Renal Allograft Fibrosis Research Articles

Expression and Significance of RANTES and MCP-1 in Renal Tissue With Chronic Renal Allograft Dysfunction.

To investigate the expression of RANTES (regulated upon activation, normal T-cell-expressed and -secreted) and monocyte chemoattractant protein-1 (MCP-1) in renal allografts with chronic renal allograft dysfunction (CRAD), and explore its relationship with interstitial fibrosis and tubular atrophy (IF/TA). An immunohistochemical assay and computer-assisted, genuine colored image analysis system were used to detect the expression of RANTES and MCP-1 in renal allografts with CRAD. The relationship among the expression level of MCP-1, RANTES, and the grade of inflammatory cell infiltration, interstitial fibrosis, and tubular atrophy in renal allografts were analyzed. Ten specimens of healthy renal tissue were used as controls. Compared to the normal tissues, the expressions of RANTES and MCP-1 were significantly higher in the renal tissues with CRAD (P< .001), and the expressions tended to increase along with the pathological grade of IF/TA. The expression of RANTES and MCP-1 were positively correlated with the pathological grades of IF/TA (r= 0.940 and 0.954 respectively, P< .001 for both). In renal allograft tissue with CRAD, the up-regulated expressions of RANTES and MCP-1 may be related to the progression of chronic renal allograft dysfunction and allograft fibrosis.

Use of Contrast-Enhanced Ultrasonography to Evaluate Chronic Allograft Nephropathy in Rats and Correlations between Time-Intensity Curve Parameters and Allograft Fibrosis

This study quantitatively analyzed changes in the hemodynamic characteristics of renal allografts at different stages in a rat chronic allograft nephropathy (CAN) model as well as the relationship between hemodynamic parameters and renal allograft fibrosis using contrast-enhanced ultrasonography (CEUS). The experimental group used a CAN rat model (n = 30), and the control group used an orthotopic syngeneic renal transplant model (n = 30). After surgery, creatinine clearance rates were regularly monitored every 2 wk. The checking times were set at 4, 12 and 24 wk after surgery, which represent early, middle and late stage of CAN, respectively. At different stages of CAN, eight rats from each group were randomly selected for CEUS examination. Time-intensity curve (TIC) parameters, including rise time, peak intensity, mean transit time, area under the curve, wash-in slope, time-to-peak and α-smooth muscle actin (α-SMA) expression; Vimentin expression; and chronic allograft damage index scores were evaluated by linear correlation analysis. Before the creatinine clearance rate showed significant abnormalities, the renal allografts in the experimental group had already presented pathologic changes associated with CAN. In the early stage after surgery, compared to the TIC curve of the control group, the experimental group showed increased rise time, mean transit time, area under the curve and time-to-peak, and decreased wash-in slope (p < 0.05). Chronic allograft damage index scores and the expression levels of α-SMA and Vimentin proteins in renal allografts were correlated with TIC parameters (p < 0.05). Compared to creatinine clearance rate, CEUS can detect CAN at earlier stages. The correlations between TIC-related parameters and the expression levels of α-SMA and Vimentin in renal allografts indicate that CEUS is a feasible way to assess the degree of renal allograft fibrosis quantitatively.

Noninvasive evaluation of renal allograft fibrosis by virtual touch tissue quantification

Objective To evaluate the diagnostic value of virtual touch tissue quantification (VTQ) in the diagnosis of renal allograft fibrosis. Methods The renal allografts of 82 patients with biopsies or nephrectomy were assessed by virtual touch quantification. The renal allograft fibrosis was categorized according to the 2005 updated Banff criteria for a G0~G3 grade.All the results were compared among four groups. Results The mean SWV values in G0~G3 were (2.39±0.31)m/s, (2.45±0.34)m/s, (2.58±0.18)m/s, (3.11±0.40)m/s, respectively. There were no significant differences in the mean SWV value between G0 and G1 group, or between G1 and G2 group(P>0.05). There were significant differences in the mean SWV value between G0~G2 and G3 group, or between G0 and G2 group(P<0.05). Stiffness of renal allograft was significantly correlated to the mean SWV value ( Spearman r=0.671, P<0.001). According to the area under the ROC curve, the sensitivity and specificity of SWV (area under ROC curve=0.847, cut-off=2.64 m/s ) for grade ≥G2 was 78.9% and 79.5% respectively. Conclusions Stiffness measured by VTQ reflects the interstitial fibrosis in renal allograft.VTQ technique might be a new tool to identify patients with chronic allograft injury. Key words: Ultrasonography; Kidney transplantation; Fibrosis; Virtual touch tissue quantification

