Manuela Della Vella

TGFβ1 induces epithelial–mesenchymal transition, but not myofibroblast transdifferentiation of human kidney tubular epithelial cells in primary culture

The origin and fate of renal interstitial myofibroblasts (MFs), the effector cells of renal fibrosis, are still debated. Experimental evidence suggests that renal MFs derive from tubular epithelial cells throughout the epithelial–mesenchymal transition (EMT) process. Primary human tubular epithelial cells (HUTECs) were cultured for 4 and 6 days on plastic or type I collagen‐coated plates with 1, 5, 10 and 50 ng/ml of transforming growth factor β1 (TGFβ1). The EMT process was monitored by morphology and immunophenotyping for αSMA, cytokeratin 8–18, E‐cadherin, vimentin and collagen III. Quantitative comparative RT/PCR and real‐time PCR were used to evaluate the expression of collagen III and IV, fibronectin, tenascin, MMP‐2, CTGF, E‐cadherin and cadherin 11 genes, as well as those of the Smad signalling pathway. TGFβ1 was found capable of reactivating the mesenchymal programme switched off during tubulogenesis, but it induced no de novo expression of αSMA gene or myofibroblast phenotype. We demonstrate that the EMT process is conditioned by the extracellular matrix and characterized by TGFβ1‐driven Smad3 downregulation. Our study results suggest that TGFβ1 could function as a classic embryonal inducer, initiating a cascade of de‐differentiating events that might be further controlled by other factors in the cellular environment.

Selective Decrease in Urinary Aquaporin 2 and Increase in Prostaglandin E2 Excretion Is Associated with Postobstructive Polyuria in Human Congenital Hydronephrosis

This study was undertaken to determine the role of aquaporin 2 (AQP2) in the impaired urinary concentrating capacity observed in patients who underwent pyeloplasty because of congenital unilateral hydronephrosis as a result of pyeloureteral junction disease. Twelve children (mean age, 8 +/- 2 mo) were examined in the study. From day 1 to day 5 after surgery, the urine was collected separately from pyelostomy draining only from the postobstructed kidney and from the bladder catheter draining mostly from the contralateral kidney used as internal control. After pyeloplasty, the postobstructed kidney was characterized by a reduced urinary excretion of AQP2 (approximately 54%) associated with polyuria that persisted from day 1 to day 5 (433 +/- 58 versus 310 +/- 74 ml/24 h at day 1; 326 +/- 44 versus 227 +/- 26 ml/24 h at day 5). In parallel, urine osmolality from the postobstructed kidney was significantly reduced compared with the contralateral kidney (111 +/- 12 versus 206 +/- 49 at day 1; 136 +/- 24 versus 235 +/- 65 mOsm/kg at day 5). Creatinine clearance from the postobstructed kidney was not significantly different compared with the contralateral kidney throughout the 4 d after surgery. However, on day 5, creatinine clearance from the postobstructed kidney became significantly lower. Prostaglandin E2 in the urine from postobstructed kidneys was found to be twofold higher than in the contralateral samples (26.0 +/- 6.7 versus 13.5 +/- 2.5 at day 5). It is concluded that (1) the selective downregulation of AQP2 in postobstructed kidney may account for the higher excretion of hypotonic urine, and (2) the local increase in prostaglandin E2 synthesis in postobstructed kidney may be involved in AQP2 downregulation and in maintaining a GFR similar to that of the contralateral kidney.

Expression of Nuclear Transcription Factor PAX2 in Renal Biopsies of Juvenile Nephronophthisis

