Andrea Onetti Muda

Coxsackie B4 virus infection of β cells and natural killer cell insulitis in recent-onset type 1 diabetic patients

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic β cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between β cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, whole-genome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of β cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect β cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect β cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.

COQ2 Nephropathy: A Newly Described Inherited Mitochondriopathy with Primary Renal Involvement

Primary coenzyme Q(10) (CoQ(10)) deficiency includes a group of rare autosomal recessive disorders primarily characterized by neurological and muscular symptoms. Rarely, glomerular involvement has been reported. The COQ2 gene encodes the para-hydroxybenzoate-polyprenyl-transferase enzyme of the CoQ(10) synthesis pathway. We identified two patients with early-onset glomerular lesions that harbored mutations in the COQ2 gene. The first patient presented with steroid-resistant nephrotic syndrome at the age of 18 months as a result of collapsing glomerulopathy, with no extrarenal symptoms. The second patient presented at five days of life with oliguria, had severe extracapillary proliferation on renal biopsy, rapidly developed end-stage renal disease, and died at the age of 6 months after a course complicated by progressive epileptic encephalopathy. Ultrastructural examination of renal specimens from these cases, as well as from two previously reported patients, showed an increased number of dysmorphic mitochondria in glomerular cells. Biochemical analyses demonstrated decreased activities of respiratory chain complexes [II+III] and decreased CoQ(10) concentrations in skeletal muscle and renal cortex. In conclusion, we suggest that inherited COQ2 mutations cause a primary glomerular disease with renal lesions that vary in severity and are not necessarily associated with neurological signs. COQ2 nephropathy should be suspected when electron microscopy shows an increased number of abnormal mitochondria in podocytes and other glomerular cells.

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).

AKT2 is essential to maintain podocyte viability and function during chronic kidney disease

In chronic kidney disease (CKD), loss of functional nephrons results in metabolic and mechanical stress in the remaining ones, resulting in further nephron loss. Here we show that Akt2 activation has an essential role in podocyte protection after nephron reduction. Glomerulosclerosis and albuminuria were substantially worsened in Akt2−/− but not in Akt1−/− mice as compared to wild-type mice. Specific deletion of Akt2 or its regulator Rictor in podocytes revealed that Akt2 has an intrinsic function in podocytes. Mechanistically, Akt2 triggers a compensatory program that involves mouse double minute 2 homolog (Mdm2), glycogen synthase kinase 3 (Gsk3) and Rac1. The defective activation of this pathway after nephron reduction leads to apoptosis and foot process effacement of the podocytes. We further show that AKT2 activation by mammalian target of rapamycin complex 2 (mTORC2) is also required for podocyte survival in human CKD. More notably, we elucidate the events underlying the adverse renal effect of sirolimus and provide a criterion for the rational use of this drug. Thus, our results disclose a new function of Akt2 and identify a potential therapeutic target for preserving glomerular function in CKD.

Receptor Activator of NF-kB (RANK) Expression in Primary Tumors Associates with Bone Metastasis Occurrence in Breast Cancer Patients

Background Receptor activator of NFkB (RANK), its ligand (RANKL) and the decoy receptor of RANKL (osteoprotegerin, OPG) play a pivotal role in bone remodeling by regulating osteoclasts formation and activity. RANKL stimulates migration of RANK-expressing tumor cells in vitro, conversely inhibited by OPG. Materials and Methods We examined mRNA expression levels of RANKL/RANK/OPG in a publicly available microarray dataset of 295 primary breast cancer patients. We next analyzed RANK expression by immunohistochemistry in an independent series of 93 primary breast cancer specimens and investigated a possible association with clinicopathological parameters, bone recurrence and survival. Results Microarray analysis showed that lower RANK and high OPG mRNA levels correlate with longer overall survival (P = 0.0078 and 0.0335, respectively) and disease-free survival (P = 0.059 and 0.0402, respectively). Immunohistochemical analysis of RANK showed a positive correlation with the development of bone metastases (P = 0.023) and a shorter skeletal disease-free survival (SDFS, P = 0.037). Specifically, univariate analysis of survival showed that “RANK-negative” and “RANK-positive” patients had a SDFS of 105.7 months (95% CI: 73.9–124.4) and 58.9 months (95% CI: 34.7–68.5), respectively. RANK protein expression was also associated with accelerated bone metastasis formation in a multivariate analysis (P = 0.029). Conclusions This is the first demonstration of the role of RANK expression in primary tumors as a predictive marker of bone metastasis occurrence and SDFS in a large population of breast cancer patients.

