Biomedical subjects
Publications and source records attributed to L Gesualdo.
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IgA nephropathy, the most common form of primary glomerulonephritis, progresses to terminal renal failure in about 25% of patients 10 years after the apparent clinical onset. Since its description in 1968 an intense research effort in order to clarify the pathogenetic mechanisms has involved the study of animal models of the disease. In this review we analyze the experimental work reported since 1979, when the first animal model of IgA nephropathy was published by Rifai et al. We also discuss the interplay between experimental data and relevant clinical observations. Finally, we report the new insights about the role played by cytokines and growth factors.
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BACKGROUND: Uremia produces a wide range of abnormalities of the immune system. Blood-membrane interaction in hemodialysis results in activation and severe dysfunction of peripheral blood mononuclear cells (PBMC). However, the question of whether the use of different dialytic membranes may improve PBMC dysfunctions remains unanswered. METHODS: To address this issue, the spontaneous interleukin (IL)-6, IL-8 and monocyte chemotactic peptide-1 (MCP-1) gene expression and protein release were studied in PBMC isolated from 7 healthy subjects, 8 uremic patients on conservative therapy and 8 uremic patients undergoing subsequent one month periods of hemodialysis with cuprophan (CU) and high-flux noncomplement activating membranes, polymethylmethacrylate (PMMA) and polyamide (PA). At the end of each period of treatment, PBMC were harvested at the beginning (T0) and after 180 minutes of dialysis (T180), and then were cultured in complete medium. IL-6, IL-8 and MCP-1 mRNA expression were studied by RT-PCR. In addition, MCP-1 gene expression was evaluated also by in situ hybridization. Cytokines released in the supernatant were measured by ELISA. RESULTS: Compared to the control group, PBMC from uremic patients on conservative therapy and treated by CU showed a clear reduction in the cytokine release, while PMMA and PA membranes were able to normalize IL-6, IL-8 and MCP-1 protein concentration, which had been reduced by CU treatment. Interestingly, at T0, mRNA expression for all three cytokines was increased in the patients treated by CU, when compared to the control group and the uremic patients on conservative therapy. A further up-regulation was observed at T180. PMMA and PA treatment, despite increasing the cytokine secretion, significantly reduced the dialysis-induced cytokine gene expression. CONCLUSION: PBMC exposure to CU membranes results in cytokine mRNA overexpression associated with a paradoxically reduced protein release. In contrast, long-term hemodialysis with synthetic high-flux membranes reduces IL-6, IL-8 and MCP-1 gene expression and improves the ability of PBMC to secrete these cytokines. The reduced cytokine secretion during bioincompatible dialysis may reflect a PBMC adaptation that protects uremic patients against the inflammatory effects of persistent cytokine release.
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Mononuclear cell infiltration is a common histopathological feature of acute renal transplant rejection, in which it seems to play a key role in the pathogenesis of tubulointerstitial lesions. Monocyte chemotactic peptide-1 (MCP-1) is a specific chemotactic and activating factor for monocytes. Thus, the present study was aimed at evaluating MCP-1 gene and protein expression in renal biopsies of kidney transplant recipients with acute deterioration of graft function, and to correlate it with the extent of monocyte infiltration. We studied 20 kidney transplant recipients with acute graft dysfunction (13 with acute rejection, seven with acute tubular damage). MCP-1 gene and protein expression were analyzed by in situ hybridization and immunohistochemistry, respectively. CD68-positive cells were identified as monocytes. CD68-positive cell number and MCP-1 expression were quantified by a computerized image analysis system. MCP-1 gene expression, undetectable in normal human kidneys, was strikingly increased in patients with acute rejection. The chemokine localized mainly to the proximal tubular cells and to mononuclear-infiltrating cells. In patients with acute tubular damage, the MCP-1 expression, even if higher than in controls, was significantly lower than in acute rejection. The expression of the chemokine strictly correlated with the number of infiltrating monocytes (r=0.87, P<0.05). Moreover, we measured MCP-1 urinary excretion by ELISA, in eight normal subjects (36+/-16 pg/mg urine creatinine), in 13 clinically stable transplant recipients (33+/-9 pg/mg, ns vs. normal patients), in 12 transplant recipients with acute rejection (250+/-46 pg/mg, P<0.01 vs. normal patients), and in five transplant recipients with acute tubular damage (97+/-33 pg/mg, P<0.05 vs. controls and patients with acute rejection). Urinary MCP-1 excretion directly correlated with renal MCP-1 gene expression (r=0.65, P=0.05). Finally, we observed a significant reduction in MCP-1 urine levels in patients with acute rejection, who responded to the antirejection treatment. In conclusion, our data suggest that MCP-1 may play a critical role in modulating monocyte influx and consequent tubulointerstitial damage in acute rejection. Therefore, an increase in urinary MCP-1 excretion may represent an early signal of ongoing acute graft rejection.
