[Show abstract][Hide abstract] ABSTRACT: In children, sporadic nephrotic syndrome can be related to a genetic cause, but to what extent genetic alterations associate with resistance to immunosuppression is unknown. In this study, we designed a custom array for next-generation sequencing analysis of 19 target genes, reported as possible causes of nephrotic syndrome, in a cohort of 31 children affected by sporadic steroid-resistant nephrotic syndrome and 38 patients who exhibited a similar but steroid-sensitive clinical phenotype. Patients who exhibited extrarenal symptoms, had a familial history of the disease or consanguinity, or had a congenital onset were excluded. We identified a genetic cause in 32.3% of the children with steroid-resistant disease but zero of 38 children with steroid-sensitive disease. Genetic alterations also associated with lack of response to immunosuppressive agents in children with steroid-resistant disease (0% of patients with alterations versus 57.9% of patients without alterations responded to immunosuppressive agents), whereas clinical features, age at onset, and pathologic findings were similar in steroid-resistant patients with and without alterations. These results suggest that heterogeneous genetic alterations in children with sporadic forms of nephrotic syndrome associate with resistance to steroids as well as immunosuppressive treatments. In these patients, a comprehensive screening using such an array may, thus, be useful for genetic counseling and may help clinical decision making in a fast and cost-efficient manner.
No preview · Article · Jul 2014 · Journal of the American Society of Nephrology
[Show abstract][Hide abstract] ABSTRACT: Identification of the genetic defect underlying early-onset diabetes is important for determining the specific diabetes subtype, which would then permit appropriate treatment and accurate assessment of recurrence risk in offspring. Given the extensive genetic and clinical heterogeneity of the disease, high-throughput sequencing might provide additional diagnostic potential when Sanger sequencing is ineffective. Our aim was to develop a targeted next-generation assay able to detect mutations in several genes involved in glucose metabolism. All 13 known MODY genes, genes identified from a genome-wide linkage study or genome-wide association studies as increasing the risk of type 2 diabetes and genes causing diabetes in animal models, were included in the custom panel. We selected a total of 102 genes by performing a targeting re-sequencing in 30 patients negative for mutations in the GCK, HNF1α, HNF4α, HNF1β and IPF1 genes at the Sanger sequencing analysis. Previously unidentified variants in the RFX6 gene were found in three patients and in two of them we also detected rare variants in WFS1 and ABCC8 genes. All patients showed a good therapeutic response to dipeptidyl peptidase-4 (DPP4) inhibitors. Our study reveals that next-generation sequencing provides a highly sensitive method for identification of variants in new causative genes of diabetes. This approach may help in understanding the molecular etiology of diabetes and in providing more personalized treatment for each genetic subtype.The Pharmacogenomics Journal advance online publication, 22 July 2014; doi:10.1038/tpj.2014.37.
Full-text · Article · Jul 2014 · The Pharmacogenomics Journal
[Show abstract][Hide abstract] ABSTRACT: Introduction and Aims: According to classical nephrology practice, pure microscopic hematuria [MH] in early life is not considered a serious problem
and up to date diagnostic studies are not always carried out. Family urine studies to confirm or rule out a familial disease
should always be carried out early and if positive, molecular genetics should be used to study the whole family and establish
the underlying genetic defect.
Methods: During the last 30 years we have searched systematically in a homogeneous population of 650,000 people for familial MH and
some 120 such families are under study so far. Molecular genetic studies during the last 10 years have led to the correct
genetic diagnosis in 57 such families with a) 28 heterozygous COL4A3/A4 mutations and TBMN, b) 23 CFHR5 and C3 nephritis and
c) 6 families with Alport syndrome.
Results: Our most striking finding refers to the COL4A3/A4 heterozygous mutations that turn out to be the commonest cause of familial
MH and have been identified in 249 carriers in 30 families. Some of these families are very big indeed and two of these mutations,
COL4A3-G1334E and COL4A3-G871C, are particularly common. Mutation COL4A3-G1334E was found in 15 families and it accounts for
174 patients. C3 nephritis with the CFHR5 Cypriot mutation appears to be the 2nd commonest cause of familial MH with 150 carriers
in 23 families. Classical XLAS, COL4A5 Alport was identified in 3 families with 9 affected males and 8 female carriers, while
ARAS was also present in 3 additional families affecting 8 patients. Of the 249 patients with COL4A3/A4 heterozygous mutations
33 have reached ESKD and of the 150 CFHR5 patients 21 have reached ESKD.
