Gianna Mazzucco

Broadening the Spectrum of Diseases Related to Podocin Mutations

A total of 179 children with sporadic nephrotic syndrome were screened for podocin mutations: 120 with steroid resistance, and 59 with steroid dependence/frequent relapses. Fourteen steroid-resistant patients presented homozygous mutations that were associated with early onset of proteinuria and variable renal lesions, including one case with mesangial C3 deposition. Single mutations of podocin were found in four steroid-resistant and in four steroid-dependent; five patients had the same mutation (P20L). Among these, two had steroid/cyclosporin resistance, two had steroid dependence, and one responded to cyclosporin. The common variant R229Q of podocin, recently associated with late-onset focal segmental glomerulosclerosis, had an overall allelic frequency of 4.2% versus 2.5% in controls. To further define the implication of R229Q, a familial case was characterized with two nephrotic siblings presenting the association of the R229Q with A297V mutation that were inherited from healthy mother and father, respectively. Immunohistochemistry with anti-podocin antibodies revealed markedly decreased expression of the protein in their kidneys. All carriers of heterozygous coding podocin mutation or R229Q were screened for nephrin mutation that was found in heterozygosity associated with R229Q in one patient. Finally, podocin loss of heterozygosity was excluded in one heterozygous child by characterizing cDNA from dissected glomeruli. These data outline the clinical features of sporadic nephrotic syndrome due to podocin mutations (homozygous and heterozygous) in a representative population with broad phenotype, including patients with good response to drugs. The pathogenetic implication of single podocin defects per se in proteinuria must be further investigated in view of the possibility that detection of a second mutation could have been missed. A suggested alternative is the involvement of other gene(s) or factor(s).

Validation of the Oxford classification of IgA nephropathy in cohorts with different presentations and treatments

The Oxford Classification of IgA Nephropathy (IgAN) identified mesangial hypercellularity (M), endocapillary proliferation (E), segmental glomerulosclerosis (S), and tubular atrophy/interstitial fibrosis (T) as independent predictors of outcome. Whether it applies to individuals excluded from the original study and how therapy influences the predictive value of pathology remain uncertain. The VALIGA study examined 1147 patients from 13 European countries that encompassed the whole spectrum of IgAN. Over a median follow-up of 4.7 years, 86% received renin–angiotensin system blockade and 42% glucocorticoid/immunosuppressive drugs. M, S, and T lesions independently predicted the loss of estimated glomerular filtration rate (eGFR) and a lower renal survival. Their value was also assessed in patients not represented in the Oxford cohort. In individuals with eGFR less than 30 ml/min per 1.73 m2, the M and T lesions independently predicted a poor survival. In those with proteinuria under 0.5 g/day, both M and E lesions were associated with a rise in proteinuria to 1 or 2 g/day or more. The addition of M, S, and T lesions to clinical variables significantly enhanced the ability to predict progression only in those who did not receive immunosuppression (net reclassification index 11.5%). The VALIGA study provides a validation of the Oxford classification in a large European cohort of IgAN patients across the whole spectrum of the disease. The independent predictive value of pathology MEST score is reduced by glucocorticoid/immunosuppressive therapy.

FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair

Chronic kidney disease (CKD) represents a major health burden. Its central feature of renal fibrosis is not well understood. By exome sequencing, we identified mutations in FAN1 as a cause of karyomegalic interstitial nephritis (KIN), a disorder that serves as a model for renal fibrosis. Renal histology in KIN is indistinguishable from that of nephronophthisis, except for the presence of karyomegaly. The FAN1 protein has nuclease activity and acts in DNA interstrand cross-link (ICL) repair within the Fanconi anemia DNA damage response (DDR) pathway. We show that cells from individuals with FAN1 mutations have sensitivity to the ICL-inducing agent mitomycin C but do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from individuals with Fanconi anemia. We complemented ICL sensitivity with wild-type FAN1 but not with cDNA having mutations found in individuals with KIN. Depletion of fan1 in zebrafish caused increased DDR, apoptosis and kidney cysts. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms contributing to renal fibrosis and CKD.