PAX2, a homeotic gene of ‘paired box family’, is a nuclear transcription factor expressed in mesenchymal/epithelial conversion during the early stages of nephrogenesis; however, its repression is necessary for terminal differentiation of mature tubular cells. Transgenic overexpression in animal model causes epithelial hyperproliferation and microcyst formation. In humans, PAX2 expression has been observed in cystic and dysplasic tubular epithelia in kidney malformation and in kidney disease. We have investigated PAX2 expression and its colocalization with cytokeratin and/or vimentin in 17 biopsies of juvenile nephronophthisis (NPH), an autosomal-recessive renal disease characterized by diffuse renal fibrosis and occasional cysts. Fourteen cases were analyzed for deletion and mutation in the NPH1 gene locus and 33% resulted to be deleted or mutated; for the remaining cases the diagnosis was based on clinical and pathological criteria. The control group included 4 congenital dysplastic kidneys, and 10 biopsies of nephropathies with secondary chronic tubulointerstitial damage. In all cases of renal dysplasia a strong nuclear positivity was observed in immature tubules surrounded by αSMA-positive mesenchymal cells. In NI biopsies the tubular epithelia were almost PAX2 negative, although tubulointerstitial damage was severe. In 14/17 NPH1 moderate-to-strong nuclear PAX2 positivity of tubular cells was observed, particularly in cystic distal tubules located at the corticomedullary junction, but also in proximal tubular sections. The PAX2 signal co-localized more with cytokeratin staining than with vimentin. Our results confirm the observation of PAX2 expression in immature dysplastic tubules and its repression in mature renal tubular cells, also in the presence of severe secondary interstitial fibrosis. PAX2 seems to be overexpressed in NPH. The genetic defect of NPH, a disease probably due to a primary defect along the cascade of mesenchymal epithelial differentiation, could generate a functionally abnormal protein involved in focal adhesion signaling and cell/matrix interaction. The failure of PAX2 repression or its reactivation in NPH could be a marker of hyperproliferation and incomplete maturation of epithelial tubular cells, probably due to a defect cell/matrix cross-talk, and involved in interstitial fibrosis and cysts formation.

Investigation of Intrarenal Viral Infections in Kidney Transplant Recipients Unveils an Association between Parvovirus B19 and Chronic Allograft Injury

BACKGROUND The relevance of viral infections in the development of allograft lesions is still unclear, although some viruses have been implicated. The present study investigated systemic and intrarenal viral infections in kidney transplant recipients and their association with the risk of acute rejection and chronic allograft injuries that are predictive of long-term dysfunction. METHODS The presence of DNA sequences of human herpesviruses, polyomaviruses, and parvovirus B19 was analyzed in renal allograft biopsy specimens obtained at baseline, after acute renal dysfunction, and during follow-up evaluation in 69 transplant recipients who were children or young adults. Results were correlated with clinical data, viral DNAemia, and results of renal function tests and allograft histology analyzed at the same time points. RESULTS Overall, viral DNA was detectable in 46% of baseline and 70% of follow-up biopsy specimens of kidney allografts, where it generally persisted. The most frequently detected viruses were B19 and human herpesvirus 6, already present in donor kidneys, and BK virus and Epstein-Barr virus, usually involving the allograft during follow-up. Among viruses, only the intrarenal persistence of B19 DNA and B19 DNAemia was associated with the development of chronic allograft injury, whereas human cytomegalovirus DNAemia was a risk factor for acute rejection. CONCLUSIONS Parvovirus B19 seems to target the kidney electively. Its intrarenal persistence is associated with chronic kidney allograft injury.

Immature Renal Structures Associated With a Novel UMOD Sequence Variant

Mutations of the UMOD gene, encoding uromodulin, have been associated with medullary cystic kidney disease 2, familial juvenile hyperuricemic nephropathy, and glomerulocystic kidney disease. We report on a 13-year-old boy presenting with chronic reduced kidney function, hyperuricemia, and impairment in urine-concentrating ability. His father was affected by an undefined nephropathy that required transplantation. The boys renal ultrasonography showed reduced bilateral kidney volumes and cortical hyperechogenicity, with 2 tiny cysts in the left kidney. Renal biopsy showed up to 60% of glomeruli featuring an enlargement of Bowman space (glomerular cysts), with mild interstitial fibrosis (alpha-smooth muscle actin [alphaSMA] positive), inflammatory infiltrate, and focal tubular atrophy at the cortical level. At the corticomedullary junction, immature tubules (some dilated) with cytokeratin- and paired box gene 2 (PAX2)-positive immunostaining were seen, surrounded by vimentin-positive mesenchymal tissue. Unlike previously reported cases, no uromodulin-positive globular aggregates within the cytoplasm of tubular cells were observed. Uromodulin urinary excretion was absent. Genetic analysis showed a novel heterozygous sequence change in the UMOD gene (NM_003361.2:c.149G-->C; p.Cys50Ser) involving the first epidermal growth factor-like domain of the protein in both the boy and his father. This novel UMOD sequence variant, which is associated with an immunohistochemical pattern different from previous reports and a histological picture characterized by immature renal structures, suggests a possible role for UMOD in renal development.