Conclusions: Our results strengthen the great significance of the heterozygous COL4A3/A4 mutations, not really well understood until after
1996, less than 20 years ago. These mutations are common, lead to TBMN and are the commonest cause of familial MH. More importantly
however, these mutations lead much later in life to proteinuria, hypertension, CRF and ESKD and long term care is mandatory
with prompt attention to the addition of proteinuria that requires urgent treatment. Heterozygous mutations COL4A3/A4 cause
twice more patients to reach ESKD compared to classical XLAS and ARAS but fortunately at a much older age. Molecular genetics
should be used more widely for an early diagnosis of this entity and more renal attention should be given to these patients
to preserve kidney function and avoid ESKD.
Full-text · Article · May 2014 · Nephrology Dialysis Transplantation
[Show abstract][Hide abstract] ABSTRACT: Introduction and Aims: Autosomal recessive polycystic kidney disease (ARPKD) occurs in 1:20,000 live births and is the most common cystic kidney
disease in childhood. Since kidney and liver manifestations progress differently in ARPKD, the decision of the right type
of transplantation may be challenging. Most often kidney-alone transplantation (KT) is performed first, followed by liver
transplantation (LT) or directly combined liver kidney transplantation (CLKT). The choice for CLKT is more and more discussed
because (1) 64-80% of the mortality occurring in ARPKD with KT patients is attributed to cholangitis/sepsis related to liver
disease; (2) liver allograft is immunologically protective of the kidney graft and (3) CLKT from the same donor probably provides
better long-term. However, there is no consensus yet and long-term outcome data are scarce. In the present study, we aimed
to analyse the characteristics and outcomes of children with ARPKD of the ESPN/ERA-EDTA registry and compare the outcome of
the different transplantation strategies.
Methods: 239 incident ARPKD patients were identified from the ESPN/ERA-EDTA registry who started RRT since 1995. Data included date
of birth, gender, age at RRT, treatment modality of RRT, type of transplantation (KT or CLKT), changes in RRT modality during
follow-up, survival and cause of death.
Results: Mean age at start of RRT was 12.8 and 11.4 years in males and females respectively. Five year patient survival on RRT was
88.1%. Patients starting RRT before the age of 1 had a 20% lower 5-year survival (72.9%). Twenty nine patients died and the
causes of death were infection (28%), cardiovascular disease (16%), hemorrhage (6.9%), and unknown (31%). There were 197 transplantations
among 179 incident patients. Median age at time of the first transplantation was 9.3 years (IQR 3.9 to 13.6), which took place
after a median of 0.4 years on dialysis (IQR 0.0-1.3). Twenty three patients (13%) received a CLKT, while 123 (69%) received
a KT and in 33 (18%) the type of transplantation was unknown. There was a significant difference in 5-year patient survival
according to the type of transplantation (78.6% for CLKT vs. 96.2% for KT) (p=0.03). Five year death censored renal graft
survival was 74.3% (71.5% and 77.0% for CLKT and KT respectively p=0.83). There were 18 patients with at least 2 transplantations
(1 patients received 3). Renal graft survival tended to be worse for the second as compared to the first renal allograft (HR
1.56 95%CI 0.71 to 3.43).
Conclusions: The need for RRT in the first year of life for ARPKD adversely impacts survival. CLKT is a significant risk factor for mortality
on short-term, and is not associated with better 5-year graft survival. Long term follow-up data are needed to better delineate
the best transplantation strategy.
Full-text · Article · May 2014 · Nephrology Dialysis Transplantation
[Show abstract][Hide abstract] ABSTRACT: In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.
No preview · Article · Aug 2013 · Journal of the American Society of Nephrology
[Show abstract][Hide abstract] ABSTRACT: Recent studies implicated the existence in adult human kidney of a population of renal progenitors with the potential to regenerate glomerular as well as tubular epithelial cells and characterized by coexpression of surface markers CD133 and CD24. Here, we demonstrate that CD133+CD24+ renal progenitors can be distinguished in distinct subpopulations from normal human kidneys based on the surface expression of vascular cell adhesion molecule 1, also known as CD106. CD133+CD24+CD106+ cells were localized at the urinary pole of Bowman's capsule, while a distinct population of scattered CD133+CD24+CD106- cells was localized in the proximal tubule as well as in the distal convoluted tubule. CD133+CD24+CD106+ cells exhibited a high proliferative rate and could differentiate toward the podocyte as well as the tubular lineage. By contrast, CD133+CD24+CD106- cells showed a lower proliferative capacity and displayed a committed phenotype toward the tubular lineage. Both CD133+CD24+CD106+ and CD133+CD24+CD106- cells showed higher resistance to injurious agents in comparison to all other differentiated cells of the kidney. Once injected in SCID mice affected by acute tubular injury, both of these populations displayed the capacity to engraft within the kidney, generate novel tubular cells, and improve renal function. These properties were not shared by other tubular cells of the adult kidney. Finally, CD133+CD24+CD106- cells proliferated upon tubular injury, becoming the predominating part of the regenerating epithelium in patients with acute or chronic tubular damage. These data suggest that CD133+CD24+CD106- cells represent tubular-committed progenitors that display resistance to apoptotic stimuli and exert regenerative potential for injured tubular tissue.
