Novel markers of graft outcome in a cohort of kidney transplanted patients: a cohort observational study.

Renal biopsy (RBx) informs about kidney transplantation (KTx) prognosis. In our observational study the prevalence of histological anomalies and the prognostic role of CD45, vimentin (VIM) and periostin (POSTN) in KTx-RBx have been evaluated. One hundred forty-six KTx-RBx (2009-2012) were analysed for general histology and in immunohistochemistry for CD45, VIM and POSTN. Clinical data of the 146-KTx patients were collected at the RBx time (T0), 6 and 12 months before and after RBx. Follow-up time was 21 ± 14 months. Glomerulosclerosis was 20% glomeruli/biopsy. Tubular atrophy (TA), Interstitial infiltrate (I-Inf) and interstitial fibrosis (IF) were slight in 21-18% and 25%, moderate in 22-30% and 26% and severe in 30-18% and 28% of patients. Fifty-eight percent of patients had lesions compatible with IF-TA. CD45, VIM and POSTN correlated to each-other and to TA, I-Inf and IF. VIM and POSTN correlated to GS. CD45 and VIM correlated directly to renal function (RF) and 25(OH)VitD, while POSTN inversely to 25(OH)VitD. Thirty patients restarted dialysis (HD+). HD+ had lower T0-eGFR, and higher CD45, VIM and POSTN than HD-. POSTN resulted the strongest in discriminate for HD+ . CD45, VIM and POSTN correlate to each-other and predict graft outcome. POSTN was the strongest in discriminate for HD+. 25(OH)VitD might influence inflammation and fibrosis in KTx.

FGF23-regulated production of Fetuin-A (AHSG) in osteocytes.

INTRODUCTION: AHSG, a serum glycoprotein with recognized anti-calcification activity, has also been suggested to modulate both bone formation and resorption. Though the bulk of AHSG is mostly synthesized in the liver, it has been claimed that also bone cells might produce it. However, the extent of the bone AHSG production and the potential controlling factors remain to be definitively proven. A relevant number of studies support the notion that FGF23, a bone-derived hormone, not only regulates the most important mineral metabolism (MM) related factors (phosphate, parathyroid hormone, vitamin D, etc.), but might be also involved in cardiovascular (CV) outcome, both in chronic kidney disease (CKD) patients and in the general population. Furthermore, in addition to some direct autocrine and paracrine effects in bone, FGF23 has been suggested to interact with AHSG. In this study we investigated if AHSG is really produced by bone cells, and if its bone production is related and/or controlled by FGF23, using cultured bone cells, according to a new method recently published by our group. RESULTS: Our data show that AHSG is consistently produced in osteocytes and to a far lesser extent in osteoblasts. Both FGF23 addition to the culture medium and its over-expression in osteocytes were associated with a consistent increase of both AHSG mRNA and protein, while FGF23 silencing was followed by opposite effects. Though most of these results were largely affected by the blockage of FGF23 receptors, the role of these receptors in the different experimental sets is still not completely clarified. In addition, we found that FGF23 and AHSG proteins co-localized both in cytoplasm and nucleus, which suggests a possible reciprocal interactivity. CONCLUSIONS: Our data not only confirm that AHSG is produced in bone, mainly in osteocytes, but show for the first time that its production is modulated by FGF23. Since both proteins play important roles in the bone and cardiovascular pathology, these results add new pieces to the puzzling relationship between bone and vascular pathology, in particular in CKD patients, prompting future investigations in this field.

SIK1 localizes with nephrin in glomerular podocytes and its polymorphism predicts kidney injury.