DNA double-strand breaks induced intractable glomerular fibrosis in renal allografts

The relationship between DNA damage and glomerular fibrosis in renal allografts remains unclear. We examined renal allograft specimens from 35 patients in which DNA double-strand breaks (DSBs) and glomerular fibrosis were detected by phospho-histone H2A.X (γ-H2AX) expression and collagen (COL) types III, IV, and VI accumulation. We also examined the in vitro relationship between DNA damage and COL accumulation by mitomycin C (MMc)-induced DNA damage in human glomerular endothelial cells (HRGEc). The γ-H2AX and COL type VI, which mainly accumulated in the subendothelial and mesangial regions, were positively correlated with the duration of the post-renal transplant (RT) period. In multiple regression analysis, the duration of the post-RT period and cg in the Banff '07 classification were identified as a significant predictor of COL type VI accumulation and γ-H2AX expression in the glomerular capillaries. In addition, the γ-H2AX-positive area was also identified as a predictor of glomerular accumulation of COL type VI. COL type VI was detected in the cytoplasm of the HRGEc, which was secreted into the supernatant after MMc stimulation with γ-H2AX expression. The number of γ-H2AX (-)/COL type VI (+) cells was inversely associated with the number of γ-H2AX (+)/COL type VI (-) cells during 24-h MMc treatment. Our findings suggest that the long-term RT induces DSBs and HRGEc-secreted COL type VI accumulation in the glomerular capillaries, which might progress to intractable glomerular fibrosis.

Evaluation of Renal Allograft Fibrosis by Transient Elastography (Fibro Scan)

BackgroundChronic allograft injury (CAI) is one of the most important factors for graft failure after renal transplantation. Protocol biopsy is the most valuable tool for revealing subclinical renal allograft failure. Transient elastography (TE) is a noninvasive technique that has shown utility for the assessment of hepatic and renal fibrosis. This study sought to evaluate whether TE was a viable and effective method for the assessment of renal allograft failure. Patients and MethodsThirty-five patients underwent TE by Fibro Scan (Echosense, Paris, France). Biopsies were performed in 27 patients, allowing classification according to Banff chronic changes in the interstitium grade 0, grade 1 or grade 2. ResultsMeasurement of parenchymal stiffness was successful in 31of 35 patients (91%). Stiffness was significantly correlated with interstitial fibrosis (P < .05) and inversely related with estimated glomerular filtration rate (eGFR; P < .05). Stiffness values of patients with eGFR > 50 mL/min were lower than those of patients with eGFR < 50 mL/min (P < .05). Patients classed as CAI Banff grade 0 had significantly less parenchymal stiffness than patients with Banff grade 1 or grade 2 CAI (P < .05). Parenchymal stiffness measured by TE reflected interstitial fibrosis in renal allograft. ConclusionAssessment of parenchymal renal allograft stiffness by TE was effective for identifying patients with CAI who may subsequently benefit from biopsy and modification of the immunosuppressive regimen. Assessment of parenchymal renal allograft stiffness can be effective for identifying patients with CAI. TE has the potential to reduce the number of renal allograft biopsies required for accurate assessment of CAI.

The Role of Macrophages in the Development of Human Renal Allograft Fibrosis in the First Year After Transplantation

