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The tricellular tight junctions are crucial for the regulation of paracellular flux at tricellular junctions, where tricellulin (MARVELD2) and angulins (ILDR1, ILDR2, or LSR) are localized. The role of ILDR2 in podocytes, specialized epithelial cells in the kidney, is still unknown. We investigated the role of ILDR2 in glomeruli and its influence on blood filtration. Western blots, single-cell RNA sequencing (scRNA-seq), and superresolution microscopy showed a strong expression of ILDR2 in podocytes that colocalized with the podocyte-specific claudin CLDN5. Co-immunoprecipitation revealed that ILDR2 interacts with CLDN5. In glomerulopathies, induced by nephrotoxic serum and by desoxycorticosterone acetate (DOCA)-salt heminephrectomy, ILDR2 was strongly up-regulated. Furthermore, Ildr2 knockout mice exhibited glomerular hypertrophy and decreased podocyte density. However, they did not develop effacement of podocyte foot processes or proteinuria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analysis of isolated glomeruli showed an increase in matrix proteins, such as fibronectin and collagens. This suggests a protective role of ILDR2 in glomerulopathies., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Angiomyolipoma (AML) are the most common benign solid renal mass. Differentiation from malignant tumours is essential. Imaging features in ultrasound may overlap between malignant lesions, especially between renal cell carcinoma (RCC) and AML. So far, sectional imaging has been necessary for reliable differentiation. The aim of this study is to evaluate the use of the ultrasound-guided attenuation parameter (UGAP), a recently established tool for assessing hepatic steatosis, in the differentiation of AMLs from other renal masses. Therefore, 27 patients with unknown solid renal masses were examined by ultrasound including UGAP. The attenuation was assessed qualitatively by attenuation map and quantitatively in comparison to the surrounding renal tissue. UGAP was applicable in 26/27 patients. Findings were compared with CT/MRI as the current imaging standard. A total of 18 AML and 9 other renal tumours were found. The diagnostic performance of B-Mode (hyperechogenic lesion) ultrasound was 77.8% in identifying AML. The diagnostic performance of the attenuation map showed a diagnostic performance of 92.6%, whereby UGAP measurements were successful in 76.9% of cases. Quantitatively, we found a significant difference ( p < 0.034) in mean measured attenuation between AML (0.764 ± 0.162 dB/cm/MHz) vs. other renal tumours (0.658 ± 0.155 dB/cm/MHz). The best performance was found by a combined parameter of a hyperechogenic lesion with a positive attenuation map with an accuracy of 95.0%. In conclusion, UGAP may represent a possibility for differentiating solid renal lesions more accurately by ultrasound, especially classic hyperechoic AMLs from other renal lesions. Further studies are needed to increase the diagnostic reliability further.

More than 40 human infections with the zoonotic Borna disease virus 1 (BoDV-1) have been reported to German health authorities from endemic regions in southern and eastern Germany. Diagnosis of a confirmed case is based on the detection of BoDV-1 RNA or BoDV-1 antigen. In parallel, serological assays such as ELISA, immunoblots, and indirect immunofluorescence are in use to detect the seroconversion of Borna virus-reactive IgG in serum or cerebrospinal fluid (CSF). As immunopathogenesis in BoDV-1 encephalitis appears to be driven by T cells, we addressed the question of whether an IFN-γ-based ELISpot may further corroborate the diagnosis. For three of seven BoDV-1-infected patients, peripheral blood mononuclear cells (PBMC) with sufficient quantity and viability were retrieved. For all three patients, counts in the range from 12 to 20 spot forming units (SFU) per 250,000 cells were detected upon the stimulation of PBMC with a peptide pool covering the nucleocapsid protein of BoDV-1. Additionally, individual patients had elevated SFU upon stimulation with a peptide pool covering X or phosphoprotein. Healthy blood donors ( n = 30) and transplant recipients ( n = 27) were used as a control and validation cohort, respectively. In this pilot study, the BoDV-1 ELISpot detected cellular immune responses in human patients with BoDV-1 infection. Its role as a helpful diagnostic tool needs further investigation in patients with BoDV-1 encephalitis.

More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study ("BoDV-1 after solid-organ transplantation") to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.