Liver vitamin D receptor, CYP2R1, and CYP27A1 expression: relationship with liver histology and vitamin D3 levels in patients with nonalcoholic steatohepatitis or hepatitis C virus

Evidence suggests an association between low serum 25‐hydroxy‐vitamin D3 [25(OH)D3] levels and the presence and prognosis of liver disease. Vitamin D receptor (VDR) has been widely detected in the liver, but its expression in the course of liver disease has never been investigated. We evaluated the hepatic expression of VDR along with that of vitamin D 25‐hydroxylases in patients with nonalcoholic steatohepatitis (NASH) or chronic hepatitis C (CHC) and its relationship with hepatic histological features and serum 25(OH)D3 levels. We evaluated 61 patients (25 NASH and 36 CHC) who had undergone liver biopsy for clinical purposes and 20 subjects without liver disease. Serum 25(OH)D3 was measured via colorimetric assay. Expression of VDR, CYP2R1, and CYP27A1 was evaluated via immunohistochemistry in hepatocytes, cholangiocytes, and liver inflammatory cells. Parenchymal and inflammatory cells from liver biopsies of patients with NASH and CHC expressed VDR, CYP2R1, and CYP27A1. In NASH patients, VDR expression on cholangiocytes was inversely correlated with steatosis severity (P < 0.02), lobular inflammation (P < 0.01), and nonalcoholic fatty liver disease score (P < 0.03). Moreover, expression of CYP2R1 in hepatocytes correlated strongly with VDR positivity on liver inflammatory cells. In CHC subjects, fibrosis stage was associated with low hepatic CYP27A1 expression, whereas portal inflammation was significantly higher in patients with VDR‐negative inflammatory cells (P < 0.009) and low VDR expression in hepatocytes (P < 0.03). Conclusion: VDR is widely expressed in the liver and inflammatory cells of chronic liver disease patients and its expression is negatively associated with the severity of liver histology in both NASH and CHC patients. These data suggest that vitamin D/VDR system may play a role in the progression of metabolic and viral chronic liver damage. (HEPATOLOGY 2012;56:2180–2187)

Expression pattern of receptor activator of NFκB (RANK) in a series of primary solid tumors and related bone metastases.

Receptor activator of NFκB ligand (RANKL), RANK, and osteoprotegerin (OPG) represent the key regulators of bone metabolism both in normal and pathological conditions, including bone metastases. To our knowledge, no previous studies investigated and compared RANK expression in primary tumors and in bone metastases from the same patient. We retrospectively examined RANK expression by immunohistochemistry in 74 bone metastases tissues from solid tumors, mostly breast, colorectal, renal, lung, and prostate cancer. For 40 cases, tissue from the corresponding primary tumor was also analyzed. Sixty‐six (89%) of the 74 bone metastases were RANK‐positive and, among these, 40 (59.5%) showed more than 50% of positive tumor cells. The median percentage of RANK‐positive cells was 60% in primary tumors and metastases, without any statistically significant difference between the two groups (P = 0.194). The same percentage was obtained by considering only cases with availability of samples both from primary and metastasis. Our study shows that RANK is expressed by solid tumors, with high concordance between bone metastasis and corresponding primary tumor. These data highlight the central role of RANK/RANKL/OPG pathway as potential therapeutic target not only in bone metastasis management, but also in the adjuvant setting. J. Cell. Physiol. 226: 780–784, 2011.

The Dri 42 gene, whose expression is up-regulated during epithelial differentiation, encodes a novel endoplasmic reticulum resident transmembrane protein