The aim of this study was to investigate the role of infiltrating macrophages in renal allograft fibrosis. Forty-six protocol renal allograft biopsies obtained 1 year after transplantation were stained with Sirius red to quantify fibrosis and double stained with CD68 and CD206 to identify the proportion of alternatively activated (M2) macrophages. Biopsies were analyzed for gene expression by microarray, which was correlated with macrophage infiltration and the severity of fibrosis. The number of infiltrating CD68+ cells strongly correlated with the percentage of interstitial fibrosis (r = 0.73, p < 0.0001). Macrophage infiltration at 1 year correlated with renal dysfunction at 1, 12 and 36 months posttransplant (estimated GFR low vs. high: 1 month 78 ± 26 vs. 54 ± 19 mL/min, p < 0.01; 12 months 87 ± 29 vs. 64 ± 19 mL/min, p < 0.05; 36 months 88 ± 33 vs. 60 ± 24 mL/min, p < 0.05). Ninety-two percent of infiltrating macrophages exhibited an M2 phenotype with CD68+ CD206+ dual staining. Gene microarrays demonstrated an alloimmune response with up-regulation of interferon-γ-response genes despite the lack of rejection or inflammatory infiltrate. Consistent with this was the presence of CXCL10 in proximal tubular cells at 3 months. This suggests that M2 macrophage proliferation, or infiltration, was associated with subclinical alloimmune inflammation, tubular injury and progression of fibrosis.

Ultrasound Strain Zero-Crossing Elasticity Measurement in Assessment of Renal Allograft Cortical Hardness: A Preliminary Observation

To determine whether ultrasound strain zero-crossing elasticity measurement can be used to discriminate moderate cortical fibrosis or inflammation in renal allografts, we prospectively assessed cortical hardness with quasi-static ultrasound elastography in 38 renal transplant patients who underwent kidney biopsy from January 2013 to June 2013. With the Banff score criteria for renal cortical fibrosis as gold standard, 38 subjects were divided into two groups: group 1 (n = 18) with ≤25% cortical fibrosis and group 2 (n = 20) with >26% cortical fibrosis. We then divided this population again into group 3 (n = 20) with ≤ 25% inflammation and group 4 (n = 18) with >26% inflammation based on the Banff score for renal parenchyma inflammation. To estimate renal cortical hardness in both population divisions, we propose an ultrasound strain relative zero-crossing elasticity measurement (ZC) method. In this technique, the relative return to baseline, that is zero strain, of strain in the renal cortex is compared with that of strain in reference soft tissue (between the abdominal wall and pelvic muscles). Using the ZC point on the reference strain decompression slope as standard, we determined when cortical strain crossed zero during decompression. ZC was negative when cortical strain did not return or returned after the reference, whereas ZC was positive when cortical strain returned ahead of the reference. Fisher's exact test was used to examine the significance of differences in ZC between groups 1 and 2 and between groups 3 and 4. The accuracy of ZC in determining moderate cortical fibrosis and moderate inflammation was examined by receiver operating characteristic analysis. The intra-class correlation coefficient and analysis of variance were used to test inter-rater reliability and reproducibility. ZC had good inter-observer agreement (ICC = 0.912) and reproducibility (p = 0.979). ZCs were negative in 18 of 18 cases in group 1 and positive in 19 of 20 cases in group 2 (p ≪ 0.001), and were positive in 18 of 20 cases in group 3 and negative in 17 of 18 cases in group 4 (p ≪ 0.001). The area under the receiver operating characteristic curve was 0.992 ± 0.010 for fibrosis and 0.988 ± 0.021 for inflammation. ZC had 100% sensitivity and 95% specificity when zero strain was used as the cutoff value to determine moderate cortical fibrosis and 94% sensitivity and 90% specificity for inflammation. ZC is a new strain marker that could be straightforward to interpret and perform, making it a potentially practical approach for monitoring progression of cortical fibrosis or inflammation in renal allografts.

Peripheral Blood miRNA Signature Predicts Progression of Kidney Fibrosis Post-Transplantation.