Plasmalemma vesicle-associated protein (PLVAP) is the main component of endothelial diaphragms in fenestrae, caveolae, and transendothelial channels. PLVAP is expressed in the adult kidney glomerulus upon injury. Glomerular endothelial injury is associated with progressive loss of kidney function in diabetic kidney disease (DKD). This study aimed to investigate whether PLVAP could serve as a marker for glomerular endothelial damage in DKD. Glomerular PLVAP expression was analyzed in different mouse models of DKD and their respective healthy control animals using automatic digital quantification of histological whole kidney sections. Transgenic mice expressing a dominant-negative GIP receptor (GIPR dn ) in pancreatic beta-cells as a model for diabetes mellitus (DM) type 1 and black and tan brachyuric (BTBR) ob/ob mice, as a model for DM type 2, were used. Distinct PLVAP induction was observed in all diabetic models studied. Traces of glomerular PLVAP expression could be identified in the healthy control kidneys using automated quantification. Stainings for other endothelial injury markers such as CD31 or the erythroblast transformation-specific related gene (ERG) displayed no differences between diabetic and healthy groups at the time points when PLVAP was induced. The same was also true for the mesangial cells marker α8Integrin, while the podocyte marker nephrin appeared to be diminished only in BTBR ob/ob mice. Glomerular hypertrophy, which is one of the initial morphological signs of diabetic kidney damage, was observed in both diabetic models. These findings suggest that PLVAP is an early marker of glomerular endothelial injury in diabetes-induced kidney damage in mice.

Background: Time-intensity curve analysis (TIC analysis) based on contrast-enhanced ultrasound (CEUS) provides quantifiable information about the microcirculation of different tissues. TIC analysis of kidney transplantations is still a field of research, and standardized study protocols are missing though being mandatory for the interpretation of TIC parameters in the clinical context. The aim of this study was to evaluate the impact of different sizes and forms of regions of interest (ROIs) on the variance of different TIC parameters and the level of interoperator variance between the different ROI methods in kidney transplantations., Methods: In 25 renal transplanted patients, 33 CEUS of the transplanted kidney were performed, and TIC analysis with ROIs sized 5 mm 2 (ROI 5 ), 10 mm 2 (ROI 10 ), and ROIs circumscribing the outlines of anatomical regions (ROI Anat ) were analyzed based on CEUS examination. The TIC analysis was repeated by a second independent operator for ROI 5 and ROI Anat ., Results: Statistical analysis revealed significant differences between TIC parameters of different ROI methods, and overall, the interoperator variance was low. But a greater ROI surface (ROI 10 ) led to higher values of the intensity parameters A and AUC compared with ROI 5 ( p < 0.05). The difference in the ROI form led to high variation of certain TIC parameters between ROI 5 and ROI Anat in the myelon [intraclass correlation coefficient (A, ICC = 0.578 (0.139-0.793); TIC parameter (TTP); and ICC = 0.679 (0.344-0.842) ( p < 0.05)]. A mean variation of 1 cm of the depth of ROI 5 in the cortex did not show significant differences in the TIC parameters, though there was an impact of depth of ROI Anat on the values of TIC parameters. The interoperator variance in the cortex was low and equal for ROI 5 and ROI Anat , but increased in the myelon, especially for ROI Anat . Furthermore, the analysis revealed a strong correlation between the parameter AUC and the time interval applied for the TIC analysis in the cortex and myelon ( r = 0.710, 0.674, p < 0.000)., Conclusion: Our findings suggest the application of multiple ROIs of 5 mm 2 in the cortex and medulla to perform TIC analysis of kidney transplants. For clinical interpretation of AUC, a standardized time interval for TIC analysis should be developed. After the standardization of the TIC analysis, the clinical predictive value could be investigated in further studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Friedl, Jung, Bergler, Tews, Banas, Banas and Putz.)