Mutant α-adducin and endogenous ouabain levels exert a causal role in hypertension by affecting renal Na-K ATPase. In addition, mutant ß-adducin is involved in glomerular damage through nephrin down-regulation. Recently, the salt-inducible kinase 1 (SIK1) has been shown to exert a permissive role on mutant α-adducin effects on renal Na-K ATPase activity involved in blood pressure (BP) regulation and a SIK1 rs3746951 polymorphism has been associated with changes in vascular Na-K ATPase activity and BP. Here, we addressed the role of SIK1 on nephrin and glomerular functional modifications induced by mutant ß-adducin and ouabain, by using congenic substrains of the Milan rats expressing either mutant α- or ß-adducin, alone or in combination, ouabain hypertensive rats (OHR) and hypertensive patients. SIK1 co-localized and co-immunoprecipitated with nephrin from glomerular podocytes and associated with caveolar nephrin signaling. In cultured podocytes, nephrin-gene silencing decreased SIK1 expression. In mutant ß-adducin congenic rats and in OHR, the podocyte damage was associated with decreased nephrin and SIK1 expression. Conversely, when the effects of ß-adducin on podocytes were blocked by the presence of mutant α-adducin, nephrin and SIK1 expressions were restored. Ouabain effects were also reproduced in cultured podocytes. In hypertensive patients, nephrinuria, but not albuminuria, was higher in carriers of mutant SIK1 rs3746951 than in wild-type, implying a more direct effect of SIK1 on glomerular damage. These results demonstrate that, through nephrin, SIK1 is involved in the glomerular effects of mutant adducin and ouabain and a direct effect of SIK1 is also likely to occur in humans.

A novel model of in vitro osteocytogenesis induced by retinoic acid treatment.

Despite recent research which more and more stresses the importance of osteocytes in regulating bone and systemic mineral metabolism, current molecular and functional knowledge of osteocyte properties are still incomplete, mostly due to limited availability of in vitro models. Osteocytes are terminally differentiated dendritic cells, and therefore are not easy to obtain and maintain in primary cultures. As an alternative, osteocyte differentiation can be induced by progressive osteoblast embedding in mineralised extracellular matrix. In this model, which is suitable for reproduction of bone development, the presence of calcified matrix prevents several cell biological methods from being used. Therefore, the osteocyte-like MLO-Y4 cell line continues to be the most widely used cellular system. Here we show that treatment of primary osteoblasts or MC3T3-E1 cells with retinoic acid generates a homogeneous population of ramified cells with osteocyte features, as confirmed by morphological and molecular analyses. The first morphological changes are detectable in primary cells after 2 days of treatment, and in the cell line after 4 days of treatment. Differentiation is complete in 5 and 10 days, respectively, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers, and up-regulation of osteocyte-specific molecules, most notably among them sclerostin. Compared to other published protocols, our method has a number of advantages. It is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in the complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Podocytes: a new player for glutamate signaling.

In the renal glomerulus, podocytes envelop the external side of the capillary basement membrane with their intertwining ramifications, and ensure elimination of metabolic waste within the urine, while proteins and important blood components are retained into the circulation. To preserve the integrity of the glomerular filter, which is constantly exposed to a high variety of stimuli, podocytes need to communicate by rapid and precise signaling, likely similar to that used by neuronal cells. In the last years, we and others have shown that podocytes are indeed molecularly equipped for communicating in a synaptic-like way, where glutamate and its receptors seem to have a pivotal role, because altering glutamatergic communication leads to podocyte damage and increased filter permeability. Major components of glutamatergic signaling are organized at foot process junctions by adhesion molecules, chiefly by nephrin, and are connected to the actin cytoskeleton, that governs the health of podocytes. Further advances in understanding podocyte physiological behavior and signaling properties have the potential to improve the knowledge of podocyte diseases, first among them idiopathic focal segmental glomerulosclerosis that still needs more precise molecular-based diagnosis and targeted treatment.

[Transcriptomics illustrate a deadly TRAIL to diabetic nephropathy]. / La transcriptómica ilustra nuevas vías letales en la nefropatía diabética.

