Gunter Wolf

TGF-β and fibrosis in different organs — molecular pathway imprints

The action of transforming-growth-factor (TGF)-beta following inflammatory responses is characterized by increased production of extracellular matrix (ECM) components, as well as mesenchymal cell proliferation, migration, and accumulation. Thus, TGF-beta is important for the induction of fibrosis often associated with chronic phases of inflammatory diseases. This common feature of TGF-related pathologies is observed in many different organs. Therefore, in addition to the description of the common TGF-beta-pathway, this review focuses on TGF-beta-related pathogenetic effects in different pathologies/organs, i. e., arthritis, diabetic nephropathy, colitis/Crohns disease, radiation-induced fibrosis, and myocarditis (including their similarities and dissimilarities). However, TGF-beta exhibits both exacerbating and ameliorating features, depending on the phase of disease and the site of action. Due to its central role in severe fibrotic diseases, TGF-beta nevertheless remains an attractive therapeutic target, if targeted locally and during the fibrotic phase of disease.

Cellular and Molecular Mechanisms of Proteinuria in Diabetic Nephropathy

One of the earliest clinically detectable abnormalities in diabetic nephropathy is microalbuminuria that eventually progresses to proteinuria. The degree of proteinuria correlates with the progression of glomerulosclerosis and tubulointerstitial fibrosis. In the glomerulus, a typical podocytopathy develops that participates in the initiation of glomerulosclerosis and the accelerated plasma protein leakage across the glomerular basement membrane (GBM) into Bowman’s space. Downstream into the tubular compartment, the proteinuria induces proinflammatory and profibrogenetic injury in tubular cells which can facilitate the development of interstitial fibrosis and tubular atrophy. It has long been held that hemodynamic changes and the loss of negatively charged proteoglycans in the GBM are important mediators of proteinuria. More recently, biopsy studies in humans with diabetic kidney disease have provided strong evidence that podocytes are injured very early in the course of nephropathy. This podocytopathy – which is characterized by decreased podocyte number and/or density, GBM thickening and altered matrix composition, and foot process effacement – correlates closely with the development and progression of albuminuria. Components of the diabetic milieu (high glucose, accumulation of glycated proteins, high intrarenal angiotensin II (ANG II), and hypertension-induced mechanical stress) result in activation of cytokine systems, the most important of which are transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor-A (VEGF-A). ANG II-stimulated podocyte-derived VEGF, through a novel autocrine signaling loop, appears to be a major cause of nephrin downregulation and the development of proteinuria. Nephrin is an important protein of the slit diaphragm with anti-apoptotic signaling properties. TGF-β1 causes podocyte apoptosis and an increase in extracellular matrix deposition. As a consequence, the denuded GBM adheres to Bowman’s capsule initiating the development of glomerulosclerosis. Good control of hyperglycemia and hypertension and maximal inhibition of ANG II are essential steps in preventing the development and progression of diabetic nephropathy.

Advanced glycation end‐products and the kidney

Eur J Clin Invest 2010; 40 (8): 742–755

Hydroxyethylstarch impairs renal function and induces interstitial proliferation, macrophage infiltration and tubular damage in an isolated renal perfusion model

IntroductionThe aim of the study was to evaluate some of the underlying pathomechanisms of hydroxyethylstarch (HES) induced adverse effects on renal function using 24 porcine kidneys in an isolated perfusion model over six hours.MethodsInfusion of either 10% HES 200/0.5, 6% HES 130/0.42 or Ringers lactate (RL) was performed to achieve an haematocrit of 20% in eight kidneys from four animals per group. Physiological and pathophysiological parameters were determined (including N-acetyl-beta-aminoglucosidase as a marker for lysosomal tubular damage). Histological investigations and immunohistological stainings of the kidneys were performed.ResultsInitially after haemodilution, HES 130/0.42 and HES 200/0.5 reduced urine output compared with RL (P < 0.01). After six hours, N-acetyl-beta-aminoglucosidase was significantly higher in HES 200/0.5 (81 ± 23 U/L) compared with HES 130/0.42 (38 ± 12 U/L) and RL (21 ± 13 U/L; P < 0.001). Osmotic nephrosis-like lesions (OL) of the tubuli were present in all groups showing a significantly lower number of OL in RL (1.1 ± 0.4; P = 0.002) compared with both HES groups (HES 200/0.5 = 2.1 ± 0.6; HES 130/0.42 = 2.0 ± 0.5). Macrophage infiltration was significantly higher in HES 200/0.5 compared with HES 130/0.42 (1.3 ± 1.0 vs. 0.2 ± 0.04; P = 0.044). There was a significant increase in interstitial cell proliferation in the HES 200/0.5 group vs. HES 130/0.42 (18.0 ± 6.9 vs. 6.5 ± 1.6; P = 0.006) with no significant difference in RL (13.5 ± 4.0).ConclusionsWe observed impaired diuresis and sodium excretion by HES and identified renal interstitial proliferation, macrophage infiltration and tubular damage as potential pathological mechanisms of HES-induced adverse effects on renal function using an isolated porcine renal perfusion model. Furthermore, we demonstrated that 10% HES 200/0.5 had more of a pro-inflammatory effect compared with 6% HES 130/0.42 and caused more pronounced tubular damage than 6% HES 130/0.42 and RL. OL were present in all groups, but to a lesser degree after RL administration.