Diabetic nephropathy (DN) remains the most common reason for end-stage renal disease and a leading cause of kidney replacement therapy. Multifactorial pathophysiological mechanisms underlie the development of DN. Among the signalling pathways involved, nuclear factor-κB (NF-κB) plays a key role in pathogenesis triggering inflammation, oxidative stress and fibrosis. Recent evidence shows that periostin, a matricellular protein, is involved in the development of renal glomerular diseases through interaction with NF-κB signalling. The aim of the present study is to investigate the contribution of periostin and its interaction with NF-κB in DN development. To this end, we used the BTBR ob/ob mice model of diabetes type 2, and we applied transcriptomic analysis, immunostaining and methods quantifying protein and mRNA expressions. We found that increased periostin expression was correlated with decreased renal function, advanced stage renal damage and fibrosis, and NF-κB activation. Subsequently, we identified novel pathways and genes regulated by the NF-κB-periostin interaction which are involved in the mechanisms of progression of DN. Some of these genes, such as FGF1 and GDF15, have the potential to be new biomarkers and/or targets for the therapy of DN.

Mesangial cells (MCs), substantial cells for architecture and function of the glomerular tuft, take a key role in progression of diabetic kidney disease (DKD). Despite long standing researches and the need for novel therapies, the underlying regulatory mechanisms in MCs are elusive. This applies in particular to long non-coding RNAs (lncRNA) but also microRNAs (miRNAs). In this study, we investigated the expression of nuclear paraspeckle assembly transcript 1 ( NEAT1 ), a highly conserved lncRNA, in several diabetes in-vitro models using human MCs. These cells were treated with high glucose, TGFβ, TNAα, thapsigargin, or tunicamycin. We analyzed the implication of NEAT1 silencing on mesangial cell migration, proliferation, and cell size as well as on mRNA and miRNA expression. Here, the miRNA hsa-miR-339-5p was not only identified as a potential interaction partner for NEAT1 but also for several coding genes. Furthermore, overexpression of hsa-miR-339-5p leads to a MC phenotype comparable to a NEAT1 knockdown. In-silico analyses also underline a relevant role of NEAT1 and hsa-miR-339-5p in mesangial physiology, especially in the context of DKD.

Due to organ shortage and rising life expectancy the age of organ donors and recipients is increasing. Reliable biomarkers of organ quality that predict successful long-term transplantation outcomes are poorly defined. The aim of this study was the identification of age-related markers of kidney function that might accurately reflect donor organ quality. Histomorphometric, biochemical and molecular parameters were measured in young (3-month-old) and old (24-month-old) male Sprague Dawley rats. In addition to conventional methods, we used urine metabolomics by NMR spectroscopy and gene expression analysis by quantitative RT-PCR to identify markers of ageing relevant to allograft survival. Beside known markers of kidney ageing like albuminuria, changes in the concentration of urine metabolites such as trimethylamine-N-oxide, trigonelline, 2-oxoglutarate, citrate, hippurate, glutamine, acetoacetate, valine and 1-methyl-histidine were identified in association with ageing. In addition, expression of several genes of the toll-like receptor (TLR) pathway, known for their implication in inflammaging, were upregulated in the kidneys of old rats. This study led to the identification of age-related markers of biological allograft age potentially relevant for allograft survival in the future. Among those, urine metabolites and markers of immunity and inflammation, which are highly relevant to immunosuppression in transplant recipients, are promising and deserve further investigation in humans.

