Circulating Fibrocytes, their role in renal fibrosis and molecular pathways involved. Possible biomarkers of fibrogenesis in chronic kidney disease

Circulating Fibrocytes (CFs) are bone marrow-derived mesenchymal progenitor cells that express a similar pattern of surface markers related to leukocytes, hematopoietic progenitor cells and fibroblasts. CFs precursor display an ability to differentiate into fibroblasts and Myofibroblasts, as well as adipocytes. Fibrocytes have been shown to contribute to tissue fibrosis in the end-stage renal disease (ESRD), as well as in other fibrotic diseases, leading to fibrogenic process in other organs including lung, cardiac, gut and liver. This evidence has been confirmed by several experimental proofs in mice models of kidney injury. In the present study, we developed a protocol for the study of CFs, by using peripheral blood monocytes cells (PBMCs) samples collected from healthy human volunteers. Thanks to a flow cytometry method, in vitro culture assays and the gene expression assays, we are able to study and characterize this CFs population. Moreover, results confirmed that these approaches are reliable and reproducible for the investigation of the circulating fibrocytes population in whole blood samples. Our final aim is to confirm the presence of a correlation between the renal fibrosis progression, and the different circulating fibrocyte levels in Chronic Kidney Disease (CKD) patients. Thanks to a protocol study presented and accepted by the Ethic Committee we are continuing the study of CFs induction in a cohort of sixty patients affected by CKD, divided in three distinct groups for different glomerular filtration rate (GFR) levels, plus a control group of thirty healthy subjects. Ongoing experiments will determine whether circulating fibrocytes represent novel biomarkers for the study of CKD progression, in the early and late phases of this disease.

Molecular genetics of inherited cystic kidney diseases: new diagnostic approaches

Background. Hhereditary cystic kidney diseases are a heterogeneous spectrum of disorders leading to renal failure. Clinical features and family history can help to distinguish the recessive from dominant diseases but the differential diagnosis is difficult due the phenotypic overlap. The molecular diagnosis is often the only way to characterize the different forms. A conventional molecular screening is suitable for small genes but is expensive and time-consuming for large size genes. Next Generation Sequencing (NGS) technologies enables massively parallel sequencing of nucleic acid fragments. Purpose. The first purpose was to validate a diagnostic algorithm useful to drive the genetic screening. The second aim was to validate a NGS protocol of PKHD1 gene. Methods. DNAs from 50 patients were submitted to conventional screening of NPHP1, NPHP5, UMOD, REN and HNF1B genes. 5 patients with known mutations in PKHD1 were submitted to NGS to validate the new method and a not genotyped proband with his parents were analyzed for a diagnostic application. Results. The conventional molecular screening detected 8 mutations: 1) the novel p.E48K of REN in a patient with cystic nephropathy, hyperuricemia, hyperkalemia and anemia; 2) p.R489X of NPHP5 in a patient with Senior Loken Syndrome; 3) pR295C of HNF1B in a patient with renal failure and diabetes.; 4) the NPHP1 deletion in 3 patients with medullar cysts; 5) the HNF1B deletion in a patient with medullar cysts and renal hypoplasia and in a diabetic patient with liver disease. The NGS of PKHD1 detected all known mutations and two additional variants during the validation. The diagnostic NGS analysis identified the patient’s compound heterozygosity with a maternal frameshift mutation and a paternal missense mutation besides a not transmitted paternal missense mutation. Conclusions. The results confirm the validity of our diagnostic algorithm and suggest the possibility to introduce this NGS protocol to clinical practice.

Simkania negevensis: clinical and laboratory studies in the population of hemodialized and kidney transplanted patients.

Simkania negevensis is a bacterium belonging to the order Chlamydiales but with certain biological characteristics different from those of chlamydia, according to which it was classified in the family Simkaniaceae. It is widespread in the environment, due to its ability to survive in amoebae also in phase cystic, for which it was hypothesized a possible transmission after contact with water in which they are present amoebae. So far it is known its role in diseases of the lower respiratory tract, such as childhood bronchiolitis and pneumonia in adults of the community, following its transmission through infected aerosols. A recent American study showed, by PCR, a high prevalence of S. negevensis in patients with lung transplant than other transplant recipients, assuming an association between the presence of the bacterium in these patients, and transplant rejection, were more frequent in lung transplant recipients infected compared to uninfected. There are no data so far analyzed in Italy relative to the population of dialysis and kidney transplant recipients relative to simkania negevensis why this study was undertaken in order to start a specific location and evaluate the scientific implications. Because its ability to assume persistent forms of infection, which may lead to a prolonged inflammatory response, Simkania negevensis, similar to other persistent bacteria or viruses, may be ivolved in pathologic complication. Sn may be a factor in graft rejection in mmunesuppressed lung transplant recipients, and further studies are planned to explore the posible association of Sn infections with various in vivo pathologies.

Evaluation of renal allograft function early after transplantation with diffusion-weighted MR imaging

To determine the inter-patient variability of apparent diffusion coefficients (ADC) and concurrent micro-circulation contributions from diffusion-weighted MR imaging (DW-MRI) in renal allografts early after transplantation, and to obtain initial information on whether these measures are altered in histologically proven acute allograft rejection (AR).

