Gerhard-Anton Müller

Immuno-MALDI-TOF MS: new perspectives for clinical applications of mass spectrometry.

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.

Inflammation and Microvasculopathy in Renal Ischemia Reperfusion Injury

Acute renal failure (ARF) severely worsens prognosis of hospitalized patients. The most frequent cause of intrarenal ARF is transient or prolonged renal hypoperfusion (ischemia). Ischemia primarily affects the function and structure of tubular epithelial cells, which, in severe cases, is characterized by epithelial cell necrosis. Nevertheless, ischemia does not exclusively lead to alterations of epithelial cells but also causes interstitial inflammation and interstitial microvasculopathy. Both inflammation and microvasculopathy are particularly important in terms of postischemic kidney repair. Postischemic microvasculopathy is characterized by endothelial cell swelling with subsequent microvascular occlusion. Thus, reperfusion is inhibited (no-reflow phenomenon). Such endothelial cell dysfunction offers new therapeutic perspectives in ischemic ARF. Newer observations point towards the role of the so-called endothelial progenitor cells (EPCs) in the treatment of ARF. Systemic administration of EPCs to mice with bilateral renal ischemia mitigates postischemic endothelial cell dysfunction and protects animals from acute renal failure.

Integrin α2-deficient mice provide insights into specific functions of collagen receptors in the kidney

BackgroundIntegrins are important cellular receptors for collagens. Within the glomerulus, podocytes regulate the integrity of the glomerular basement membrane (GBM) by sensing the presence of collagen and regulating collagen IV synthesis. The present study evaluates the role of integrin α2 (ITGA2) in cell-matrix interaction.Methods and ResultsITGA2-deficient mice had normal renal function but moderate proteinuria and enhanced glomerular and tubulointerstitial matrix deposition. Electron microscopy demonstrated irregular podocyte-matrix interaction, causing pathological protrusions towards the urinary (podocyte) side of the GBM. These characteristic subepithelial bulges mimic the renal phenotype of mice, which are deficient in another collagen receptor, discoidin domain receptor (DDR)1. Using immunogold staining, ITGA2 expression was found to localize to the basolateral site of the podocyte foot processes. ITGA2-deficient mice overexpressed transforming growth factor (TGF)β and connective tissue growth factor (CTGF) compared with wild-type mice. Using in situ hybridization, tubular cells were found to be the primary site of TGFβ synthesis and podocytes the source of CTGF in ITGA2-deficient mice.ConclusionThese findings support our hypothesis that both these collagen receptors (ITGA2 and DDR1) play a similar role within the kidney. Further, cell-matrix interaction via collagen receptors seems to be crucial for maintenance of normal GBM architecture and function. Targeting collagen receptors such as ITGA2 might be a new form of treatment for progressive fibrotic diseases.

Renal Protective Effects of Aliskiren Beyond Its Antihypertensive Property in a Mouse Model of Progressive Fibrosis

BACKGROUND The direct renin inhibitor aliskiren is known to exhibit a strong antihypertensive effect. However, the organoprotective potential of aliskiren beyond its antihypertensive properties is less clear. This study investigates the antifibrotic nephroprotective effects of aliskiren in a nonhypertensive mouse model for progressive renal fibrosis. METHODS COL4A3(-/-) mice received aliskiren via osmotic minipumps. Placebo-treated animals served as controls. Therapy was initiated in 6-week-old animals already showing renal damage (proteinuria ~1 g/l, starting renal fibrosis) and lasted for 4 weeks. Six animals were sacrificed after 9.5 weeks; serum urea and proteinuria were measured. Kidneys were further investigated using histological, immunohistological, and western blot techniques. Survival until end-stage renal failure was monitored in the remaining animals. RESULTS COL4A3(-/-) mice did not develop hypertension. Aliskiren serum levels were in the therapeutic range (288 ± 44 ng/ml). Therapy significantly prolonged lifespan until death from renal failure by 18% compared with placebo-treated controls (78.6 ± 8.2 vs. 66.6 ± 4.9 days, P < 0.05). Similarly, therapy reduced the amount of proteinuria and serum urea. Compared with placebo-treated controls, the accumulation of extracellular matrix and renal scarring and the levels of transforming growth factor-β (TGFβ) and connective tissue growth factor (CTGF) were decreased in treated mice. CONCLUSIONS Despite the late onset of therapy, our results indicate nephroprotective effects of the renin inhibitor aliskiren beyond its antihypertensive property in this animal model of progressive renal fibrosis. In addition to the recognized antihypertensive action of aliskiren, its antifibrotic, antiproteinuric effects demonstrated in the present study indicate that aliskiren may have potential as an important therapeutic option for chronic fibrotic diseases in humans.