Progressive renal fibrosis leading to decline in renal function remains the predominant cause of renal allograft loss. Current methodologies based on clinical and pathological parameters fail to identify grafts at risk for loss prior to the development of irreversible injury. The aim of this study was to identify a miRNA signature to predict progression of renal allograft fibrosis. We performed miRNA profiling using Nanostring technology on peripheral blood from a cohort of 48 kidney transplant patients from the Genomics of Chronic Allograft Rejection (GoCAR) study. Spearman correlation analysis of miRNA expression profiles with 3 and 12 month CADI scores identified a set of 69 miRNAs specifically associated with m12 CADI from which penalized-logistic regression combined with repeat analysis (1000-time) was then applied to identify and further refine the set to 6 miRNA. This set accurately predicted the high (≥2) and low (<2) CADI-12 (AUC 0.974), and was significantly better than clinical parameters alone (AUC 0.73). The PPV and NPV were 0.81 and 0.86, respectively. Moreover, the 6 miRNA set could be used to accurately differentiate between progressors (n=24) and non-progressors (n=24), progression being defined as ΔCADI>3 from 3 to 12 month (AUC=0.969). Validation of 6 miRNA set by qPCR in a large independent cohort is in progress. In summary, our miRNA profiling revealed a molecular signature from peripheral blood which accurately predicted the future development of kidney allograft fibrosis. This data suggests that peripheral miRNA profiling may be used as surveillance to stratify patients at risk for fibrosis and graft loss, obviating the need for allograft biopsy, and identifying those who may benefit from early interventions to prevent chronic allograft loss.

Noninvasive evaluation of renal allograft fibrosis by virtual touch tissues quantification

To explore the value of virtual touch tissue quantification (VTQ) in the diagnosis of renal allograft fibrosis and analyze its advantages and limitations. The renal allografts of 54 patients with renal allograft biopsies or nephrectomy were assessed by Virtual Touch quantification of Siemens-Acuson S2000. Stiffness of renal allograft was significantly correlated to the value of VTQ (Spearman r=0.796, P<0.05). VTQ is a new technique in the assessment of renal allograft fibrosis.

Urinary Connective Tissue Growth Factor Is Associated with Human Renal Allograft Fibrogenesis

Connective tissue growth factor (CTGF) is a key mediator of tissue fibrogenesis in kidney disease. Its involvement in renal allograft fibrosis was recently demonstrated in a mouse model. We prospectively studied the association between urinary CTGF (CTGFu) levels and renal allograft fibrosis during the first 2 years after transplantation. Histologic and biochemical data were collected from 315 kidney transplant recipients enrolled in a protocol biopsy-based clinical program. At 3, 12, and 24 months after transplantation, CTGFu levels were independently associated with the degree of interstitial fibrosis in protocol biopsies, scored according to the revised 1997 Banff criteria. In a subgroup of 164 patients with pristine biopsies at 3 months, higher CTGFu levels at 3 months were associated with moderate and severe interstitial fibrosis developed at 24 months after transplantation. As it is readily quantifiable in urine, a role for CTGFu as a noninvasive candidate biomarker and predictor of human renal allograft fibrogenesis deserves further study.

Renal Transplant Elasticity Ultrasound Imaging: Correlation Between Normalized Strain and Renal Cortical Fibrosis

After transplantation, over a widely variable time course, the cortex of the transplanted kidney becomes stiffer as interstitial fibrosis develops and renal function declines. Elasticity ultrasound imaging (EUI) has been used to assess biomechanical properties of tissue that change in hardness as a result of pathologic damage. We prospectively assessed the hardness of the renal cortex in renal transplant allograft patients using a normalized ultrasound strain procedure measuring quasi-static deformation, which was correlated with the grade of renal cortical fibrosis. To determine cortical strain, we used 2-D speckle-tracking software (EchoInsight, Epsilon Imaging, Ann Arbor, MI, USA) to perform offline analysis of stored ultrasound loops capturing deformation of renal cortex and its adjacent soft tissue produced by pressure applied using the scanning transducer. Normalized strain is defined as the mean developed strain in the renal cortex divided by the overall mean strain measured in the soft tissues from the abdominal wall to pelvic muscles. Using the Banff scoring criteria for renal cortical fibrosis as the gold standard, we classified 20 renal transplant allograft biopsy tissue samples into two groups: group 1 (n = 10) with mild (<25%) renal cortical fibrosis and group 2 (n = 10) with moderate (26%–50%) renal cortical fibrosis. An unpaired two-tailed t-test was used to determine the statistical difference in strains between patients with mild and those with moderate renal cortical fibrosis. Receiver operating characteristic curve analysis was performed to assess the accuracy of developed strain and normalized strain in predicting moderate renal cortical fibrosis. The reference strain did not significantly differ between the two groups (p = 0.10). However, the developed renal cortical strain in group 1 with mild fibrosis was higher than that in group 2 with moderate fibrosis (p = 0.025). The normalized strain in group 1 was also higher than that in group 2 (p = 0.0014). The areas under receiver operating characteristic curves for developed strain and normalized strain were 0.78 and 0.95, respectively. The optimal cutoff for distinguishing moderate renal cortical fibrosis was −0.08 for developed strain (sensitivity = 0.50, specificity = 1.0) and 2.5 for normalized strain (sensitivity = 0.80, specificity = 1.0). In summary, renal cortex strain is strongly correlated with grade of renal cortical fibrosis. Normalized strain is superior to developed strain in distinguishing moderate from mild renal cortical fibrosis. We conclude that free-hand real-time strain EUI may be useful in assessing the progression of cortical fibrosis in renal transplant allografts. Further prospective study using this method is warranted.