Apparent preferential loss of heterozygosity at TSC2 over TSC1 chromosomal region in tuberous sclerosis hamartomas

To investigate the molecular mechanisms of tuberous sclerosis (TSC) histopathologic lesions, we have tested for loss of heterozygosity the two TSC loci (TSC1 and TSC2) and seven tumor suppressor gene‐containing regions (TP53, NF1, NF2, BRCA1, APC, VHL, and MLM) in 20 hamartomas from 18 TSC patients. Overall, eight angiomyolipomas, eight giant cell astrocytomas, one cortical tuber, and three rhabdomyomas were analyzed. Loss of heterozygosity at either TSC locus was found in a large fraction of the informative patients, both sporadic (7/14) and familial (1/4). Interestingly, a statistically significant preponderance of loss of heterozygosity at TSC2 was observed in the sporadic group (P < 0.01). Among the possible explanations considered, the bias in the selection for TSC patients with the most severe organ impairment seems particularly appealing. According to this view, a TSC2 defect might confer a greater risk for early kidney failure or, possibly, a more rapid growth of a giant cell astrocytoma. None of the seven antioncogenes tested showed loss of heterozygosity, indicating that the loss of either TSC gene product may be sufficient to promote hamartomatous cell growth. Finally, the observation of loss of heterozygosity at different markers in an astrocytoma and in an angiomyolipoma from the same patient might suggest the multifocal origin of the second‐hit mutation. Genes Chromosom Cancer 15:18–25 (1996).

Thickness of multiwalled carbon nanotubes affects their lung toxicity.

Two samples of highly pure multiwalled carbon nanotubes (MWCNTs) similar in hydrophobicity and surface reactivity were prepared with similar length, <5 μm, but markedly different diameter (9.4 vs 70 nm). The samples were compared for their cytotoxic activity, uptake, and ability to induce oxidative stress (ROS production and intracellular GSH depletion) in vitro in murine alveolar macrophages (MH-S). The in vivo toxicity was evaluated by measuring biochemical (LDH activity and total proteins) and cellular responses in bronchoalveolar lavage (BAL) after intratracheal instillation in rats. Both samples were internalized in MH-S cells. However, thin MWCNTs appeared significantly more toxic than the thicker ones, both in vitro and in vivo, when compared on a mass-dose basis. The data reported herein suggest that the nanotube diameter is an important parameter to be considered in the toxicological assessment of CNTs.

Oxidative Stress and Galactose-Deficient IgA1 as Markers of Progression in IgA Nephropathy

BACKGROUND AND OBJECTIVES We assessed the activation of the oxidative stress pathway in patients with IgA nephropathy (IgAN), while evaluating the classic marker of the disease (galactose-deficient serum IgA1). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Sera from 292 patients and 69 healthy controls from Italy and the United States were assayed for advanced oxidation protein products (AOPPs), free sulfhydryl groups on albumin (SH-Alb), and IgA1 with galactose-deficient hinge-region O-glycans (Gd-IgA1). Gd-IgA1 was detected by binding to Helix aspersa agglutinin (HAA) and expressed as total Gd-IgA1 or as degree of galactose deficiency relative to a standard Gd-IgA1 myeloma protein (%HAA). RESULTS Sera from IgAN patients showed higher levels of Gd-IgA1, %HAA, and AOPPs, but lower levels of SH-Alb in comparison to that from healthy controls. Serum levels of AOPPs significantly correlated with serum Gd-IgA1 and %HAA. The relationship between these biomarkers and clinical features at sampling and during follow-up was assessed in 62 patients with long-term follow-up. AOPPs and %HAA correlated with proteinuria at sampling and independently associated with subsequent proteinuria. Levels of AOPPs correlated with rate of decline in renal function after sampling. The combination of a high level of AOPPs and a high level of %HAA associated with decline in estimated GFR. CONCLUSIONS Serum levels of aberrantly glycosylated IgA1 are elevated and oxidative stress pathways are activated in patients with IgAN; the intensity of the stress correlated with expression and progression of the disease. We speculate that oxidative stress may modulate the nephrotoxicity of aberrantly glycosylated IgA1 in IgAN.

Mitochondrial Localization of Vitamin D Receptor in Human Platelets and Differentiated Megakaryocytes

Background Like other steroid hormones, vitamin D elicits both transcriptional events and rapid non genomic effects. Vitamin D receptor (VDR) localization and mechanisms of VDR-triggered non genomic responses are still controversial. Although anticoagulant effects of vitamin D have been reported and VDR signalling has been characterized in monocytes and vascular cells, nothing is known about VDR expression and functions in human platelets, anucleated fragments of megakaryocytes which are known targets of other steroids. Methodology/Principal Findings In this study we characterized the expression and cellular localization of VDR in human platelets and in a megakaryocyte lineage. Human platelets and their TPA-differentiated precursors expressed a classical 50 kDa VDR protein, which increased with megakaryocytes maturation. By biochemical fractionation studies we demonstrated the presence of the receptor in the soluble and mitochondrial compartment of human platelets, and the observation was confirmed by immunoelectron microscopy analysis. Similar localization was found in mature megakaryocytes, where besides its classical nuclear localization the receptor was evident as soluble and mitochondria resident protein. Conclusions The results reported here suggest that megakaryocytopoiesis and platelet activation, which are calcium-dependent events, might be modulated by a mitochondrial non genomic activity of VDR. These data open challenging future studies on VDR physiological role in platelets and more generally in mitochondria.