Background: In an earlier monocentric study, we have developed a novel non-invasive test system for the prediction of renal allograft rejection, based on the detection of a specific urine metabolite constellation. To further validate our results in a large real-world patient cohort, we designed a multicentric observational prospective study (PARASOL) including six independent European transplant centers. This article describes the study protocol and characteristics of recruited better patients as subjects. Methods: Within the PARASOL study, urine samples were taken from renal transplant recipients when kidney biopsies were performed. According to the Banff classification, urine samples were assigned to a case group (renal allograft rejection), a control group (normal renal histology), or an additional group (kidney damage other than rejection). Results: Between June 2017 and March 2020, 972 transplant recipients were included in the trial (1,230 urine samples and matched biopsies, respectively). Overall, 237 samples (19.3%) were assigned to the case group, 541 (44.0%) to the control group, and 452 (36.7%) samples to the additional group. About 65.9% were obtained from male patients, the mean age of transplant recipients participating in the study was 53.7 ± 13.8 years. The most frequently used immunosuppressive drugs were tacrolimus (92.8%), mycophenolate mofetil (88.0%), and steroids (79.3%). Antihypertensives and antidiabetics were used in 88.0 and 27.4% of the patients, respectively. Approximately 20.9% of patients showed the presence of circulating donor-specific anti-HLA IgG antibodies at time of biopsy. Most of the samples (51.1%) were collected within the first 6 months after transplantation, 48.0% were protocol biopsies, followed by event-driven (43.6%), and follow-up biopsies (8.5%). Over time the proportion of biopsies classified into the categories Banff 4 (T-cell-mediated rejection [TCMR]) and Banff 1 (normal tissue) decreased whereas Banff 2 (antibody-mediated rejection [ABMR]) and Banff 5I (mild interstitial fibrosis and tubular atrophy) increased to 84.2 and 74.5%, respectively, after 4 years post transplantation. Patients with rejection showed worse kidney function than patients without rejection. Conclusion: The clinical characteristics of subjects recruited indicate a patient cohort typical for routine renal transplantation all over Europe. A typical shift from T-cellular early rejections episodes to later antibody mediated allograft damage over time after renal transplantation further strengthens the usefulness of our cohort for the evaluation of novel biomarkers for allograft damage., Competing Interests: SN, VR, AS, and ES report personal fees from numares AG, outside the submitted work. In addition, SN has a patent WO2018167157A1 pending that is directly related to this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Banas, Böhmig, Viklicky, Rostaing, Jouve, Guirado, Facundo, Bestard, Gröne, Kobayashi, Hanzal, Putz, Zecher, Bergler, Neumann, Rothe, Schwäble Santamaria, Schiffer and Banas.)

Background: To validate a novel method for post-transplant surveillance to detect kidney allograft rejection via a characteristic constellation of the urine metabolites alanine, citrate, lactate, and urea investigated by nuclear magnetic resonance (NMR) spectroscopy a first prospective, observational study was performed., Methods: Within the UMBRELLA study 986 urine specimens were collected from 109 consecutively enrolled renal transplant recipients, and metabolite constellations were analyzed. A metabolite rejection score was calculated and compared to histopathological results of corresponding indication and protocol allograft biopsies (n = 206)., Findings: The metabolite constellation was found to be a useful biomarker to non-invasively detect acute allograft rejection (AUC = 0.75; 95% confidence interval (CI) 0.68-0.83; based on 46 cases and 520 control samples). Combined analysis of the metabolite rejection score and the estimated glomerular filtration rate (eGFR) at the time of urine sampling further improved the overall test performance significantly (AUC = 0.84; 95% CI 0.76-0.91; based on 42 cases and 468 controls). Regarding the time course analysis in patients without rejection episodes the test results remained well below a diagnostic threshold associated with high risk of acute rejection. In other cases, a marked increase above this threshold indicated acute allograft rejection already six to ten days before diagnostic renal biopsies were performed., Interpretation: A combination of an NMR-based urine metabolite analysis and eGFR is promising as a non-invasive test for post-transplant surveillance and to support decision making whether renal allografts need histopathological evaluation., (Copyright © 2019. Published by Elsevier B.V.)

The prevalence of type 2 diabetes mellitus (T2DM) and by association diabetic nephropathy (DN) will continuously increase in the next decades. Nevertheless, the underlying molecular mechanisms are largely unknown and studies on the role of new actors like long non-coding RNAs (lncRNAs) barely exist. In the present study, the inherently insulin-resistant mouse strain "black and tan, brachyuric" (BTBR) served as T2DM model. While wild-type mice do not exhibit pathological changes, leptin-deficient diabetic animals develop a severe T2DM accompanied by a DN, which closely resembles the human phenotype. We analyzed the glomerular expression of lncRNAs from wild-type and diabetic BTBR mice (four, eight, 16, and 24 weeks) applying the "GeneChip Mouse Whole Transcriptome 1.0 ST" array. This microarray covered more lncRNA gene loci than any other array before. Over the observed time, our data revealed differential expression patterns of 1746 lncRNAs, which markedly differed from mRNAs. We identified protein-coding and non-coding genes, that were not only co-located but also co-expressed, indicating a potentially cis-acting function of these lncRNAs. In vitro-experiments strongly suggested a cell-specific expression of these lncRNA-mRNA-pairs. Additionally, protein-coding genes, being associated with significantly regulated lncRNAs, were enriched in various biological processes and pathways, that were strongly linked to diabetes.