The German Chronic Kidney Disease (GCKD) study: design and methods

BACKGROUND Chronic kidney disease (CKD) is increasingly recognized as a global health problem. The conditions leading to CKD, the health impact of CKD and the prognosis differ markedly between affected individuals. In particular, renal failure and cardiovascular mortality are competing risks for CKD patients. Opportunities for targeted intervention are very limited so far and require an improved understanding of the natural course of CKD, of the risk factors associated with various clinical end points and co-morbidities as well as of the underlying pathogenic mechanisms. METHODS The German Chronic Kidney Disease (GCKD) study is a prospective observational national cohort study. It aims to enrol a total of 5000 patients with CKD of various aetiologies, who are under nephrological care, and to follow them for up to 10 years. At the time of enrolment, male and female patients have an estimated glomerular filtration rate (eGFR) of 30-60 mL/min×1.73 m2 or overt proteinuria in the presence of an eGFR>60 mL/min×1.73 m2. Standardized collection of biomaterials, including DNA, serum, plasma and urine will allow identification and validation of biomarkers associated with CKD, CKD progression and related complications using hypothesis-driven and hypothesis-free approaches. Patient recruitment and follow-up is organized through a network of academic nephrology centres collaborating with practising nephrologists throughout the country. CONCLUSIONS The GCKD study will establish one of the largest cohorts to date of CKD patients not requiring renal replacement therapy. Similarities in its design with other observational CKD studies, including cohorts that have already been established in the USA and Japan, will allow comparative and joint analyses to identify important ethnic and geographic differences and to enhance opportunities for identification of relevant risk factors and markers.

Prevalence of the diabetic foot syndrome at the primary care level in Germany : a cross-sectional study

Aims  The diabetic foot syndrome (DFS) is an important complication of diabetes mellitus resulting in amputations, disability and reduced quality of life. DFS is preventable. The aim was to investigate the prevalence of the DFS at the primary care level in Germany.

Advanced glycation endproducts influence the mRNA expression of RAGE, RANKL and various osteoblastic genes in human osteoblasts.

Abstract Glycation reactions resulting in the generation and accumulation of advanced glycation endproducts (AGEs) are potential mechanisms by which bone protein may be altered in vivo. AGEs accumulate in the bone increasingly with age come into close contact with osteoblasts or osteoclasts. The direct effect of AGEs on bone cells has not been thoroughly investigated. This study aimed to examine whether glycated bovine serum albumin (AGE – BSA) as an AGE modulate the mRNA expression of various genes in primary human osteoblast cultures. The following parameters were included: RAGE (receptor for AGEs), alkaline phosphatase, osteocalcin, osterix and RANKL (receptor activator of nuclear factor-κB ligand). Primary human osteoblast cultures were obtained from bone specimens of six patients with osteoarthrosis. Human osteoblasts were treated in AGE – BSA or control-BSA (non-glycated BSA) containing medium (5 mg/ml each) over a time course of seven days. After RT–PCR the mRNA expression was measured by real-time PCR. Related to control – BSA exposure, the mRNA expression of RAGE, RANKL and osterix increased during AGE – BSA treament. For alkaline phosphatase and osteocalcin a tendency of down-regulation was found. In summary, the study presents evidence that advanced glycation end products accumulated in bone alter osteoblasts by activation the AGE – RAGE pathway (RAGE mRNA up-regulation), inducing enhanced osteoclastogenesis (RANKL mRNA up-regulation) and impaired matrix mineralization (down-regulation of alkaline phosphatase and osteocalcin mRNA). Thus, AGEs may play a functional role in the development of bone diseases (e.g. osteoporosis).