Diabetic nephropathy is the most common cause of endstage renal disease. Approaches targeting angiotensin II significantly delay its progression. However, many patients still need renal replacement therapy. High throughput techniques such as unbiased gene expression profiling and proteomics may identify new therapeutic targets. Cell death is thought to contribute to progressive renal cell depletion in chronic nephropathies. A European collaborative effort recently applied renal biopsy transcriptomics to identify novel mediators of renal cell death in diabetic nephropathy. Twenty-five percent of cell death regulatory genes were upor downregulated in diabetic kidneys. TNF-related apoptosisinducing ligand (TRAIL) and osteoprotegerin had the highest level of expression. In diabetic nephropathy, tubular cells and podocytes express TRAIL. Inflammatory cytokines, including MIF via CD74, upregulate TRAIL. A high glucose environment sensitized renal cells to the lethal effect of TRAIL, while osteoprotegerin is protective. These results suggest that, in addition to glucose levels, inflammation and TRAIL are therapeutic targets in diabetic nephropathy.

Glomerular barrier dysfunction in glomerulosclerosis- resistant Milan rats with experimental diabetes: the role of renal haemodynamics.

Rats of the Milan hypertensive strain (MHS) are resistant to both hypertensive and diabetic renal disease. Genetically determined hypertrophy of intrarenal arteries has been suggested as the putative mechanism preventing transmission of systemic hypertension to the glomerular microcirculation or diabetes-induced loss of autoregulation, which lead to glomerular hypertension and consequent podocyte injury and proteinuria. This study aimed to investigate glomerular barrier function and structure in ageing and diabetic MHS rats under basal conditions and after injection of 2.5 g of bovine serum albumin (BSA) causing increased workload and possibly removing haemodynamic protection by inducing renal cortical vasodilatation. Genetically related rats of the Milan normotensive strain (MNS) served as a proteinuric counterpart. No change in renal function or structure was detected in diabetic MHS rats, whereas MNS rats developed diabetic nephropathy superimposed on that occurring spontaneously in this strain. Diabetic, but not non-diabetic, MHS rats showed significantly reduced synaptopodin and nephrin expression, though to a lesser extent than non-diabetic and diabetic MNS rats, together with unchanged podocyte number, density and structure and no proteinuria. Agrin expression was significantly altered in diabetic versus non-diabetic MHS animals, whereas collagen I was expressed only in diabetic MHS rats and collagen IV content did not change significantly between the two groups. Upon BSA injection, proteinuria increased markedly and abundant BSA was detected only in kidneys from diabetic MHS rats. BSA injection was associated with changes in intrarenal arteries suggesting vasodilatation, without any influx of inflammatory cells. These data indicate that while MNS rats show marked changes in the glomerular filtration barrier with either age or diabetes, glomerulosclerosis-resistant MHS rats develop only minor diabetes-induced podocyte (and extracellular matrix) alterations, which are not associated with proteinuria unless they are unmasked by an increased workload or removal of the haemodynamic protection.

Apoptosis in the kidneys of patients with type II diabetic nephropathy.

The occurrence and extent of apoptosis in the kidneys of patients with diabetic nephropathy is largely unknown. We evaluated apoptosis in renal biopsies obtained from patients with early or advanced type II diabetic nephropathy. Apoptosis was about 6- and 3-fold higher, respectively, in glomeruli and tubules in kidneys of patients with early nephropathy than in the normal kidney and this was not further increased in advanced diabetic nephropathy. Glomerular apoptosis was related directly to hemoglobin A1(c) and systolic blood pressure, whereas tubular cell apoptosis correlated to diabetes duration and low-density lipoprotein-cholesterol. Fas, Fas ligand, and p38 mitogen-activated protein kinase expressions were enhanced in glomeruli and tubules; however, this did not correlate with apoptosis. In patients with proteinuria, apoptosis was associated with the subsequent loss of kidney function. When these parameters were subjected to multivariate analysis, only glomerular apoptosis retained a significant independent predictive value. Our findings suggest that apoptosis might be a clinically relevant mechanism of glomerular and tubular cell loss in proteinuric type II diabetic patients.

