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Forschungsbericht 2010

 

Forschungsprofil

Die Forschungsaktivitäten der Abteilung für Nephrologie umfassen die klinischen Gebiete Nierentransplantation, Mechanismen der Proteinurie, diabetische Nephropathie, Hypertonie und Vaskulitis. Besondere Schwerpunkte sind Gefäßveränderungen insbesondere die Endothelzellfunktion und die Differenzierung glatter Gefäßmuskelzellen klinischen und klinisch-experimentellen Forschung sowie Forschung auf dem Gebiet der Fibrose.

 

 

Mechanismen der chronischen Organschädigung

Die Forschungsprojekte in den jeweiligen Bereichen sind in klinische Forschung, klinisch-experimentelle Forschung und experimentelle Forschung gegliedert.

Die klinische Forschung beschäftigt sich in erster Linie mit pathophysiologischen Untersuchungen an Patienten und Probanden sowie der Durchführung interventioneller Therapiestudien. Es kommen dabei neue avancierte Methoden der klinischen Forschung wie quantitative RT-PCR, FACS-Analytik und „Proteomics" zum Einsatz. Die Untersuchungen konzentrieren sich auf Störungen der Endothelzellfunktion bei den verschiedenen Erkrankungen. Ein weiterer Schwerpunkt der Abteilung ist die farbkodierte Duplexsonographie zur Analyse renaler Gefäßveränderungen.

Die Abteilung für Nephrologie hat zusammen mit dem Institut für Pathologie (Prof. Kreipe) erfolgreich ein Protokollbiopsieprogramm nach Nierentransplantationen eingerichtet. Dort werden in mehrmonatigen Abständen nach Transplantation regelmäßige Nierenbiopsien durchgeführt, um die Ursachen der chronischen Transplantatnephropathie zu untersuchen.

Die klinisch-experimentelle Forschung beschäftigt sich im Wesentlichen mit Tiermodellen menschlicher Erkrankungen. Es werden Untersuchungen an transgenen Ratten und Mäusen vorgenommen. Vor allem die physiologische Analyse von genveränderten Mäusen ist ein Schwerpunkt der Abteilung. Hier sind die Nierentransplantation in der Maus und die die diabetische Nephropathie hervorzuheben. Ein wichtiges Forschungsprojekt sind außerdem die Mechanismen der Nierenregeneration. Diese Untersuchungen werden an Haifischen und Zebrafischen in unserem Labor in Bar Harbor, Maine, USA durchgeführt.

In den experimentellen Forschungsprojekten werden zelluläre und molekulare Untersuchungen durchgeführt. Hier sind die Schwerpunkte der Abteilung die intrazelluläre Signaltransduktion, Analysen der Zellmigration und Zell-Zell-Interaktionen des Endothels. Viele dieser Untersuchungen werden mit Hilfe der konfokalen Lasermikroskopie und GFP-Fusionsproteinen vorgenommen. Ein weiterer Schwerpunkt der zellulären Forschung der Abteilung für Nephrologie ist die Regulation und Wirkung von Proteasen. Hier wird insbesondere das Urokinase-abhängige Plasminogensystem (uPA) untersucht.

 

 

Forschungsprojekte

The podocyte as a direct target of immunosuppressive agents

 

Nephrotic syndrome is common in adults and is one of the most common kidney diseases in children [1]. The majority of non-genetic nephrotic syndromes are caused by membranous nephropathy or focal segmental glomerulosclerosis (FSGS) in adults and minimal change disease (MCD) in children [1,2], In all these diseases the podocyte, which is considered to be terminally differentiated, is the primary target of injury [3]. Recently, a possible autoantigen of idiopathic membranous nephropathy (MN) was identified and the presence of autoantibodies was documented in 70% of patients with idiopathic MN [4]. However, the different pathophysiologies of idiopathic FSGS and MCD are still ongoing subjects of debate and not fully understood. Podocyte foot process effacement and disruption of the glomerular slit diaphragm is a common phenotype observed in almost all glomerular diseases associated with nephrotic range proteinuria. The common concepts of podocyte foot process effacement involve dedifferentiation, direct injury of the slit diaphragm or the actin cytoskeleton as well as changes in the glomerular basement membrane and podocyte interaction [5]. The resulting loss of glomerular barrier function leads to proteinuria. However, sclerosis and adhesion of the glomerular tuft to the Bowman’s capsule are restricted to FSGS and absent in MCD.