Living donor kidney transplantation from relatives with mild urinary abnormalities in Alport syndrome: long-term risk, benefit and outcome

BACKGROUND Alport syndrome is a hereditary nephropathy leading to renal failure during adolescence. This study evaluates the outcome of living donor transplantation (Tx) from heterozygous mothers to their affected children. METHODS Seven mothers were evaluated, and donation was refused in one because of proteinuria. RESULTS All of the remaining six donors had microhaematuria, and one had proteinuria. Renal function was monitored after Tx (average 6.7 years in donors and 5.3 years in acceptors). Three of six donors developed new onset hypertension, and two new onset of proteinuria. Renal function declined significantly in four donors: (1) -35% after 2 years; (2) -25% after 3 years; (3) -30% after 4 years and (4) -60% after 14 years versus before Tx. However, creatinine clearance remained >40 ml/min in all donors. All transplanted kidneys worked well 1 and 5 years after Tx, and one failed after 10 years. One patient died from meningitis, and the remaining four remained stable. CONCLUSION Living donor Tx from relatives in Alport families is an ambivalent option. Proteinuria should be an exclusion criterion. Yet, even in donors with isolated microhaematuria, families and their physicians should be aware of an increased risk of renal failure in donor and recipient. This risk might be minimized by careful donor evaluation including biopsy and nephroprotective strategies after Tx in both donor and recipient.

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice.

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3(-/-)/ITGA2(-/-) double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3(-/-)/ITGA2(+/-) and COL4A3(-/-)/ITGA2(+/+) mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3(-/-) Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3(-/-)/ITGA2(+/+) Alport mice, but not in COL4A3(+/+)/ITGA2(-/-) mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell-matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.

Therapeutic Apheresis in Pediatric Patients with Acute CNS Inflammatory Demyelinating Disease

Background/Aims: In adults, plasma exchange (PE) has been shown to be an efficient treatment for severe relapses of acute inflammatory CNS demyelinating diseases. The aim of this study was to evaluate the safety and efficacy of this treatment in pediatric patients. Methods: We retrospectively analyzed a single-center cohort of pediatric patients with inflammatory CNS demyelinating disorders who underwent apheresis between 2007 and 2011. Results: Ten patients (mean age: 11.6 ± 3.4 years) with an acute relapse of multiple sclerosis (n = 5), neuromyelitis optica (n = 2) or acute disseminated encephalomyelitis were included. All received methylprednisolone prior to treatment with either PE (n = 5) or immunoadsorption (n = 5). Apheresis-related side effects were either self-limiting or easily managed. EDSS (Expanded Disability Status Scale) improved in 7 of 8 patients during apheresis and in all patients within 30 days from a median of 7.5 to 1 (p < 0.01). The visual acuity initially worsened during the procedure in 3 of 7 affected eyes (mean 0.09), but improved in all at follow-up (mean: 0.5; p = 0.008). Conclusions: Apheresis was well tolerated and associated with a favorable outcome in all pediatric patients similar to reports in adults.