[Show abstract][Hide abstract] ABSTRACT: The contribution of microRNA (miRNA) to the pathogenesis of renal fibrosis is not well understood. Here, we investigated whether miRNA modulates the fibrotic process in Munich Wistar Fromter (MWF) rats, which develop spontaneous progressive nephropathy. We analyzed the expression profile of miRNA in microdissected glomeruli and found that miR-324-3p was the most upregulated. In situ hybridization localized miR-324-3p to glomerular podocytes, parietal cells of Bowman's capsule, and most abundantly, cortical tubules. A predicted target of miR-324-3p is prolyl endopeptidase (Prep), a serine peptidase involved in the metabolism of angiotensins and the synthesis of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). In cultured tubular cells, transient transfection with a miR-324-3p mimic reduced Prep protein and activity, validating Prep as a target of this miRNA. In MWF rats, upregulation of miR-324-3p associated with markedly reduced expression of Prep in both glomeruli and tubules, low urine Ac-SDKP, and increased deposition of collagen. ACE inhibition downregulated glomerular and tubular miR-324-3p, promoted renal Prep expression, increased plasma and urine Ac-SDKP, and attenuated renal fibrosis. In summary, these results suggest that dysregulation of the miR-324-3p/Prep pathway contributes to the development of fibrosis in progressive nephropathy. The renoprotective effects of ACE inhibitors may result, in part, from modulation of this pathway, suggesting that it may hold other potential therapeutic targets.
No preview · Article · Jul 2012 · Journal of the American Society of Nephrology
[Show abstract][Hide abstract] ABSTRACT: • To compare the frequency of T regulatory cells (Tregs) in peripheral blood of patients (pPB) affected by renal cell carcinoma (RCC) both with the frequency of Tregs found in PB of healthy donors (hPB) and that of Tregs present in tumour infiltrating lymphocytes (TILs). To verify in vitro the inhibitory activity of tumour isolated Tregs on the effector T cells and, finally, to assess the prognostic role of Treg frequency determination.
• Treg frequency in hPB, pPB and TILs was evaluated in 30 patients and 20 healthy controls by measuring both membrane-CD25 and intracytoplasmic-Foxp3 expression by flow cytometry. • Treg inhibitory activity was evaluated by an in vitro proliferation assay performed on total, CD25-depleted mononuclear cells (MNC) and CD25-depleted MNC cultured in the presence of purified CD25(+) Tregs. • Finally, Treg frequency in pPB and TIL were correlated with conventional prognostic factors and scores of University of California Los Angeles and Kattan predictive models.
• Treg frequency was higher in TILs than in pPB (P= 0.002), whereas there were no important differences between hPB and pPB. CD25(+) cells isolated either from PB and tumours showed the ability to significantly suppress in vitro both proliferation and interferon-γ production by CD25-depleted MNC, thus demonstrating that they are active Tregs. • Treg frequency was found to significantly correlate both with pathological stage (pPB, P= 0.03; TIL, P= 0.04) and nuclear grade (TIL, P= 0.005), both for UCLA and Kattan models (all: P < 0.05 for both pPB and TIL).
• Treg frequency is significantly higher in TIL than in pPB of patients with RCC. Tregs showed in vitro an inhibitory activity on effector T cells isolated from kidney tumours. The increase in both peripheral and intratumoral Tregs in subjects affected with RCC were associated with worse prognosis.
Full-text · Article · May 2011 · BJU International
[Show abstract][Hide abstract] ABSTRACT: Glomerular diseases account for 90% of end-stage kidney disease. Podocyte loss is a common determining factor for the progression toward glomerulosclerosis. Mature podocytes cannot proliferate, but recent evidence suggests that they can be replaced by renal progenitors localized within the Bowman's capsule. Here, we demonstrate that Notch activation in human renal progenitors stimulates entry into the S-phase of the cell cycle and cell division, whereas its downregulation is required for differentiation toward the podocyte lineage. Indeed, a persistent activation of the Notch pathway induced podocytes to cross the G(2)/M checkpoint, resulting in cytoskeleton disruption and death by mitotic catastrophe. Notch expression was virtually absent in the glomeruli of healthy adult kidneys, while a strong upregulation was observed in renal progenitors and podocytes in patients affected by glomerular disorders. Accordingly, inhibition of the Notch pathway in mouse models of focal segmental glomerulosclerosis ameliorated proteinuria and reduced podocyte loss during the initial phases of glomerular injury, while inducing reduction of progenitor proliferation during the regenerative phases of glomerular injury with worsening of proteinuria and glomerulosclerosis. Taken altogether, these results suggest that the severity of glomerular disorders depends on the Notch-regulated balance between podocyte death and regeneration provided by renal progenitors.