Persistent proteinuria is a prognostic marker for the progression to end stage renal disease [6]. Patients presenting with long-term nephrotic-range proteinuria and without partial or complete remission progress to end stage renal disease over the course of 3-6 years [7]. Acquired podocytopathies like idiopathic FSGS and MCD are historically considered as immunological diseases [8]. Therefore, immunosuppressive agents such as steroids and calcineurin inhibitors are the commonly used treatment strategies. More than 50% of nephrotic adults and about 80% of children respond to an induction therapy with glucocorticoids within a range of a few days to several months and maintenance treatment with glucocorticoids will prevent relapses [9, 10]. The response to corticosteroids is still the best prognostic factor for maintaining renal function in idiopathic nephrotic syndrome, irrespective of the histopathology. In steroid resistant nephrotic syndromes several other immunosuppressive agents were successfully used as rescue therapy.

After kidney transplantation proteinuria is highly prevalent and associated with decreased patient and allograft survival irrespectively of the underlying primary renal disease. Depending on the definition up to 45% of patients develop pathological proteinuria mostly due to recurrent glomerulonephritis, chronic allograft nephropathy, de novo transplant glomerulopathy or acute rejection [11]. Moreover, there is an ongoing debate about several immunosuppressive agents causing allograft proteinuria as it was shown for rapamycin and cyclosporine A [12, 13].

 

Glucocorticoids

Glucocorticoids bind to the glucocorticoid receptor in the cytoplasm, form dimers, and translocate to the nucleus where they bind to glucocorticoid response elements on the DNA or interact with other transcription factors [14]. Glucocorticoid receptors have been described to be expressed in human podocytes and translocate to the nucleus upon treatment with dexamethasone [15]. Therefore, a direct effect of glucocorticoids on podocytes in the course of nephrotic syndrome seems likely. The proteom of differentiated, cultured podocytes is particularly rich in actin cytoskel-etal proteins, annexins, and stress-associated proteins such as heat shock proteins and antioxidant enzymes. Ransom et al. demonstrated that dexamethasone treatment of podocytes leads to increased expression of ciliary neurotrophic factor (CNTF), an interleukin-6 (IL-6) type cytokine [16], increased expression of alpha B-crystallin, and the heat shock protein 27 (hsp27) [17]. Both are molecular chaperones inducing thermotolerance [18, 19]. Furthermore Smoyer and colleagues observed the importance of hsp27 for the regulation of the morphological and actin cytoskeletal response of podocytes by regulating actin polymerization in a podocyte injury model, using puromycin aminonucleosid (PAN) [20]. This goes along with findings that dexamethasone enhances the stability of actin filaments against disruption by cytochalasin D, latrunculin A, or PAN by increasing the total amount of cellular polymerized actin and an increased activity of the actin-regulating GTPase RhoA. It was also previously demonstrated that these effects are specific to glucocorticoids compare d to other classes of steroid hormones [21]. However, treatment with spironolacton, an aldosteron antagonis t, reduced albuminuria, renal tissue renin-angiotensin activity, and increased AKT phosphorylation, thereby improving podocyte structural integrity in the transgenic Ren2 rat model with increased tissue renin-angiotensin activity [22]. Wada at al. demonstrated rescued podocyte viability in a PAN cell culture model upon treatment with dexamethasone by blocking p53 expression, lowering the proapoptotic Bax expression and increasing the expression of the antiapoptotic Bcl-xL [23, 24]. Bax belongs to the Bcl2 family and is known to mediate podocyte apoptosis induced by TGF-beta [25]. Interestingly, dexamethasone failed to prevent podocyte apoptosis induced by UV light or TGF-beta, which is primarily caused by caspase-3 activation [24]. Moreover, Wada and colleagues have shown that dexamethasone prevents the reduced ERK phosphorylation in PAN treated podocytes. Interestingly, when ERK was directly inhibited in this cell model dexamethasone exerted a pro-apoptotic effect which was associated with translocation of AIF [24], indicating that the ERK pathway itself has important impact in podocyte survival.

In normal glomeruli vascular endothelial growth factor (VEGF) is exclusively expressed by podocytes and is up-regulated in minimal change nephropathy [26, 27]. VEGF plays an important role in vasculogenesis and angiogenesis and induces vascular leakage and vasodilation. Treatment with dexamethasone led to a down-regulated VEGF expression in an immortalized human podocyte cell line [28]. However, these changes in VEGF-expression affect different VEGF-isoforms and how this contributes to a clinical remission of disease remains controversial, since we recently could demonstrate that expression of VEGF-A and VEGF-C is important for podocyte survival [29] and VEGF-ablation therapy in patients leads to proteinuria and podocyte loss [30]. The ability of cytokine production links the podocyte to the immune system. Next to VEGF and TGF-beta podocytes produce the interleukins IL-6 and IL-8 [28, 29]. Similarly they express a variety of functional CC and CXC receptors [31].