Oral Paricalcitol Reduces the Prevalence of Posttransplant Hyperparathyroidism: Results of an Open Label Randomized Trial

Postkidney transplant hyperparathyroidism is a significant problem. Vitamin D receptor agonists are known to suppress parathyroid hormone (PTH) secretion. We examined the effect of oral paricalcitol on posttransplant secondary hyperparathyroidism by conducting an open label randomized trial in which 100 incident kidney transplant recipients were randomized 1:1 to receive oral paricalcitol, 2 μg per day, for the first year posttransplant or no additional therapy. Serial measurements of serum PTH, calcium and bone alkaline phosphatase, 24-h urine calcium and bone density were performed. The primary endpoint was the frequency of hyperparathyroidism 1-year posttransplant. Eighty-seven patients completed the trial. One-year posttransplant, 29% of paricalcitol-treated subjects had hyperparathyroidism compared with 63% of untreated patients (p = 0.0005). Calcium supplementation was discontinued in two control and 15 treatment patients due to mild hypercalcemia or hypercalcuria. Paricalcitol was discontinued in four patients due to hypercalcuria/hypercalcemia and in one for preference. Two subjects required decreasing the dose of paricalcitol to 1 μg daily. Hypercalcemia was asymptomatic and reversible. Incidence of acute rejection, BK nephropathy and renal function at 1 year were similar between groups. Moderate renal allograft fibrosis was reduced in treated patients. Oral paricalcitol is effective in decreasing posttransplant hyperparathyroidism and may have beneficial effects on renal allograft histology.

Distribution and Expression of Fibroblast-Specific Protein Chemokine CCL21 and Chemokine Receptor CCR7 in Renal Allografts

We aimed to characterize the expression and distribution of the fibroblast surface protein (FSP), the chemokine CC-ligand 21 (CCL21) secondary lymphoid tissue chemokine CC-chemokine receptor 7 (CCR7) in renal allograft biopsy specimens obtained from patients after transplantation. We recruited 165 patients who received renal transplants at our center for this study. Histological examination of the renal allograft biopsy specimens was performed using hematoxylin-eosin, periodic acid–Schiff, and Masson's trichrome staining. Distribution and expression of FSP, CCL21, and CCR7 were determined using immunohistochemistry staining. Serum creatinine levels were evaluated using an enzymatic sarcosine oxidase method. FSP was mainly localized in the cytoplasm and nucleus of renal interstitial fibroblasts and tubular epithelial cells. Compared with the normal group, an elevated number of FSP-positive fibroblasts were observed in patients with acute/active cellular rejection and chronic/sclerosing allograft nephropathy (P < .05). Patients with chronic/sclerosing allograft nephropathy also showed increased total fibroblasts as compared with borderline changes (P < .05). In a multiple regression analysis, CCR7-positive expression was a strong protective factor for acute/active cellular rejection and recurrent nephropathy (odds ratio [OR] = 0.12, P = .034, and OR = 0.08; P = .036, respectively). In contrast, CCL21-positive expression led to a high susceptibility to recurrent nephropathy among renal transplant patients (OR = 10.41, P = .029). Moreover, FSP and CCL21, or CCL21 and CCR7 were localized in the interstitial fibroblasts and renal tubular epithelium cells. In addition, FSP and CCL21 expression positively correlated with serum creatinine levels. Our results suggested that the CCL21/CCR7 signaling pathway is involved in renal fibrosis in kidney transplant patients. An increased number of FSP-positive fibroblasts may be a risk factor for acute/active cellular rejection and chronic/sclerosing allograft nephropathy after renal transplantation. These findings may help understanding of renal allograft fibrosis.