Role of CX3C-chemokine CX3C-L/fractalkine expression in a model of slowly progressive renal failure

BACKGROUND The chemokine/chemokine receptor pair CX(3)C-L/CX(3)C-R is suspected to play a role in renal fibrogenesis. The aim of this study was to investigate their function in an animal model of slowly progressive chronic renal failure. METHODS Functional data were analysed in folic acid nephropathy (FAN) at different time points (up to day 142 after induction). Immunostaining for CX(3)C-L, CD3, S100A4, collagen type I, fibronectin, alpha-smooth muscle actin, Tamm-horsfall protein, aquaporin 1 and 2 as well as quantitative real-time PCR (qRT-PCR) for CX(3)C-L, CX(3)C-R and fibroblast-specific protein 1 (FSP-1) were performed. Additionally, regulatory mechanisms and functional activity of CX(3)C-L in murine proximal and distal tubular epithelial cells as well as in fibroblasts were investigated. RESULTS CX(3)C-L/GAPDH ratio was upregulated in FAN 3.4-fold at day 7 further increasing up to 7.1-fold at day 106. The expression of mRNA CX(3)C-L correlated well with CX(3)C-R (R(2) = 0.96), the number of infiltrating CD3+ cells (R(2) = 0.60) and the degree of tubulointerstitial fibrosis (R(2) = 0.56) and moderately with FSP-1 (R(2) = 0.33). Interleukin-1beta, tumour necrosis factor-alpha, transforming growth factor-beta as well as the reactive oxygen species (ROS) H(2)O(2) were identified by qRT-PCR as inductors of CX(3)C-L/fractalkine (FKN) in tubular epithelial cells. Functionally, CX(3)C-L/FKN chemoattracts peripheral blood mononuclear cells, activates several aspects of fibrogenesis and induces the mitogen-activated protein kinases in renal fibroblasts. CONCLUSIONS In FAN, there is a good correlation between the expression of CX(3)C-L with markers of interstitial inflammation and fibrosis which may result from upregulation by pro-inflammatory and pro-fibrotic cytokines as well as by ROS in tubular epithelial cells. The FKN system may promote renal inflammation and renal fibrogenesis.

Plasma exchange therapy for steroid-refractory superimposed relapses in secondary progressive multiple sclerosis

