characterized by very thick (1,0004,000 microm) sclerotic tissue involving the whole peritoneal wall, often with inflammatory infiltrates, microabscesses, giant cells of macrophagic origin, calcifications and severe vascular alterations. Intermediate stages between simple sclerosis and sclerosing peritonitis have rarely been detected. Simple sclerosis and sclerosing peritonitis also seem to be distinct with respect to frequency, etiology, reproducibility in animal models and clinical manifestations.

Pathophysiology and morphological clinical correlation in experimental and peritoneal dialysis-induced peritoneal sclerosis.

The definition of peritoneal sclerosis encompasses a vast range of peritoneal alterations induced by peritoneal dialysis. The morphology, clinical correlations and pathophysiology of, and experimental animal models for, this condition show such striking differences from case to case as to suggest the existence of two nosological entities: simple sclerosis and sclerosing peritonitis.

Peritoneal sclerosis: one or two nosological entities?

The frequency, pathology, animal models, pathogenesis, clinical manifestations, diagnostic criteria, therapy and prevention of peritoneal sclerosis are reviewed. Many of these aspects have a bimodal configuration which suggests that peritoneal sclerosis, usually considered a single pathology in peritoneal dialysis, is actually two distinct nosological entities: simple sclerosis and sclerosing peritonitis. The former is very frequent, with minor anatomical alterations and low clinical impact; it is reproducible in animals by means of peritoneal dialysis, and is clearly due to the poor biocompatibility of peritoneal dialysis solutions. The latter is rare, with radical anatomical alterations and high mortality requiring valid methods of diagnosis, therapy and prevention; it can only be reproduced in animal models by means other than peritoneal dialysis and seems to be due to factors both related and unrelated to peritoneal dialysis.

Extracorporeal blood oxygenation and ozonation (EBOO) in man. preliminary report.

Autohemotherapy with ozone has been used for four decades with encouraging results but, owing to the lack of clinical studies, it has never been adopted by orthodox medicine. Confident of the valid principles of ozone therapy, we have endeavoured to increase its therapeutic efficacy. Over a ten-year period we have developed an apparatus that makes it possible to treat large quantities of blood with ozone in extracorporeal circulation (extracorporeal blood oxygenation and ozonation EBOO). One of us volunteered to test the system and after six treatments noted the disappearance of two lipomas. This prompted us to treat a patient with Madelung disease and several patients with atherosclerotic vasculopathy. Besides showing therapeutic effects, the preliminary results indicate that EBOO is clinically valid, without side-effects and worthy of testing in various diseases.

Ozonation of blood during extracorporeal circulation. I. Rationale, methodology and preliminary studies.

We investigated whether exposure of blood ex-vivo to oxygen-ozone (O2-O3) through a gas exchanger is feasible and practical. We first evaluated the classical dialysis-type technique but we soon realized that semipermeable membranes are unsuitable because they are hydrophilic and vulnerable to O3. We therefore adopted a system with hydrophobic O3-resistant hollow fibers enclosed in a polycarbonate housing with a membrane area of about 0.5 m2. First we tested the system with normal saline, determining the production of hydrogen peroxide (H2O2) at O3 concentrations from 5 to 40 microg/ml. We then evaluated critical parameters by circulating swine blood in vitro; this revealed that heparin is not an ideal anticoagulant for this system. Finally, we performed several experiments in sheep and defined optimal anticoagulant dose (sodium citrate, ACD), priming solution, volume of blood flow per min, volume and concentration of O2-O3 mixture flowing countercurrent with respect to blood and the time necessary for perfusion in vivo. The biochemical parameters showed that an O3 concentration as low as 10 microg/ml is effective; this means that gas exchange and O3 reactivity are rapid and capable of inducing biological effects. The sheep showed no adverse effects even after 50 min of extracorporeal circulation at higher O3 concentrations (20 to 40 microg/ml) but the exchanger became less effective (low pO2 values) due to progressive clogging with cells.

Peritoneal sclerosis.

The term "peritoneal sclerosis" encompasses a vast range of peritoneal alterations, from the manifestations of low clinical impact constantly associated with peritoneal dialysis, to dramatic thickening of the peritoneal membrane, which is rare, but often life-threatening. The epidemiology, pathology, etiopathogenesis, clinical manifestations, diagnostic criteria, therapy and prevention of peritoneal sclerosis are reviewed.

Peritoneal sclerosis--an overview.

The term peritoneal sclerosis encompasses a vast range of peritoneal alterations, from the low clinical impact manifestations associated with chronic peritoneal dialysis, to dramatic thickening of the peritoneal membrane, which is rare, but often life-threatening. The frequency, pathology, etiopathogenesis, clinical manifestations, diagnostic criteria, therapy, and prevention of peritoneal sclerosis are reviewed. Preliminary observations from the Italian Registry of Peritoneal Sclerosis, established in the framework of a program of the Italian Society of Nephrology, are reported.

Biocompatibility of a peritoneal dialysis solution with amino acids: histological evaluation in the rabbit.

OBJECTIVE: To determine the biocompatibility of a peritoneal dialysis (PD) solution containing amino acids compared to PD solutions containing glucose. DESIGN: The biocompatibility of three dialysis solutions containing 1.1% amino acids, 1.36% glucose, and 3.86% glucose, respectively, was evaluated in vivo in rabbits. METHODS: After 60 days of PD, peritoneal histological changes in rabbits were investigated by light and transmission electron microscopy. The parameters investigated were: (1) mesothelial damage; (2) submesothelial edema; (3) submesothelial cell infiltration; (4) submesothelial fibrosis; and (5) vascular alterations. Semiquantitative evaluations were performed for all the above alterations; quantitative morphometric evaluation was performed for mesothelial damage (cubic transformation of the mesothelium, areas devoid of mesothelium, submesothelial edema) and thickness of peritoneal arteriole walls. RESULTS: (1) Mesothelial damage was practically nonexistent in rabbits dialyzed with the solution containing amino acids, and intermediate and severe with low-glucose and high-glucose solutions, respectively. Both controls and rabbits dialyzed with amino acid solution showed flat continuous mesothelium; rabbits dialyzed with low-glucose solution showed cubic continuous mesothelium; and rabbits dialyzed with high-glucose solution showed cubic discontinuous mesothelium. Cytopathic mesothelial effects were slight with the solution containing amino acids and severe with both the low- and high-glucose solutions. Duplication and thickening of mesothelial basement membrane were never observed. (2) Submesothelial edema showed a worsening trend from controls to rabbits dialyzed with solution containing amino acids, low glucose, and high glucose. (3) No difference in submesothelial infiltration was found between groups. (4) Submesothelial fibrosis was never observed. (5) Vascular alterations were never observed. CONCLUSION: These results are evidence that PD solution with amino acids is more biocompatible than high- and also low-glucose solutions.