Assessment of renal allograft fibrosis by transient elastography

Transient elastography (TE, Fibroscan) has been established as a noninvasive assessment tool of liver fibrosis. We evaluated potentials and limitations of TE for identifying renal allograft fibrosis. The technical possibility of kidney examination by TE was assessed in two 10-week-old German landrace pigs and kidney stiffness (KS) was evaluated in 164 renal transplant patients. KS could be determined in all animals at the pole and pars media (29±10kPa vs. 31±17kPa). In human renal allografts KS was successfully performed in 94.5% of the test series with reliable results in 72% of the measurements. Mean KS at the pole or pars media were comparable (35.0±19.9kPa vs. 33.2±18.6kPa). Significantly higher KS was detected in renal allografts with histologically confirmed advanced fibrosis. Body-mass-index, skin-allograft distance, and peri or intrarenal fluid accumulation were important confounders of successful KS measurements (BMI: r=-0.31; P<0.001; distance: r=-0.50; P<0.001). Notably, KS did not correlate with renal function. TE represents a noninvasive approach in selected transplant recipients to identify allografts with severe fibrosis. The heterogeneous kidney morphology and several other confounding factors negatively affect measurability of KS by TE. Further technical modifications are required to improve applicability of TE for kidney assessment.

Discovery and Validation of a Molecular Signature for the Noninvasive Diagnosis of Human Renal Allograft Fibrosis

Tubulointerstitial fibrosis (fibrosis), a histologic feature associated with a failing kidney allograft, is diagnosed using the invasive allograft biopsy. A noninvasive diagnostic test for fibrosis may help improve allograft outcome. We obtained 114 urine specimens from 114 renal allograft recipients: 48 from 48 recipients with fibrosis in their biopsy results and 66 from 66 recipients with normal biopsy results. Levels of messenger RNAs (mRNAs) in urinary cells were measured using kinetic, quantitative polymerase chain reaction assays, and the levels were related to allograft diagnosis. A discovery set of 76 recipients (32 with allograft fibrosis and 44 with normal biopsy results) was used to develop a diagnostic signature, and an independent validation set of 38 recipients (16 with allograft fibrosis and 22 with normal biopsy results) was used to validate the signature. In the discovery set, urinary cell levels of the following mRNAs were significantly associated with the presence of allograft fibrosis: vimentin (P<0.0001, logistic regression model), hepatocyte growth factor (P<0.0001), α-smooth muscle actin (P<0.0001), fibronectin 1 (P<0.0001), perforin (P=0.0002), plasminogen activator inhibitor 1 (P=0.0002), transforming growth factor β1 (P=0.0004), tissue inhibitor of metalloproteinase 1 (P=0.0009), granzyme B (P=0.0009), fibroblast-specific protein 1 (P=0.006), CD103 (P=0.02), and collagen 1A1 (P=0.04). A four-gene model composed of the levels of mRNA for vimentin, NKCC2, and E-cadherin and of 18S ribosomal RNA provided the most accurate, parsimonious diagnostic model of allograft fibrosis with a sensitivity of 93.8% and a specificity of 84.1% (P<0.0001). In the independent validation set, this same model predicted the presence of allograft fibrosis with a sensitivity of 77.3% and a specificity of 87.5% (P<0.0001). Measurement of mRNAs in urinary cells may offer a noninvasive means of diagnosing fibrosis in human renal allografts.