A search for novel genes that are up-regulated during development and differentiation of the epithelial cells of the intestinal mucosa led us to the isolation of the Dri 42 cDNA clone (Dri, differentially expressed in rat intestine). The nucleotide sequence of the full-length cDNA has shown that it encodes a 35.5-kDa protein with one consensus sequence for N-linked glycosylation and alternating hydrophilic and hydrophobic domains. To determine the intracellular localization of Dri 42 we have raised polyclonal antibodies in hens against a bacterially produced Dri 42-glutathione S-transferase fusion protein. Immunofluorescence detection with these antibodies has shown specific staining of the endoplasmic reticulum (ER) in the relatively undifferentiated fetal rat intestinal cell line FRIC B and in sections of rat small intestine. ER membrane localization of Dri 42 was confirmed by laser confocal microscopy of polarized Madin-Darby canine kidney cells overexpressing a Dri 42-chloramphenicol acetyltransferase (CAT) fusion protein by transfection. Pulse labeling experiments on transiently transfected cells demonstrated that the protein does not acquire Golgi modifications up to 4 h after synthesis, thus indicating that Dri 42 is an ER resident protein. The transmembrane disposition of Dri 42 was studied using in vitro insertion of Dri 42-CAT fusion proteins into microsomal membranes. The fusion proteins consisted of several different lengths of truncated Dri 42 and a reporter protein, CAT, that was linked in-frame after each hydrophobic segment. We found that hydrophobic segments H1, H3, and H5 had a signal/anchor function, and that membrane insertion of Dri 42 was achieved co-translationally by the action of a series of alternating insertion signals and halt transfer signals, resulting in the exposure of both termini of the protein to the cytosolic side. The functional implications of the structure and localization of Dri 42, whose primary sequence does not share significant homology to any previously described protein, are discussed.

Podocin Inactivation in Mature Kidneys Causes Focal Segmental Glomerulosclerosis and Nephrotic Syndrome

Podocin is a critical component of the glomerular slit diaphragm, and genetic mutations lead to both familial and sporadic forms of steroid-resistant nephrotic syndrome. In mice, constitutive absence of podocin leads to rapidly progressive renal disease characterized by mesangiolysis and/or mesangial sclerosis and nephrotic syndrome. Using established Cre-loxP technology, we inactivated podocin in the adult mouse kidney in a podocyte-specific manner. Progressive loss of podocin in the glomerulus recapitulated albuminuria, hypercholesterolemia, hypertension, and renal failure seen in nephrotic syndrome in humans. Lesions of FSGS appeared after 4 wk, with subsequent development of diffuse glomerulosclerosis and tubulointerstitial damage. Interestingly, conditional inactivation of podocin at birth resulted in a gradient of glomerular lesions, including mesangial proliferation, demonstrating a developmental stage dependence of renal histologic patterns of injury. The development of significant albuminuria in this model occurred only after early and focal foot process effacement had progressed to diffuse involvement, with complete absence of podocin immunolabeling at the slit diaphragm. Finally, we identified novel potential mediators and perturbed molecular pathways, including cellular proliferation, in the course of progression of renal disease leading to glomerulosclerosis, using global gene expression profiling.

Glial fibrillary acidic protein as an early marker of hepatic stellate cell activation in chronic and posttransplant recurrent hepatitis C

Activated alpha‐smooth muscle actin (α‐SMA)–positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. In the rat, increased GFAP expression in the acute response to injury and down‐regulation in the chronic response have been observed, whereas reports concerning GFAP expression in human liver are still conflicting. We investigated the utility of GFAP compared to α‐SMA as an immunohistochemical marker of early activated HSCs in chronic and posttransplant recurrent hepatitis C and correlated GFAP expression with vascular remodeling and fibrosis progression. With immunohistochemistry and a semiquantitative scoring system, the expression of GFAP and α‐SMA in HSCs and the microvessel density were analyzed in biopsies from normal livers obtained from cadaveric donors [donor liver (DL); n = 21] and from livers from posttransplant hepatitis C virus recurrent hepatitis (HCV‐PTR) patients (n = 19), hepatitis C virus chronic hepatitis (HCV‐CH) patients, (n = 12), and hepatitis C virus cirrhosis (HCV‐C) patients (n = 16). The percentage of α‐SMA–positive HSCs was significantly higher in the HCV‐PTR, HCV‐CH, and HCV‐C groups compared to the DL group (P < 0.01). The percentage of GFAP‐positive HSCs was significantly higher in the HCV‐PTR group compared to the DL, HCV‐C (P < 0.01), and HCV‐CH (P < 0.05) groups and in the HCV‐CH group compared to the DL group (P < 0.01), inversely correlating with the extent of fibrosis and microvessel density (P < 0.01). In the HCV‐PTR group, the percentage of GFAP‐positive HSCs correlated with fibrosis progression (P < 0.01). In conclusion, GFAP could represent a useful marker of early activation of HSCs in HCV‐CH and seems to predict fibrosis progression in HCV‐PTR. Liver Transpl 14:806–814, 2008.