Background: Although diabetic nephropathy (DN) is the most common cause for end-stage renal disease in western societies, its pathogenesis still remains largely unclear. A different gene pattern of diabetic and healthy kidney cells is one of the probable explanations. Numerous signalling pathways have emerged as important pathophysiological mechanisms for diabetes-induced renal injury., Methods: Glomerular cells, as podocytes or mesangial cells, are predominantly involved in the development of diabetic renal lesions. While many gene assays concerning DN are performed with whole kidney or renal cortex tissue, we isolated glomeruli from black and tan, brachyuric (BTBR) obese/obese (ob/ob) and wildtype mice at four different timepoints (4, 8, 16 and 24 weeks) and performed an mRNA microarray to identify differentially expressed genes (DEGs). In contrast to many other diabetic mouse models, these homozygous ob/ob leptin-deficient mice develop not only a severe type 2 diabetes, but also diabetic kidney injury with all the clinical and especially histologic features defining human DN. By functional enrichment analysis we were able to investigate biological processes and pathways enriched by the DEGs at different disease stages. Altered expression of nine randomly selected genes was confirmed by quantitative polymerase chain reaction from glomerular RNA., Results: Ob/ob type 2 diabetic mice showed up- and downregulation of genes primarily involved in metabolic processes and pathways, including glucose, lipid, fatty acid, retinol and amino acid metabolism. Members of the CYP4A and ApoB family were found among the top abundant genes. But more interestingly, altered gene loci showed enrichment for processes and pathways linked to angioneogenesis, complement cascades, semaphorin pathways, oxidation and reduction processes and renin secretion., Conclusion: The gene profile of BTBR ob/ob type 2 diabetic mice we conducted in this study can help to identify new key players in molecular pathogenesis of diabetic kidney injury.

Impaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of CMV reactivation and associated complications in solid-organ transplantation. Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of T-Track ® CMV, a novel IFN-γ ELISpot assay based on the stimulation of peripheral blood mononuclear cells with pp65 and IE-I CMV proteins, to monitor CMV-CMI following kidney transplantation. A prospective longitudinal multicenter study was conducted in 86 intermediate-risk renal transplant recipients. CMV-CMI, CMV viral load, and clinical complications were monitored over 6 months post-transplantation. Ninety-five percent and 88-92% ELISpot assays were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection, indicating the ability of the ELISpot assay to monitor patients' immunosuppressive state. Interestingly, median pp65-specific response was ninefold higher in patients with self-clearing viral load compared to antivirally treated patients prior to first viral load detection (P < 0.001), suggesting that reactivity to pp65 represents a potential immunocompetence marker. Altogether, T-Track ® CMV is a highly sensitive IFN-γ ELISpot assay, suitable for the immunomonitoring of CMV-seropositive renal transplant recipients, and with a potential use for the risk assessment of CMV-related clinical complications (ClinicalTrials.gov Identifier: NCT02083042)., (© 2017 The Authors. Transplant International published by John Wiley & Sons Ltd on behalf of Steunstichting ESOT.)

The homeostatic chemokine receptor CCR7 serves as key molecule in lymphocyte homing into secondary lymphoid tissues. Previous experiments from our group identified CCR7 also to be expressed by human mesangial cells. Exposing cultured human mesangial cells to the receptor ligand CCL21 revealed a positive effect on these cells regarding proliferation, migration, and survival. In the present study, we localized CCR7 and CCL21 during murine nephrogenesis. Analyzing wild-type and CCR7 deficient (CCR7 -/- ) mice, we observed a retarded glomerulogenesis during renal development and a significantly decreased mesangial cellularity in adult CCR7 -/- mice, as a consequence of less mesangial cell proliferation between embryonic day E17.5 and week 5 postpartum. Cell proliferation assays and cell-wounding experiments confirmed reduced proliferative and migratory properties of mesangial cells cultured from CCR7 -/- kidneys. To further emphasize the role of CCR7 as important factor for mesangial biology, we examined the chemokine receptor expression in rats after induction of a mesangioproliferative glomerulonephritis. Here, we demonstrated for the first time that extra- and intraglomerular mesangial cells that were CCR7-negative in control rats exhibited a strong CCR7 expression during the phase of mesangial repopulation and proliferation.