Membranoproliferative glomerulonephritis (MPGN) may be a component of a generalized vasculitis as well as a component of the clinical expression of type-II mixed cryoglobulinemia (MC). Several studies have established a striking association between hepatitis C virus (HCV) infection and MC. The potential role of HCV in the pathogenesis of MPGN, which occurs in almost half of the cases of MC patients, has not been fully investigated, and the demonstration of HCV proteins as the antigenic constituent of the glomerular immune deposits has remained elusive. Kidney biopsy specimens were obtained from 12 HCV RNA, antibody to HCV (anti-HCV)-positive patients with MPGN and type-II MC, and from 8 controls (3 HCV RNA, anti-HCV-negative patients with MPGN and MC and 5 with noncryoglobulinemic "idiopathic" MPGN). Murine monoclonal antibodies developed against c22-3, E2/NS1, c33c, c100-3, and NS5 proteins were used to detect HCV-related antigens by indirect immunohistochemistry. Acid electroelution of tissue sections was performed to enhance the sensitivity of the immunohistochemical method. Specific HCV-related proteins were detected in glomerular and tubulo-interstitial vascular structures in 8 (66.7%) HCV-positive MC patients and in none of the HCV RNA, anti-HCV-negative controls. HCV immunoreactive deposits displayed the following two major patterns: 1) a linear, homogeneous deposition along glomerular capillary walls, including endothelial cells and sub-endothelial spaces; and 2) a granular bead-like appearance with distinct deposits in mesangial and paramesangial cells. Immunoglobulin G (IgG) and M (IgM) and C3 fraction deposition in adjacent kidney sections displayed features comparable with those found for HCV deposits. Patients with granular deposits showed more pronounced renal impairment and severe proteinuria. These findings indicate that in MC patients with HCV-associated MPGN, kidney deposits consist of HCV-containing immune complexes that are likely to play a direct pathogenetic role in the renal damage.
Monocyte chemotactic peptide-1 (MCP-1) belongs to a large family of cytokines known as chemokines. It is a potent mediator of inflammatory response and is thought to play a major role in recruiting monocytes into the site of inflammation. Mixed cryoglobulinemia is a systemic vasculitis characterized in 10 to 30% of the cases by renal involvement. Monocyte infiltration into the glomerulus, and in the periglomerular and perivascular areas is a common histopathological feature of this form of glomerulonephritis. We sought to determine, by in situ hybridization and immunohistochemistry, the renal gene and protein expression of MCP-1 in cryoglobulinemic glomerulonephritis compared to normal kidney, and to correlate it with macrophage infiltration. Kidney biopsy specimens were obtained from 9 patients with cryoglobulinemic glomerulonephritis and 9 control kidneys. The distribution and intensity of MCP-1 gene and protein expression, and the macrophage infiltration (CD68 positive cells) were evaluated and quantitated by a computerized image analysis system. In normal kidneys, MCP-1 was weakly expressed, both at the gene as well as at the protein level. In diseased kidneys, a statistically significant (P < 0.001) up-regulation of MCP-1 gene and protein expression was found, particularly within the areas of tubulointerstitial damage and the glomeruli. By means of CD68 positive cells, a significant correlation (P < 0.001) was found between glomerular, tubulointerstitial macrophage infiltration and MCP-1 expression. Moreover, by combining immunohistochemistry and in situ hybridization, we observed the presence of CD68 positive cells mainly, if not exclusively, around the cells expressing MCP-1 mRNA. Interestingly, a striking increase in MCP-1 urinary concentration was found in cryoglobulinemic patients. In conclusion, our data suggest that MCP-1 may play a major role in modulating the inflammatory process observed in cryoglobulinemic glomerulonephritis.