[The renal biopsy in the post-genomic era.]. / L'approccio alla biopsia renale e le nuove possibilita' diagnostiche: cosa cambia nell'era postgenomica.

Histological and immunohistological examination of renal biopsy material is the method of choice for the diagnosis of glomerular and interstitial renal disease. However, our understanding of renal damage is still largely incomplete because of the limited knowledge of the etiology and pathogenesis of numerous kidney diseases. For this reason, we still provide unspecific treatment to kidney patients, which is generally aimed at counteracting inflammatory alterations and slowing progression towards renal failure, without intervening directly in the cause of the disease. The recent development of the ''omics'' (genomics, proteomics, metabolomics) following the enormous progress of high-throughput technologies and information technology tools is profoundly transforming our knowledge in every biomedical field, including nephrology. It is expected that in a very short time a better understanding of both physiological and pathological events in the kidney will translate into different therapeutic strategies, possibly targeted to individual needs. Nephrologists and renal pathologists must take these changes into account and realize that a new approach to renal biopsy is urgently required. Renal biopsy material has in fact an enormous importance in the generation of new knowledge and in the validation of experimental results from high-throughput technologies and animal models. Furthermore, it is conceivable that a new classification of renal diseases will be needed soon as a result of the improved knowledge. For these reasons, renal biopsy material should be adequately processed and preserved according to modern methods, and collaborative projects should be fostered to achieve standardized methods and avoid a waste of energy in singular efforts.

Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

Histopathological atlas of renal diseases: anti-glomerular basement membrane antibody disease.

The main diagnostic feature of anti-glomerular basement membrane (anti-GBM) antibody disease is represented by the immunofluorescence pattern of intense and diffuse linear IgG deposition along the glomerular basement membrane. By light microscopy several histological patterns can be observed.

Cortical fibroblast culture from human biopsies.

BACKGROUND: Tubulointerstitial fibrosis is an integral part of progressive renal disease. Human cortical fibroblasts are believed to be key effector cells in fibrogenesis. Thus, a reliable culture of these cells is necessary for studies of their pathophysiology. METHODS: Cortical fibroblast culture from routine kidney biopsies were analyzed and the cells were characterized. Indirect immunofluorescence staining was done after the first passage for cytokeratin, vimentin, alpha-smooth muscle actin, CD 44, CD 54, CD 68, collagen types I, III, and HLA-DR. We then assessed the utility of the putative fibroblast markers CD 90, prolyl-4-hydroxylase (P4H) and F1b in simultaneous stainings of tubular epithelial cells. RESULTS: During the study period, 49 biopsy cores were cultured and cortical fibroblasts could be successfully established in 21 cases (42.9%). There was no relation between the success rate of culture and the degree of interstitial fibrosis, but an association was seen with the time of completion of the first passage. There was a negative correlation between the extent of scarring and the percentage of cytokeratin positive cells (r = -0.66, p < 0.001). All primary fibroblasts were negative for factor VIII, HLA-DR, CD 68, and cytokeratin. They expressed alpha-smooth muscle actin and collagen types I and III to variable degrees. There was a robust correlation between the percentage of alpha-smooth muscle actin positive cells and interstitial scarring but no such association with collagen type I or type III positive cells. The three putative fibroblast markers did not prove useful in differentiating between tubular epithelial cells and fibroblasts. However, since only fibroblasts stained positive for CD 90 and negative for cytokeratin, these two markers may suffice to distinguish fibroblasts from other renal cellular elements. CONCLUSIONS: Cortical renal fibroblasts can be easily cultured from kidney biopsy cores, though the success rate of pure cultures is below 50%. Staining for CD 90 and cytokeratin may suffice for initial characterization of these cells.

Idiopathic IgA nephropathy with segmental necrotizing lesions of the capillary wall.