Objective: Despite substantial progress in recent years, graft survival beyond the first year still requires improvement. Since modern immunosuppression addresses mainly T-cell activation and proliferation, we studied macrophage infiltration into the allografts of 103 kidney transplant recipients during acute antibody and T-cell mediated rejection. Macrophage infiltration was correlated with both graft function and graft survival until month 36 after transplantation., Results: Macrophage infiltration was significantly elevated in antibody-mediated and T-cell mediated rejection, but not in kidneys with established IFTA. Treatment of rejection with steroids was less successful in patients with more prominent macrophage infiltration into the allografts. Macrophage infiltration was accompanied by increased cell proliferation as well as antigen presentation. With regard to the compartmental distribution severity of T-cell-mediated rejection was correlated to the amount of CD68+ cells especially in the peritubular and perivascular compartment, whereas biopsies with ABMR showed mainly peritubular CD68 infiltration. Furthermore, severity of macrophage infiltration was a valid predictor of resulting creatinine values two weeks as well as two and three years after renal transplantation as illustrated by multivariate analysis. Additionally performed ROC curve analysis showed that magnitude of macrophage infiltration (below vs. above the median) was a valid predictor for the necessity to restart dialysis. Having additionally stratified biopsies in accordance to the magnitude of macrophage infiltration, differential CD68+ cell infiltration was reflected by striking differences in overall graft survival., Conclusion: The differences in acute allograft rejection have not only been reflected by different magnitudes of macrophage infiltration, but also by compartment-specific infiltration pattern and subsequent impact on resulting allograft function as well as need for dialysis initiation. There is a robust relationship between macrophage infiltration, accompanying antigen-presentation and resulting allograft function.

We tested the hypothesis that increased urinary cytokine concentrations may indicate an acute kidney transplant rejection. Eight patients with an early rejection in their protocol biopsy about 14days after transplantation (group A), 9 patients with a biopsy proven rejection 2-3months after transplantation (group B) and 18 patients without acute rejection in their protocol biopsies both at 14days and 3months (group C, represents the control group) were chosen for this study. At the time of biopsy, the mean urinary concentration of interleukin 6 (IL6), soluble IL6 receptor (sIL6R), tumor necrosis factor receptor 1 (TNFR1), and soluble vascular cell adhesion molecule -1 (sVCAM-1) were significantly higher in patients with an early acute transplant rejection, i.e. in group A compared to patients in the control group (p<0.01). Additionally we found already 14days after transplantation significantly higher concentrations of urinary sIL6R and sVCAM-1 in group B patients who suffered of late acute rejection compared to patients with no acute rejection (group C, p<0.05). No significant correlation could be shown for interleukin 1 receptor antagonist (IL1ra), TNF, and TNFR2. In conclusion, elevated urinary concentrations of IL6, sIL6R, TNFR1 and sVCAM-1 clearly indicate an early acute transplant rejection. Especially sVCAM-1 may also serve as an early marker of an upcoming late rejection. However, further studies are warranted to verify the value of individual cytokine profiles to predict acute rejection episodes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Platelet-derived growth factor-D (PDGF-D), normally expressed in podocytes, mediates mesangial cell proliferation in vivo. To study this further, we created transgenic mice with podocyte-specific overexpression of PDGF-D. Hemizygous mice were grossly indistinguishable from wild-type littermates through 11 months of age; however, hemizygous mice older than 4 weeks commonly exhibited increased cell proliferation within the glomerular tuft. Many hemizygous mice also developed widespread segmental glomerulosclerosis and focal extracapillary proliferation with fibrin/fibrinogen deposition, extensive tubulointerstitial damage, proteinuria, and renal insufficiency. Electron microscopy found focal foot process effacement. Renal mRNA expression of podocin and nephrin, as well as the number of glomerular WT-1-positive cells, were significantly reduced in hemizygous compared to wild-type mice, indicating loss and/or dedifferentation of podocytes. PDGF-A, -B, and both PDGF receptor chain mRNAs, fibronectin, type IV collagen, RANTES, MCP-1, and CCR-2 mRNAs were all increased in the renal cortex of PDGF-D transgenic mice. Only 8.5% of newborn mice were homozygous overexpressors exhibiting a mortality rate of 37% at 4 weeks. Thus, podocyte-specific overexpression of PDGF-D caused mesangioproliferative disease, glomerulosclerosis, and crescentic glomerulonephritis. Hence, podocyte-specific growth factor overexpression can induce paracrine mesangial cell proliferation upstream of the filtration flow.