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BACKGROUND: Despite marked improvements in the success of solid organ transplantation, a significant percentage of transplanted organs is lost due to recurrent episodes of acute cellular rejection. The mechanisms that govern allograft rejection likely include a complex regulatory network of multiple cytokines and growth factors. DESIGN AND METHOD: This study investigated the kidney gene (in situ hybridization) and protein (immunohistochemistry) expression and the urinary excretion rate of IL-6 and EGF in 29 renal transplant recipients: 16 with acute cellular rejection (AR) and 13 with acute tubular damage/cyclosporine toxicity (ATD). RESULTS: AR patients displayed a 4-fold increase of renal IL-6 expression, which localized chiefly to proximal tubular cells and monocytes/macrophages, whereas EGF signal was extremely weak or even absent. In ATD patients, EGF expression was markedly reduced, while IL-6 specific signal was unchanged. In all the patients examined the renal expression of IL-6 and EGF strictly correlated with their urinary excretion rate (r:0.459, P:0.001). Thus, urinary IL-6/EGF ratio was markedly increased in the former group (> 20-fold at day 1), where it paralleled the modifications of plasma creatinine over time (r:0.603, P < 0.0001), and was only slightly increased in the latter group (< 3-fold). CONCLUSION: Kidney transplanted patients with acute cellular rejection or acute tubular damage/CyA nephrotoxicity exhibit a distinctly different pattern of intragraft expression of IL-6 and EGF, which is closely reflected by their rate of urinary excretion.
Glomerular C3 deposits are commonly found in immunoglobulin A (IgA) nephropathy. Renal gene expression and protein synthesis of complement components have been shown in settings of tissue inflammation. In this study, the pathogenetic involvement of locally produced C3 in IgA nephropathy was analyzed. C3 gene expression was analyzed by reverse transcription, polymerase chain reaction, and in situ hybridization techniques. C3 mRNA was detected in 56% of cases, with a significantly higher percentage in patients with moderate-to-severe lesions than in those with mild lesions (P < 0.01). By in situ hybridization, C3 transcript was predominantly expressed by tubular cells and some interstitial cells. C3 mRNA was also observed on glomerular parietal epithelial cells. Immunoreactive native C3 was detected on cortical tubuli by an anti-C3c immunoalkaline-phosphatase technique. A significant correlation was found between renal C3 transcription and glomerulosclerosis, intracapillary proliferation (both P < 0.005) and markers of interstitial damage, including tubular atrophy (P < 0.05), interstitial infiltration (P < 0.05), and fibrosis (P < 0.005). Proteinuria (P < 0.05), but not serum creatinine, at the time of renal biopsy correlated with C3 mRNA. In conclusion, it was demonstrated that the C3 gene was expressed primarily in proximal tubular cells and occasionally in glomerular crescents, and that its expression correlated with clinical and histologic markers of severity and poor outcome of IgA nephropathy. Thus, a pathogenetic involvement of the local transcription and translation of the C3 gene in IgA nephropathy was suggested.