BACKGROUND: Segmental glomerular necrosis has been described in the biopsy material in a minority of patients with idiopathic IgA nephropathy in the oldest studies on this disease, but this marker of active capillaritis has received little attention in the subsequent literature, and its significance and relevance for the clinical outcome is still unknown. METHODS: Thirty-five out of 340 patients (10.3%) biopsied in our division at the San Carlo Hospital since 1974 showed active segmental necrotizing lesions. The morphological features and the natural history of this group of patients were compared with those of a control group of 229 patients who had comparable serum creatinine and extent of glomerular sclerosis, but who lacked active segmental necrosis. RESULTS: Patients with the necrotic variant showed a significantly more marked extracapillary proliferation and interstitial accumulation of monocytes and T lymphocytes and, in the segmental areas of necrotizing and extracapillary lesions, infiltration of monocytes, deposition of fibrinogen, and expression of the adhesion molecule vascular cell adhesion molecule-1. No difference was found in the presenting clinical syndrome. The clinical course was frequently characterized by acute flare ups, and the progression to end-stage renal failure was more frequent, although actuarial renal survival was not significantly worse (P = 0.07). The aggressive treatment with steroids and cyclophosphamide, carried out in 20 of the 35 patients, has probably been beneficial, justifying the multicenter controlled trial that recently has been initiated. CONCLUSIONS: Vasculitic lesions of the glomerular capillaries, with histologic and immunohistological features similar to those of Henoch-Schönlein purpura and antineutrophil cytoplasmic antibody-positive renal vasculitis, were found in 10% of patients with idiopathic IgAN. Clinical features at presentation did not differ from those of the other patients with IgAN, and despite of the more frequent occurrences of recurrent acute flare ups, rapid progression to end-stage renal failure was a rare phenomenon, even in untreated patients.

Morphological features in IgA nephropathy.

The hallmark of Berger's disease is the mesangial and/or mesangioparietal deposition of IgA as the predominant or sole immunoglobulin. Despite similar appearance to the immunohistological pattern, morphological glomerular lesions differ in that they are wide ranging and variable, making precise and uniform classificatory approaches extremely difficult. The most characteristic and frequent abnormality is mesangial enlargement, which is produced by various combinations of excess of matrix and of hypercellularity, ranging from minimal to very extensive. In some cases the mesangial lesions are more severe giving a pattern of membrano-proliferative glomerulonephritis. The concomitant presence of necrotizing alterations of glomerular tuft with segmental extracapillary proliferation, similar to capillaritis of Henoch-Schönlein purpura and ANCA associated vasculitis, is now well documented and recognized by researchers. This similarity with vasculitic lesions is confirmed by the strong positivity of fibrinogen, of VCAM-1, and of the accumulation of monocytes within the same areas of segmental necrotic glomerular lesions. These active lesions appear to play a crucial role in the progression of the disease due to repeat formations of necrotizing/extracapillary alterations with subsequent glomerular sclerosis and fibrous adhesions. In the last decade, many groups of investigators have focused their attention on tubulo-interstitial lesions in IgA nephropathy, in particular, on leukocyte infiltration and intersitial fibrosis, demonstrating that the impairment of the glomerular filtration rate and the progression of the disease correlate better with tubulo-interstitial damage than with the degree of glomerular damage. This has also been confirmed by studies with repeat biopsies. Moreover, the recent availability of immunohistochemical and in situ hybridation methods that allow more precise evaluations of infiltrating cells and of numerous factors secreted by these cells (chemokines, cytokines, adhesion molecules etc ...) offers us incredible opportunities to expand our knowledge on mechanisms involved in the inflammatory process and in the progression of the disease.

Transforming growth factor-beta, endothelin-1, and c-fos expression in necrotizing/crescentic IgA glomerulonephritis.