There remains a need for robust mouse models of diabetic nephropathy (DN) that mimic key features of advanced human DN. The recently developed mouse strain BTBR with the ob/ob leptin-deficiency mutation develops severe type 2 diabetes, hypercholesterolemia, elevated triglycerides, and insulin resistance, but the renal phenotype has not been characterized. Here, we show that these obese, diabetic mice rapidly develop morphologic renal lesions characteristic of both early and advanced human DN. BTBR ob/ob mice developed progressive proteinuria beginning at 4 weeks. Glomerular hypertrophy and accumulation of mesangial matrix, characteristic of early DN, were present by 8 weeks, and glomerular lesions similar to those of advanced human DN were present by 20 weeks. By 22 weeks, we observed an approximately 20% increase in basement membrane thickness and a >50% increase in mesangial matrix. Diffuse mesangial sclerosis (focally approaching nodular glomerulosclerosis), focal arteriolar hyalinosis, mesangiolysis, and focal mild interstitial fibrosis were present. Loss of podocytes was present early and persisted. In summary, BTBR ob/ob mice develop a constellation of abnormalities that closely resemble advanced human DN more rapidly than most other murine models, making this strain particularly attractive for testing therapeutic interventions.

Imatinib is a receptor tyrosine kinase inhibitor that blocks the activity of c-Abl, c-Kit, and PDGF receptors. We tested the protective effects of imatinib in thymic stromal lymphopoietin transgenic mice, a model of cryoglobulinemia and associated membranoproliferative glomerulonephritis (MPGN), in which some glomerular manifestations likely result from PDGF receptor activation. Surprising, administration of imatinib beginning at weaning suppressed production of cryoglobulin, attenuating both the renal injury and systemic features of cryoglobulinemia. Flow cytometry suggested that inhibition of B cell development in the bone marrow likely caused the reduction in cryoglobulin production. In addition, administration of imatinib to thymic stromal lymphopoietin transgenic mice with established MPGN also diminished cryoglobulin production and reversed the renal and systemic lesions. These data suggest that treatment with imatinib may be a novel therapeutic approach for cryoglobulinemia and MPGN in humans.

Background: Smoking is the most important remediable cardiovascular risk factor, and an independent risk factor for the progression of renal diseases. To date, only limited information about changes in cigarette-smoking habits before and after renal transplantation is available., Methods: In a comprehensive cross-sectional single centre study, we analysed smoking habits of patients registered on the waiting list for renal transplantation and patients that had received an allograft., Results: Of 230 patients (76.1%), 175 on the waiting list and of 264 allograft recipients (87.5%), 231 were non-smokers at the time of investigation (P <0.01). Among the non-smoking waiting list patients, only 71 (30.9%) had never smoked, whereas 108 (40.9%) patients of the allograft recipients were never-smokers. Of former smoking patients, 27.6% (n = 34) had stopped smoking after transplantation. Patients <55 years of age and females were more likely to stop smoking than patients >55 years of age or males. A data analysis revealed that smokers had a significantly lower probability to attain renal transplantation., Conclusion: We conclude that renal transplantation is a strong incentive for patients to stop smoking. Reasons for changes in smoking behaviour after renal transplantation may be an intense contact of the patients with their physicians, the fear of a premature loss of the transplanted organ with continued smoking and the psychological support during post-transplantation patient care.