Asymmetrical (ADMA) and symmetrical dimethylarginine (SDMA) as potential risk factors for cardiovascular and renal outcome in chronic kidney disease – possible candidates for paradoxical epidemiology?

Summary.Background: Asymmetrical dimethylarginine (ADMA) is an inhibitor of nitric-oxide synthase. It has been linked to atherosclerotic risk in the general population as well as in end-stage renal disease patients (ESRD), whereas symmetrical dimethylarginine (SDMA) is thought to be biological inactive. Prospective data concerning the role of both dimethylarginines are rare in patients with chronic kidney disease.Methods: 200 patients with chronic kidney disease (mean age 57.6 ± 13.0 years, 69 female, 131 male); 82 with chronic renal failure (CRF), 81 on maintenance haemodialysis (HD) and 37 renal transplant recipients (RTR) were prospectively followed for 24 months. ADMA and SDMA were measured by HPLC. The relation of plasma levels of ADMA and SDMA together with conventional risk factors for the cardiovascular and renal outcome was investigated with Cox proportional hazards model.Results: Mean serum levels of SDMA were significantly increased in all groups compared to the control group (P ≤ 0.0005), ADMA was increased only in HD and RTR (P ≤ 0.004). Forty-seven cardiovascular events (CVE) occurred during follow-up, 35 patients died, and 39 patients reached ESRD. Multivariate analysis showed diabetes (RR 3.072, P = 0.01), ESRD (RR 11.915, P < 0.0005), elevated CRP levels (RR 3.916, P < 0.0005) and surprisingly a lower ADMA level (RR 0.271, P = 0.008) as independent risk factors for CVE. Serum creatinine (RR 11.378, P = 0.001), haemoglobin (RR 0.710, P = 0.038 for an increment of 1 mmol/l), and SDMA levels (RR 1.633, P = 0.006, per 1 µmol/l increment) were predictors for the progression to ESRD.Conclusions: Data from a heterogeneous group of patients with chronic kidney disease provide evidence that conventional risk factors seem to play a more important role than elevated serum levels of ADMA or SDMA for cardiovascular events. Increasing serum SDMA concentration seems to play an additive role for the renal outcome besides serum creatinine and haemoglobin levels. Whether ADMA might possibly be a candidate for the phenomenon of “paradoxical epidemiology” in chronic kidney disease needs further investigation.

Glycaemic control is positively associated with prevalent fractures but not with bone mineral density in patients with Type 1 diabetes

Diabet. Med. 28, 872–875 (2011)

Angiotensin II Upregulates RAGE Expression on Podocytes: Role of AT2 Receptors

Background: Advanced glycation end products (AGEs) play an important role in diabetic nephropathy. The receptor for AGEs, called RAGE, is present on podocytes. We investigated whether angiotensin II (ANG II) modulates RAGE expression on cultured differentiated podocytes. Results: Cultured podocytes expressed AT1 and AT2 receptors. Surprisingly, ANG II induced RAGE mRNA and protein expression through AT2 receptors. ANG II had no influence on proliferation or protein content of podocytes. The increase in RAGE expression depended on stimulated transcriptional activity. Using various mutant reporter constructs of the RAGE promoter region, it was shown that a NF-κB binding site at –1519 was essential for ANG II-induced transcriptional activity. Preincubation with ANG II increased the expression of tumor necrosis factor-α mRNA and protein expression induced by AGE, indicating that the ANG II-mediated upregulation of RAGE has functional consequences. AGE-BSA was incorporated into cells as measured by Western blots for Nε-carboxymethyllysine, but ANG II did not influence this process. ANG II in the absence or presence of AGE-BSA did not induce apoptosis of podocytes. Conclusion: Our study revealed aninteraction between the renin-angiotensin system and the AGE/RAGE axis in podocytes. Since intraglomerular ANG II levels are increased in diabetic nephropathy, this interaction may have pathophysiological consequences for podocyte injury and inflammation associated with the development of diabetic nephropathy.