BACKGROUND: Among our cases of IgA glomerulonephritis (IgAGN), 10% show necrotizing/extracapillary lesions involving a small percentage of glomeruli and associated with a certain degree of inflammation in absence of glomerular and interstitial scarring. In our experience, also in repeat biopsies, these cases of IgAGN have a worse prognosis probably because necrotizing/extracapillary lesions can repeat and accumulate, leading to the progression of damage. As it is well known that transforming growth factor-beta (TGF-beta) and endothelin-1 (ET-1) are key-factors in the progression of glomerulonephritis, aim of the study was to examine their expression in renal biopsies of primary IgAGN with necrotizing/crescentic lesions in complete absence of interstitial fibrosis. To obtain information about the mitogenic effect of ET-1, the expression of c-fos, whose upregulation by ET-1 has been established in culture, was also studied. METHODS: Eighteen renal biopsies of patients with necrotizing/crescentic IgAGN were examined by immunohistochemistry with antibodies against TGF-beta, ET-1 and c-fos. The results were compared with those obtained on 22 cases of IgAGN characterized only by pure mesangial proliferation and 25 IgAGN biopsies with advanced, not active, glomerulointerstitial lesions. RESULTS: In necrotizing/crescentic IgAGN glomerular TGF-beta appeared more positive than in cases characterized only by pure mesangial proliferation and was especially expressed on cellular crescents. In the interstitium, TGF-beta, ET-1 and c-fos were expressed by infiltrating leukocytes, tubules, and small vessels. This positivity, although similar as localization, was less diffuse than in biopsies with advanced interstitial damage, but significantly greater than in cases with pure mesangial proliferation. CONCLUSIONS: Positivity of TGF-beta on cellular crescents is similar to that observed from other authors in different types of necrotizing/crescentic human glomerulonephritis and supports our hypothesis that this is a peculiar type of IgAGN. Moreover, interstitial expression of TGF-beta, ET-1 and c-fos in biopsies with glomerular active lesions but complete absence of interstitial fibrosis may potentially represent a signal of activation of mechanisms that induce and amplify the damage leading to further progression of the disease.

Adhesion molecules expression in noncrescentic acute post-streptococcal glomerulonephritis.

Clinicomorphological features of 11 cases of non-crescentic acute post-streptococcal glomerulonephritis (APSGN) were reviewed. Intraglomerular and interstitial leukocytes and their possible correlation with the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial-leukocyte adhesion molecule-1 (ELAM-1/E-selectin) were investigated by an immunohistochemical method. Intraglomerular leukocytes were primarily granulocytes (11.4 +/- 10 cells/glomerular cross-section) and monocytes-macrophages (13.4 +/- 19.4 cells/glomerular cross-section). The granulocytes outnumbered monocytes-macrophages in 7 of 11 specimens. The number of intraglomerular leukocytes correlated with proteinuria at the time of renal biopsy. Intraglomerular ICAM-1 staining was strongly positive in all biopsies, especially when intraglomerular monocytes-macrophages prevailed. Expression of intraglomerular VCAM-1 and E-selectin in diseased kidneys did not differ from that in normal kidneys. Interstitial leukocytes were primarily monocytes-macrophages (158.9 +/- 96.8 cells/mm2) and T lymphocytes (102.2 +/- 63.9 cells/mm2). The number of interstitial leukocytes, especially monocytes-macrophages, correlated with serum creatinine level at the time of biopsy. Interstitial ICAM-1 staining was strongly positive on tubules, peritubular capillaries, and small vessels. The tubular positivity for ICAM-1 correlated with the number of interstitial monocytes-macrophages. Interstitial VCAM-1 and E-selectin were expressed as in normal kidney tissues. The data from this study demonstrate that APSGN is characterized by the presence of both intraglomerular and interstitial leukocyte infiltration, correlating respectively with proteinuria and serum creatinine at the time of renal biopsy. Among the adhesion molecules studied, ICAM-1 seems the most involved in leukocyte recruitment, especially in that of monocytes-macrophages.