Toll-like receptors (TLR) classically recognize pathogen-associated danger signals but are also activated via endogenous ligands. For evaluation of their role in inflammatory kidney disease, the function of TLR was analyzed in two mouse models of cryoglobulinemic membranoproliferative glomerulonephritis (MPGN; mice transgenic for thymic stromal lymphopoietin [TSLP], with or without deletion of the Fcgamma receptor IIb). Expression of TLR1 through 9 and TLR11 mRNA was detectable in whole kidneys and in isolated glomeruli of wild-type mice, with TLR3 and TLR4 having the highest absolute levels of expression. TLR1, 2, and 4 were increased in TSLP transgenic mice and even higher in TSLP transgenic FcgammaRIIb-deficient mice. TLR5 through 9 and 11 were upregulated to similar degrees in TSLP transgenic and TSLP transgenic FcgammaRIIb-deficient mice. Immunohistochemical studies of nephritic glomeruli localized TLR4 protein to podocytes. Cultured podocytes also expressed TLR4, and stimulation with TLR4-specific ligands resulted in a marked induction of chemokines; this was reduced by specific knockdown of TLR4 with siRNA. Fibrinogen, a potential endogenous TLR4 ligand, was shown to induce a similar profile of chemokines. In conclusion, it was demonstrated that TLR4 is constitutively expressed by podocytes and is upregulated in MPGN, where it may mediate glomerular injury by modulating expression of chemokines; therefore, TLR4 may link podocytes with the innate immune system to mediate MPGN triggered by the deposition of immune complexes.

Background: Transgenic (tg) mice overexpressing thymic stromal lymphopoietin (TSLP) develop mixed cryoglobulinaemia with renal disease closely resembling human cryoglobulinaemic membranoproliferative glomerulonephritis (MPGN), as well as systemic inflammation involving lung, liver and skin as a result of cryoglobulin deposits. We assessed the effect of all-trans-retinoic acid (ATRA), a powerful anti-inflammatory agent, on this model of cryoglobulinaemic MPGN., Methods: Groups of male TSLP tg mice and wild-type controls were treated with either ATRA (20 mg/kg) or vehicle 3 times weekly by intraperitoneal injection for 4 or 8 weeks, when mice were then sacrificed. Routine histology and immunohistochemistry for collagen IV, alpha-smooth muscle actin, Mac-2 and Ki67 were performed. Immunoglobulin levels were measured by enzyme-linked immunosorbent assay., Results: ATRA unexpectedly exacerbated renal injury in TSLP tg mice with increased glomerular extracellular matrix, mesangial cell activation, glomerular cell proliferation, glomerular macrophage influx and immune complex deposition. Systemic injuries involving liver and lung, and the amount of circulating cryoglobulins were all worsened by ATRA treatment. Furthermore, ATRA resulted in increased IgG1 and IgM levels, the main components of the cryoglobulins in TSLP tg mice, and a manifestation of an enhanced Th2 immune response., Conclusions: ATRA is not protective but instead aggravates cryoglobulinaemic MPGN and its systemic manifestations in TSLP tg mice. We speculate these findings may be due to augmented production of pathogenic immunoglobulins and/or an enhanced systemic Th2 response. Although disappointing, our results also suggest caution in the application of retinoid therapy to human disease based on the largely positive animal data reported to date.

Smad proteins are signaling intermediates of the TGF-beta superfamily and are involved in a range of biological activities including development and immune responses. We studied the expression of TGF-beta-receptor activated Smads (Smad2 and Smad3), the common partner Smad (Smad4), an inhibitory Smad (Smad7), and the activated (phosphorylated) Smad2 (pSmad2) in developing and adult kidneys of humans and mice. These studies demonstrate associated expression of these Smads in multiple renal cell types in all developmental stages and in mature non-diseased kidneys. Smad expression is in general most widespread at the earliest stages of nephron development and diminishes as components of the nephrons become more differentiated. Paucity of Smad expression in mesangial cells in contrast to widespread expression of these Smads in glomerular visceral epithelial cells in both developing and mature kidneys was remarkable. Divergent and less extensive expression of Smad4, compared with other Smad proteins, was also demonstrated in tubules of human kidneys. Based on the observed expression patterns, these findings demonstrate, for the first time, expression of the TGF-beta-receptor-activated Smad2 and Smad3, the common mediator Smad4, and the inhibitory Smad7 in the developing human fetal kidney, extending observations previously made in rodent systems to humans.