Incidence of biopsy-proven primary glomerulonephritis in an Italian Province

Between January 1, 1970, and December 31, 1994, 1,926 cases of biopsy-proven primary glomerulonephritis (PGN) were diagnosed in an adult population (> 15 years of age) in a northwestern region of Italy with approximately 3.5 million inhabitants. The principal long-term changes were an increase in the absolute number of biopsies per year, an increase in the mean age of patients undergoing biopsy (from 29.3 +/- 12.2 years to 47.0 +/- 17.8 years), an increase in the percentage of patients older than 65 years (from 1.7% to 20.4%), and an increase in the percentage of isolated urinary abnormalities as an indication for biopsy (from 3.5% to 29.6%). In the total biopsy material, immunoglobulin A glomerulonephritis (IgA-GN) is the most frequent type (26%), followed by membranous glomerulonephritis (MGN; 20%). An incidence study was begun in 1990; this survey was restricted to the population of the province of Torino (approximately 2 million inhabitants) as only this area completely refers to the nephrologic centers that entered patients into this study. The overall incidence of PGN is 4.68 new cases/yr/10(5) population with a predominance of males (> 2:1); IgA-GN is the most common type (1.47/yr/10(5) population [34.5%]) in the overall population. In the elderly, cases of PGN are twice as high as in adults (8.19/yr/10(5) population v 4.02/yr/10(5) population in the 65 to 74 year and 45 to 54 year age groups, respectively); MGN mainly accounts for this high incidence (3.4/yr/10(5) population), while the nephrotic syndrome is the most common indication for biopsy (53.8%). A comparison with the incidence in the same area in the early 1970s is evaluable only for PGN, which was mainly registered in the age groups for which an unrestricted biopsy policy was already in place (15 to 35 years). In contrast with a misleading increase of all types of PGN, which is in reality due to the extension of the biopsy policy to older and asymptomatic patients, membranoproliferative glomerulonephritis type I shows a countercurrent decrease from 0.43 to 0.13/yr/10(5) population. Evidence of a simultaneous decrease in severe cardiac valvulopathy, due to rheumatic fever, is also provided. We feel that before epidemiologic conclusions can be reached, a clear understanding of ones own biopsy policy is essential. An apparent change in the PGN rate in our region over the last 25 years mainly depends on modifications in our biopsy policy, most probably coupled with a change in the threshold of detection of symptoms in the general population. At present, according to our experience, IgA-GN is the most common type of PGN in the total bioptic material, as demonstrated in other European countries, while the elderly show a peculiar pattern with a higher PGN incidence, mainly represented by MGN and heralded by the nephrotic syndrome. We also confirm that membranoproliferative glomerulonephritis type I is indeed decreasing in parallel with changes in the microbiologic environment.

Intertubular capillary changes in kidney allografts: an ultrastructural study in patients with transplant glomerulopathy.

Fifteen kidney allograft specimens from 14 patients with transplant glomerulopathy were investigated by electron microscopy. Intertubular capillaries of all patients showed splitting and multilayering of the basement membranes, a change recalling that observed in the glomerular basement membranes in transplant glomerulopathy. Although the severity of the lesions varied from case to case, it was, to a certain extent, constant in each specimen and correlated well with the severity of the glomerular basement membrane changes in the same patient. The similarity of the two lesions suggests a possible common pathogenetic mechanism. The constant finding of intertubular capillary splitting and multilayering in patients with transplant glomerulopathy leads to the suspicion of this condition when such changes are found in kidney samples in which glomeruli are lacking. Therefore electron microscopy could achieve a specific diagnostic relevance in this field of nephropathology.

The Brush Border of Proximal Tubules of Normal Human Kidney Activates the Alternative Pathway of the Complement System In Vitroa a

The aim of this investigation was to study complement fixation by normal human kidney tissue. C fixation was assessed on acetone-fixed sections of frozen human kidney. In addition, C consumption following incubation of normal fresh human serum with tubular or glomerular fractions of human kidney was measured. The results are consistent with the interpretation that the brush border of proximal tubules of human kidney activates the C system via the alternative pathway. It is suggested that this activation may occur in vivo in patients with a non-selective proteinuria.