The introduction of echography represented a focal step in the management of renal biopsy, resulting in easier and faster procedures. Chiefly, echography allows the diagnosis and monitoring of complications of bioptic procedure. Here we present a series of 722 consecutive echo-guided renal biopsies, carried out from 1990 to 1995, 97 of which on kidney allografts. Echographic examination, performed 24 to 48 hours after renal biopsy, enable to diagnose the presence of perirenal hematoma in 30% of patients. Of these, only 10% presented with clinical symptoms and/or signs (reduction of Hct and arterial pressure, local pain). Our protocol comprises an echographic follow-up to control the evolution of hematoma, that usually resolves within 15-40 days, according to the initial size of the lesion. One time we observed an intra-parenchimal hematoma, which resulted in kidney rupture and consequent nephrectomy. 14% of all patients complained with macrohematuria: in 10% of these cases, echography showed the presence of coaguli in the urinary tract, which was associated with the clinical features of renal colic pain. Only in two cases of persistent macrohematuria, the echography together with echo- and color-doppler, allowed the diagnosis of the arterio-venous fistula. In conclusion, our experience demonstrates that the echographic examination allows not only to simplify the bioptic procedure, but also to early diagnose the complications due to this invasive manouvre.
Interleukin 6 (IL-6) is produced by human mesangial and tubular cells, and its urinary levels has been proposed as a marker of mesangial proliferation and tubulointerstitial damage. Epidermal growth factor (EGF) is expressed within the Henle's loop and the distal tubule and has been shown to accelerate recovery from renal injury. In the present study we have defined renal gene and protein expression of IL-6 and EGF in 10 normal, 10 nonproliferative glomerulonephritis (NPGN) and 30 IgA nephropathy (IgAN) human kidneys by RT-PCR, in situ hybridization and immunohistochemical techniques. Moreover, urinary IL-6 and EGF levels were measured in 41 patients with IgAN and in 20 normal subjects (N). In normal kidneys, EGF was localized in Henle's loop and distal convoluted tubule whereas IL-6 was mainly located in the proximal tubule and, less, within the glomerulus. In IgAN patients, EGF was decreased whereas IL-6 expression was upregulated. These modifications paralleled the degree of tubulointerstitial damage. Moreover, IgAN patients as a whole exhibited a reduction of EGF and an increase of IL-6 urinary concentration (EGF values: N, 12.96 +/- 1.15; IgAN Grades 1-2, 20.05 +/- 2.64; Grades 3-4 7.60 +/- 1.70: Grade 5, 3.14 +/- 0.71, ng/mg urinary creatinine. IL-6 values: N, 2.04 +/- 0.51; IgAN Grades 1-2, 3.26 +/- 0.38; Grades 3-4, 5.67 +/- 0.92; Grade 5, 27.20 +/- 9.70 pg/mg urinary creatinine), that correlated with the degree of histological lesions, the presence of hypertension and serum creatinine level. Interestingly, patients with the highest urinary IL-6/EGF ratio showed a worse evolution in a three year follow-up. In conclusion, our data show that: (1) renal IL-6 and EGF expression are strictly correlated to the degree of tubulointerstitial damage; and (2) urinary IL-6/EGF ratio might be a valuable prognostic marker for the progression of the renal damage in IgAN.
The kidney is one of the major sites of EGF production and there it seems to play several biological functions, such as modulation of cell growth, renal repair following injury, regulation of cellular metabolism and glomerular haemodinamics. The present study was first aimed at localizing EGF and its receptor (R) in normal human kidney by immunohistochemical and in situ hybridization techniques. Then, the distribution of the growth factor and its R was explored in biopsy specimens from eight patients with acute tubulointerstitial damage. In the normal human kidney, both EGF immunoreactivity and EGF mRNA were localized in tubular profiles corresponding to Henle's loop and, although to a lesser intensity, to distal convoluted tubule. EGF immunostaining was remarkable mainly at the apical surface of tubular cells. EGF-R protein expression was detected in glomerular endothelial cells, in peritubular capillaries and arteriolar walls, as well as along the thick ascending limb of Henle's lop and distal convoluted tubule, where it colocalized with Tamm-Horsfall protein. Immunohistochemical analysis of tubular profiles revealed that EGF-R was located especially along the basolateral membrane of tubular cells and within the basal part of cytoplasm. Endogenous alkaline phosphatase and CHIP28 positive tubules did not show any signal for EGF and its receptor. Kidneys with acute tubulointerstitial injury exhibited a dramatic decrease of EGF expression, whereas EGF-R showed only minor modifications. Interestingly, EGF-R was localized to both apical and antiluminal membranes of positive tubular cells. It is concluded that EGF-EGF receptor loop may be relevant in the pathogenesis of acute tubulointerstitial damage and recovery from tubular injury, while its role in the physiological renewal of the urothelium remains speculative.