Tissue elasticity estimated by acoustic radiation force impulse quantification depends on the applied transducer force: an experimental study in kidney transplant patients

Acoustic radiation force impulse (ARFI) quantification estimates tissue elasticity by measuring shear-wave velocity (SWV) and has been applied to various organs. We evaluated the impact of variations in the transducer force applied to the skin on the SWV ultrasound measurements in kidney transplant cortex and ARFI's ability to detect fibrosis in kidney transplants. SWV measurements were performed in the cortex of 31 patients with kidney allografts referred for surveillance biopsies. A mechanical device held the transducer and applied forces were equal to a compression weight of 22, 275, 490, 975, 2,040 and 2,990g. SWV group means were significantly different by repeat measures ANOVA [F(2.85,85.91) = 84.75, P < 0.0005 for 22, 275, 490, 975 and 2,040g compression weight] and also by pairwise comparisons. Biopsy specimens were sufficient for histological evaluation in 29 of 31 patients. Twelve had grade 0, 11 grade 1, five grade 2 and one grade 3 fibrosis. One-way ANOVA showed no difference in SWV performed with any of the applied transducer forces between grafts with various degrees of fibrosis. SWV measurements in kidney transplants are dependent on the applied transducer force and do not differ in grafts with different grades of fibrosis. • Acoustic radiation force impulses (ARFI) can quantify tissue elasticity during ultrasound examinations. • Elasticity estimated by ARFI in kidney transplants depends on applied transducer force. • ARFI quantification cannot detect renal allograft fibrosis. • ARFI elasticity estimates may in general vary with applied transducer force.

A genetic polymorphism in the CAV1 gene associates with the development of bronchiolitis obliterans syndrome after lung transplantation

BackgroundCaveolin 1 (Cav-1) is the primary structural component of cell membrane invaginations called 'caveolae'. Expression of Cav-1 is implicated in the pathogenesis of pulmonary fibrosis. Genetic polymorphisms in the CAV1 gene influence the function of Cav-1 in malignancies and associate with renal allograft fibrosis. Chronic allograft rejection after lung transplantation, called 'bronchiolitis obliterans syndrome' (BOS), is also characterised by the development of fibrosis.In this study, we investigated whether CAV1 genotypes associate with BOS and whether Cav-1 serum levels are influenced by the CAV1 genotype and can be used as a biomarker to predict the development of BOS.MethodsTwenty lung transplant recipients with BOS (BOSpos), ninety without BOS (BOSneg) and four hundred twenty-two healthy individuals donated DNA samples. Four SNPs in CAV1 were genotyped. Serial Cav-1 serum levels were measured in a matched cohort of 10 BOSpos patients and 10 BOSneg patients. Furthermore, single-time point Cav-1 serum levels were measured in 33 unmatched BOSneg patients and 60 healthy controls.ResultsHomozygosity of the minor allele of rs3807989 was associated with an increased risk for BOS (odds ratio: 6.13; P = 0.0013). The median Cav-1 serum level was significantly higher in the BOSpos patients than in the matched BOSneg patients (P = 0.026). Longitudinal analysis did not show changes in Cav-1 serum levels over time in both groups. The median Cav-1 serum level in the group of 43 BOSneg patients was lower than that in the healthy control group (P = 0.046).In lung transplant recipients, homozygosity of the minor allele of rs3807989 and rs3807994 was associated with increased Cav-1 serum levels.ConclusionIn lung transplant recipients, the CAV1 SNP rs3807989 was associated with the development of BOS and Cav-1 serum levels were influenced by the CAV1 genotype.

Non-invasive assessment of renal allograft fibrosis by dynamic sonographic tissue perfusion measurement

Chronic allograft nephropathy (CAN) characterized by interstitial fibrosis and tubular atrophy is a major cause of renal transplant failure. The diagnosis can currently only be verified by a graft biopsy. To evaluate whether non-invasive dynamic color Doppler sonographic parenchymal perfusion measurements are different in grafts with various degrees of biopsy proven renal transplant fibrosis. Forty-nine adult patients were prospectively included. Four patients were excluded. Color Doppler videos from the renal cortex were recorded. Perfusion in the renal cortex was evaluated using a software package which calculates color pixel area and flow velocity, encoded by each pixel inside a region of interest of a video sequence. The software calculates parameters that describe tissue perfusion numerically. Two of these, the perfusion intensity and tissue pulsatility index, were compared to grade of interstitial fibrosis (0-3) in biopsies. Observer agreement was evaluated in a subset of 12 patients. Of the 45 patients analyzed, 18 patients had grade 0, 18 had grade 1, seven had grade 2 and two had grade 3 fibrosis. The mean perfusion intensity of grade 0 was significantly higher than that of grade 2 and 3 fibrosis in the proximal cortical layer (1.65 m/s vs. 0.84 m/s, P = 0.008). No significant difference was found between grade 0 and grade 1 fibrosis. Perfusion intensity was correlated to estimated glomerular filtration rate (Pearson r 0.51, P = 0.001, R(2) = 0.26 and 0.46, P = 0.001, R(2) = 0.22 in the distal and proximal cortex, respectively). Inter-observer agreement of the perfusion intensity, expressed as intraclass correlation coefficient was 0.69 in the proximal part of the cortex. Intra-observer agreement was 0.85 for observer 1 and 0.82 for observer 2. Perfusion intensity assessed by dynamic color Doppler measurements is significantly reduced in allografts with grade 2 and 3 fibrosis compared to allografts without fibrosis. Further studies involving longitudinal assessment of allografts undergoing protocol biopsies would be of interest.