Sirs: High dose corticosteroids are the mainstay of relapse treatment in multiple sclerosis (MS). Yet, this regimen does not lead to an improvement of symptoms in all cases. If repeated application of high dose corticosteroids does not result in a significant recovery, plasma exchange therapy (PE) may be considered on an empirical basis. As shown in a placebo controlled cross-over study, PE is efficient for steroid refractory relapses in about 40–50% of cases classified as acute central nervous system inflammatory demyelinating disease [1]. Subsequent open case series summarizing patients with severe optic neuritis [2] but also motor impairment or severe ataxia revealed a significant improvement of symptoms in about 70% of cases [3]. This is also relevant for patients with a clinically isolated syndrome. Recently, the positive response to PE could be correlated with humoral immune mechanisms in MS lesions, i.e. antibody mediated damage and complement deposition (subtype II according to the histopathological classification by Lassmann, Bruck and Lucchinetti [4]). This correlation gave rise to the notion that efficacy of PE may result from the elimination of humoral factors which at first lead only to conduction block rather than irreversible tissue damage. Further evidence for this concept stems from the observation that a good response to PE is only observed in a period of less than six weeks after the onset of symptoms [5]. So far, efficacy of PE was only shown for patients with relapsing– remitting MS. Here we report on two cases with beneficial effects also for treatment of superimposed relapses in secondary progressive MS. The first patient is a 52-year-old female suffering from secondary chronic progressive MS with superimposed relapses since 40 months. Diagnosis was established with MS-typical MRI and typical CSF. Despite initial treatment with glatiramer acetate, she developed gradually progressing left leg palsy. EDSS slowly increased from 3.5 (1 year after the initial diagnosis) to EDSS 6.0 (15 months later), when she was still able to walk 50 m without aid. Over night, she then suffered a sudden worsening of the preexisting tetraparesis. Thus, the patient needed bilateral aid for further ambulation with a walking distance of a few meters (EDSS 7.0). Infection was ruled out by laboratory tests. Despite treatment with two intravenous methylprednisolone (MP) pulses of first 3 · 1000 mg and then 5 · 1000 mg the patient still required bilateral assistance for a walking distance of 150 m. 5 weeks after the onset of the new symptoms the patient underwent PE (50 ml/ kg body weight, BW, replacement fluid albumin). After three cycles, a significant improvement of symptoms, including the weakness of the right leg was noted. After completion of five PE cycles, the patient was again ambulatory with only unilateral aid for nearly 40 m. Latencies of motor evoked potentials to the upper limbs improved by 3 ms. She was further treated with mitoxantrone (see Table). The second patient is a 49-yearold female with a long standing history of MS for 30 years and a secondary progressive course since 10 years with superimposed relapses under treatment with interferon beta, mitoxantrone and at last 3 months MP pulses. At baseline, she was ambulatory for 30 m with bilateral aid (EDSS 6.5). Before admission, she suffered another relapse with an increasing paraparesis unfolding over 4 days thus requiring a wheelchair for distances longer than 5 m (EDSS 7.0). Subsequent application of two MP pulses with 5 · 1000 mg and then 4 · 1000 mg did not lead to any improvement of symptoms. Before PE, a urinary tract infection R. A. Linker, MD Æ A. Chan, MD R. Gold, MD Dept. of Experimental and Clinical Neuroimmunology Institute for MS research Medical Faculty University of Göttingen and Gemeinnützige Hertie-Stiftung 37073 Göttingen, Germany

Antifibrotic, nephroprotective effects of paricalcitol versus calcitriol on top of ACE-inhibitor therapy in the COL4A3 knockout mouse model for progressive renal fibrosis

BACKGROUND The COL4A3-/- mouse serves as animal model for progressive renal fibrosis. Using this animal model, the present study investigates the nephroprotective effects of Paricalcitol versus Calcitriol alone and on top of ACE-inhibitor therapy. METHODS Eighty six mice were divided into six groups: (PC) with Paricalcitol 0.1 mcg/kg, (CA) Calcitriol 0.03 mcg/kg (dose equipotent), (PLAC) vehicle 0.1 mL i.p. five times per week, (ACE + PC) Paricalcitol plus Ramipril, (ACE + CA) Calcitriol plus Ramipril and (ACE + PLAC) vehicle plus Ramipril 10 mg/kg/day p.o. ACE therapy started pre-emptively in Week 4, PC/CA therapy was initiated in 6-week-old animals with ongoing renal fibrosis and lasted for 8 weeks. Four to six animals were sacrificed after 9.5 weeks and kidneys were further investigated using histological, immunohistological and Western-blot techniques. Survival until end-stage renal failure was determined in the remaining animals. RESULTS PC, but not CA, prolonged lifespan until renal failure by 13% compared with untreated controls (P = 0.069). ACE-inhibition prolonged lifespan by >50%. Added on top of ACE inhibition, ACE + PC (but not ACE + CA) even further prolonged lifespan by additional 18.0% (P < 0.01 versus ACE + PLAC) and improved renal function (blood urea nitrogen; P < 0.05 versus ACE + CA). Accumulation of extracellular matrix and renal scarring was decreased in PC and ACE + PC-treated mice. CONCLUSIONS The present study demonstrated a substantial nephroprotective and antifibrotic effect of the vitamin D-receptor activator Paricalcitol on top of early ACE inhibition in the COL4A3-/- model of progressive kidney fibrosis. The synergistic effect of Paricalcitol on top of RAAS-blockade might as well be valuable in other chronic kidney diseases.