Plasmapheresis-resistant acute humoral rejection successfully treated with anti-C5 antibody

Even if kidney graft survival has improved during the last decades, sensitized pediatric patients are an emerging problem. We describe a 17‐yr‐old male who lost his first graft due to chronic rejection becoming hyperimmunized (CDC PRA 99.61%). A desensitization protocol based on high‐dose IVIG, PP, and two Mabthera® infusions was performed with minor response (CDC PRA post‐desensitization 80%). One month after his second non‐living transplant, he developed a biopsy‐proven AMR; post‐transplant immunological monitoring showed the presence of donor‐specific anti‐DQ5 antibodies (DSA, MFI 20.000). He received methylprednisolone pulses and 45 PP sessions without clinical response; eculizumab was then used to salvage a kidney undergoing severe PP‐resistant rejection. A biopsy performed after the fourth eculizumab infusion showed complete resolution of AMR. Eculizumab infusions were then continued for the first year post‐transplantation. Two yr after transplantation, graft function is stable. Anti‐C5 therapy may represent an effective therapeutic option in pediatric patients with PP‐resistant AMR.

Decreased expression and promoter methylation of the menin tumor suppressor in pancreatic ductal adenocarcinoma

Loss of menin, a tumor suppressor coded by the MEN1 gene, is a key factor in the pathogenesis of multiple endocrine neoplasia type I and in a percentage of sporadic endocrine tumors of the pancreas and parathyroid glands. This study investigated expression of the menin protein in the normal exocrine pancreas and in pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic tumor. Immunofluorescence (IF) analyses showed that menin is expressed at high levels in normal acinar and duct cells. Examination of 24 clinical samples of PDAC revealed a pronounced decrease in menin expression in all tumors examined. To identify alterations underlying this defect, we searched for disruption and epigenetic silencing of the MEN1 gene. Analysis of nine laser‐microdissected tumors revealed loss of heterozygosity of intragenic (one tumor) or adjacent (three tumors) MEN1 microsatellite markers. Methylation of CpG sites in the MEN1 promoter was documented in five of 24 tumors. IF analyses also revealed low to undetectable menin expression in the PDAC cell lines MiaPaCa‐2 and Panc‐1. Ectopic expression of menin in these cells resulted in a marked alteration of the cell cycle, with an increase in the G1/S+G2 ratio. These findings represent the first evidence that the MEN1 gene is a target of mutation and methylation in PDAC and that menin influences the cell cycle profile of duct cells.

Detection of viral DNA in kidney graft preservation and washing solutions is predictive of posttransplant infections in pediatric recipients.

BACKGROUND In pediatric kidney transplant recipients, viral infections occur soon after transplant and may be transmitted from the graft. METHODS This study of 75 pediatric kidney transplants investigated whether genome sequences of parvovirus B19, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and BK polyomavirus (BKV) could be detected in kidney graft samples (graft biopsy samples and preservation and washing solutions) collected before implantation and whether their presence was a risk factor for infections in the recipient. RESULTS B19 DNA was detected in approximately 30% of graft biopsy samples, preservation solutions, and washing solutions; EBV DNA was detected in approximately 20% of preservation and washing solutions but rarely in biopsy samples; and HCMV DNA and BKV DNA were rarely detected in graft biopsy samples. Seronegative recipients of B19 DNA-positive and EBV DNA-positive grafts had a significantly higher risk of infection during the early posttransplant period than did recipients of negative grafts. In particular, none of the B19-seronegative recipients of B19 DNA-negative grafts experienced infection soon after transplant, whereas most recipients of B19 DNA-positive grafts experienced infection within the first month after transplant. CONCLUSIONS Molecular testing of donor grafts for viruses that infect circulating and resident cells in the graft-such as B19 in the kidney-could be useful (in association with donor/recipient serostatus) for identifying recipients at high risk for posttransplant infections.