Validity of an automatic measure protocol in distal femur for allograft selection from a three-dimensional virtual bone bank system

Osteoarticular allograft is one possible treatment in wide surgical resections with large defects. Performing best osteoarticular allograft selection is of great relevance for optimal exploitation of the bone databank, good surgery outcome and patient’s recovery. Current approaches are, however, very time consuming hindering these points in practice. We present a validation study of a software able to perform automatic bone measurements used to automatically assess the distal femur sizes across a databank. 170 distal femur surfaces were reconstructed from CT data and measured manually using a size measure protocol taking into account the transepicondyler distance (A), anterior-posterior distance in medial condyle (B) and anterior-posterior distance in lateral condyle (C). Intra- and inter-observer studies were conducted and regarded as ground truth measurements. Manual and automatic measures were compared. For the automatic measurements, the correlation coefficients between observer one and automatic method, were of 0.99 for A measure and 0.96 for B and C measures. The average time needed to perform the measurements was of 16 h for both manual measurements, and of 3 min for the automatic method. Results demonstrate the high reliability and, most importantly, high repeatability of the proposed approach, and considerable speed-up on the planning.

Lymphangiogenesis is upregulated in kidneys of patients with multiple myeloma

Neolymphangiogenesis has recently been demonstrated in transplanted kidneys as well as in chronic interstitial nephritis and IgA nephropathy. However, its significance in kidney disease remains to be defined and a systematic study of renal lymphangiogenesis is warranted. We investigated patients with multiple myeloma (MM) presenting in the great majority with acute renal insufficiency. Controls were allograft kidney donors and patients with renal insufficiency due to acute renal failure (ARF). Lymph vessel length density (LVD) was quantified immunohistochemically by means of antipodoplanin staining followed by computer-assisted stereology. The mean LVD in kidneys of patients with MM (23.19 mm(-2)) was higher when compared with allograft donors (7.42 mm(-2), P = 0.0003) and patients with ARF (6.78 mm(-2), P = 0.0002). The higher LVD was significantly associated with interstitial inflammation, and the newly formed lymph vessels were accompanied by diffuse and nodular interstitial infiltrates composed mainly of CD20(+) B cells and CD27(+) plasma cells. The infiltrates in patients with MM also displayed a higher expression of the B-cell chemoattractant CXCL13. These results demonstrate for the first time that lymphangiogenesis is a prominent feature in MM kidneys and that it is associated with a significant accumulation of macrophages, CD20(+) and CD27(+) B lymphocytes. Further studies should clarify whether these changes represent a beneficial or detrimental factor in the progression of the myeloma-related kidney damage.

Pretransplant pulmonary hypertension and long-term allograft right ventricular function

Graft right ventricular (RV) function is compromised directly posttransplant, especially in heart transplantation (HTx) recipients with pretransplant pulmonary hypertension (PH). Graft RV size and systolic function, and the effect of the recipient's pulmonary haemodynamics on the graft extracellular matrix are not well characterised in the patients long-term after HTx.

Urinary proteomics before and after extracorporeal circulation in patients with and without acute kidney injury

Acute kidney injury is a well-known complication with high morbidity and mortality after cardiopulmonary bypass. Cardiopulmonary bypass-associated acute kidney injury is still poorly understood.

Mapping of a new locus for congenital anomalies of the kidney and urinary tract on chromosome 8q24

Congenital anomalies of the kidney and urinary tract (CAKUT) account for the majority of end-stage renal disease in children (50%). Previous studies have mapped autosomal dominant loci for CAKUT. We here report a genome-wide search for linkage in a large pedigree of Somalian descent containing eight affected individuals with a non-syndromic form of CAKUT.

Impact of daclizumab, low-dose cyclosporine, mycophenolate mofetil and steroids on renal function after kidney transplantation

Early and long-term use of cyclosporine A (CsA) leads to increased risks of renal toxicity. We hypothesized that administration of daclizumab in combination with mycophenolate mofetil (MMF) allows a relevant reduction in the dose of CsA.

In vivo nuclear translocation of mineralocorticoid and glucocorticoid receptors in rat kidney: differential effect of corticosteroids along the distal tubule

Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that inactivates 11β-hydroxy glucocorticoids. High expression levels of 11β-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCl loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11β-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11β-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function.

Diffusion-weighted MR imaging of native and transplanted kidneys

Applications of diffusion-weighted (DW) magnetic resonance (MR) imaging outside the brain have gained increasing importance in recent years. Owing to technical improvements in MR imaging units and faster sequences, the need for noninvasive imaging without contrast medium administration, mainly in patients with renal insufficiency, can be met successfully by applying this technique. DW MR imaging is quantified by the apparent diffusion coefficient (ADC), which provides information on diffusion and perfusion simultaneously. By using a biexponential fitting process of the DW MR imaging data, these two entities can be separated, because this type of fitting process can serve as an estimate of both the perfusion fraction and the true diffusion coefficient. DW MR imaging can be applied for functional evaluation of the kidneys in patients with acute or chronic renal failure. Impairment of renal function is accompanied by a decreased ADC. Acute ureteral obstruction leads to perfusion and diffusion changes in the affected kidney, and renal artery stenosis results in a decreased ADC. In patients with pyelonephritis, diffuse or focal changes in signal intensity are seen on the high-b-value images, with increased signal intensity corresponding to low signal intensity on the ADC map. The feasibility and reproducibility of DW MR imaging in patients with transplanted kidneys have already been demonstrated, and initial results seem to be promising for the assessment of allograft deterioration. Overall, performance of renal DW MR imaging, presuming that measurements are of high quality, will further boost this modality, particularly for early detection of diffuse renal conditions, as well as more accurate characterization of focal renal lesions.