Adenovirus-mediated anti-sense ERK2 gene therapy inhibits tubular epithelial–mesenchymal transition and ameliorates renal allograft fibrosis

Purpose Epithelial–mesenchymal transition (EMT) plays an important role in progress of renal allograft fibrosis. The adenovirus-mediated anti-sense extracellular signal-regulated kinase 2 (Adanti-ERK2) gene therapy was used to block ERK signaling pathway, and its effect on EMT and renal allograft fibrosis both in vivo and in vitro was explored. Methods We first generated an in vitro EMT model by connective tissue growth factor (CTGF) stimulation in a HK-2 cell culture system, and then applied Adanti-ERK2 gene therapy on it. The transition of epithelial marker (E-cadherin) to mesenchymal markers (α-SMA, Vimentin) and the cell mobility function alteration were monitored for the observation of EMT progress. In vivo, a rat renal transplant model with Fisher–Lewis combination was employed and the Adanti-ERK2 gene therapy was given. The tubular EMT changes and pathology of allograft fibrosis were examined. Results In vitro, Adanti-ERK2 gene therapy inhibited CTGF-induced tubular EMT and attenuated the cell motility function induced by CTGF. In vivo, Adanti-ERK2 gene therapy attenuated tubular EMT, modulated the infiltration of macrophages and CD8 +, CD4 +T lymphocytes, and ameliorated fibrosis effectively in the renal allografts 24 weeks after transplantation . Conclusions Adanti-ERK2 gene therapy inhibits tubular EMT and attenuates renal allograft fibrosis. It is possible to develop promising molecular drug(s) in the future based on ERK signaling pathway.

The Influence of Tubular Phenotypic Changes on the Development of Diffuse Interstitial Fibrosis in Renal Allografts

It has been reported that myofibroblasts are the major cells in the development of interstitial fibrosis (IF) and, therefore, chronic dysfunction in renal allografts. Our aim was two fold: first, to understand the key markers controlling tubular and glomerular epithelial-to-mesenchymal transition (EMT); second, to show the role of tubular EMT on the development of interstitial fibrosis (IF) and the role of glomerular EMT on the development of proteinuria and, therefore, graft survival. For this purpose we evaluated the vinculin- and paxillin-containing adhesion complexes and α-smooth muscle actin (α-SMA) expression on tubular cells and glomerular podocytes in first year renal allograft biopsy specimens of 74 patients. We established the proteinuric state at the time of the biopsy of all patients. Follow-up biopsy specimens of all patients were evaluated for the development of diffuse IF (≥50% of the biopsy specimen). Among 74 patients, 21 showed grade 1 and 9 showed grade 2 tubular EMT. Only 25/74 cases showed glomerular EMT. The incidence of the development of diffuse IF at 18 and 24 months after the initial biopsy and the graft loss at 5 years were higher among subjects with tubules and glomerular podocytes showing EMT ( P < .001 for all). The development of IF and graft loss was significantly earlier in cases with grade 2 compared with grade 1 or no tubular EMT ( P < .001 for all). The proteinuric state at the time of the biopsy showed a significant positive correlation with the glomerular EMT ( P < .001). In conclusion, our results showed that renal tubular cells and glomerular podocytes can undergo epithelial-to-mesenchymal differentiation. The transformed cells with reorganized cytoskeletal features have an important role in renal allograft survival by inducing the development of diffuse IF and proteinuria.