Tubulointerstitial damage is a common histopathological feature of acute and chronic renal diseases and a prognostic indicator of renal function outcome. Monocytes infiltrating the interstitium, through the release of cytokines and/or growth factors, may play a key role in the pathogenesis of tubulointerstitial damage. Monocyte chemotactic peptide-1 (MCP-1) is a specific and powerful chemoattractant and activating factor for monocytes. This study investigated MCP-1 expression and its correlation with monocyte infiltration and tubulointerstitial damage in biopsies of patients with acute interstitial nephritis (AIN) and a chronic glomerulonephritis, namely immunoglobulin. A nephropathy (IgAN), often characterized by tubulointerstitial involvement. Six patients with AIN and 20 patients with IgAN, nine with mild (G1 to 2) and 11 with moderate to severe histologic lesions (G3 to 5), were studied. MCP-1 gene and protein expression were evaluated by in situ hybridization and immunohistochemistry. Infiltrating CD68-positive cells were identified as monocytes. MCP-1, weakly expressed in normal kidneys, was clearly upregulated in AIN biopsies. The gene and the protein expression were primarily localized in tubular and glomerular parietal epithelial cells, as well as in infiltrating mononuclear cells. In IgAN, a striking increase in MCP-1 mRNA and protein expression was observed only in the biopsies with moderate to severe lesions, with a pattern of expression similar to AIN. The MCP-1 expression strictly correlated with monocyte infiltrates and tubulointerstitial damage. In addition, the urinary excretion of this chemokine was studied in 17 IgAN patients. MCP-1 protein concentration was higher, compared with healthy subjects, in IgAN patients, especially in the G3 to 5 group, and directly correlated with the renal MCP-1 gene expression. In conclusion, these data suggest that production of MCP-1 in the tubulointerstitial compartment may play a key role in modulating monocytes influx and, consequently, tubulointerstitial damage.
High glucose concentration has been shown to induce the overexpression of transforming growth factor (TGF)-beta 1 mRNA and protein in different cell types, including murine mesangial cells, thus possibly accounting for the expansion of mesangial extracellular matrix observed in diabetic glomerulopathy. In the present study, we evaluated platelet-derived growth factor (PDGF) B-chain and PDGF-beta receptor gene expression in human mesangial cells (HMCs) exposed to different concentrations of glucose and then sought a possible relationship between a PDGF loop and the modulation of TGF-beta 1 expression. HMC [3H]thymidine incorporation was upregulated by 30 mmol/L glucose (HG) up to 24 hours, whereas it was significantly inhibited at later time points. Neutralizing antibodies to PDGF BB abolished the biphasic response to HG, whereas anti-TGF-beta antibodies reversed only the late inhibitory effect of hyperglycemic medium. HG induced an early and persistent increase of PDGF B-chain gene expression, as evaluated by reverse transcriptase polymerase chain reaction, whereas PDGF-beta receptor mRNA increased by twofold after 6 hours, thereafter declining at levels 70% lower than in controls after 24 hours. 125I-Labeled PDGF BB binding studies in HMCs exposed to HG for 24 hours confirmed the decrease of PDGF-beta receptor expression. TGF-beta 1-specific transcripts showed 43 and 78% increases after 24 and 48 hours of incubation in HG, respectively, which was markedly diminished by anti-PDGF BB neutralizing antibodies or suramin. We conclude that HG induces an early activation of a PDGF loop that, in turn, causes an increase of TGF-beta 1 gene expression, thus modulating both HMC proliferation and mesangial matrix production.
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