 

Calcineurin Inhibitors

Calcineurin is a serine/threonine phosphatase that is ubiquitiously expressed in all mammalian tissues and tightly regulated by Ca2+/calmodulin [30]. Calcineurin dephosphorylates the nuclear factor of activated T cells (NFAT) family members, leading to nuclear translocation and activation of early genes of the T cell driven immune response, e.g., cytokines as IL-2 and IL-4. The immunosuppressive action of calcineurin inhibitors such as cyclosporin A (CsA) or tacrolimus (FK506) is due to the inhibition of the NFAT signaling in T cells by binding to the cytosolic cyclophilins or FK- binding proteins and subsequently inhibiting the phosphatase activity of calcineurin. Recent evidence supports that the podocyte itself is a target of CsA. Faul et al. analyzed the consequence of CsA treatment on the actin cytoskeleton of podocytes [31, 32]. Treatment of podocytes with CsA leads to a stabilization of the actin cytoskeleton and stress fibers, while calcineurin mediates dephosphorylation of synaptopodin, an actin-organizing protein in podocytes. By blocking calcineurin the phosphorylation of synaptopodin promotes binding to the chaperone-like protein 14-3-3. Subsequently, synaptopodin is protected against cathepsin L mediated cleavage and degradation. Thereby, CsA has a stabilizing effect on the actin cytoskeleton. Moreover, calcineurin is tightly regulated by intracellular calcium levels. The podocyte cell membrane associated transient potential cation channel 6 (TRPC 6) mediates calcium influx and gain of function mutations are known to be causal for genetic forms of FSGS [33]. High levels of intracellular calcium would lead to an activation of calcineurin and thereby loss of synaptopodin and stress fibers and to an activation of the NFAT signalling pathway as a further potential mediator of FSGS [34]. Both pathways can be inhibited by treatment with CsA and FK506 [33, 35, 36]. Furthermore, CsA and steroids were reported to effectively treat early onset nephrotic syndrome due to a mutation in the gene coding for phospholipase C epsilon [37]. Phospholipase C is an important intracellular mediator of TRPC 6 activity [33] and mutations are known to interfere with glomerular development and probably with glomerular repair processes as well [37].

The anti-inflammatory and immunosuppressive action of glucocorticoids and calcineurin inhibitors may only play a minor role in modulation of podocyte biology and promotion of glomerular repair mechanisms. Instead, these drugs have direct effects on podocytes through regulation of some cytokines and several signaling pathways relevant for cytoskeletal stability, cell maturation and survival. Furthermore, the expression and distribution of key components of the slit diaphragm and the cytoskeleton are regulated. However, data on direct effects of immunosuppressive agents on proteinuria induced by podocyte dysfunction remain controversial and more research is necessary to differentiate the multifactorial effects especially regarding time and dose of treatment and the effects according to the type of glomerular pathology.

References

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2. Haas M, Spargo BH, Coventry S. Increasing incidence of focal-segmental glomerulosclerosis among adult nephropathies: a 20-year renal biopsy study. Am J Kidney Dis 1995; 26: 740-750

3. Marshall CB, Shankland SJ. Cell cycle regulatory proteins in podocyte health and disease. Nephron Exp Nephrol 2007; 106: e51-59

4. Beck LH, Jr., Bonegio RG, Lambeau G et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 2009; 361: 11-21

5. Kwoh C, Shannon MB, Miner JH, Shaw A. Pathogenesis of nonimmuneglomerulopathies. Annu Rev Pathol 2006; 1: 349-374

6. Korbet SM, Schwartz MM, Lewis EJ. Primary focal segmental glomerulosclerosis: clinical course and response to therapy. Am J Kidney Dis 1994; 23: 773-783

7. Korbet SM. Treatment of primary focal segmental glomerulosclerosis. Kidney Int2002; 62: 2301-2310

8. Shalhoub RJ. Pathogenesis of lipoid nephrosis: a disorder of T-cell function. Lancet 1974; 2: 556-560

9. Ponticelli C, Villa M, Banfi G et al. Can prolonged treatment improve the prognosis in adults with focal segmental glomerulosclerosis? Am J Kidney Dis 1999; 34: 618-625

10. Wong W. Idiopathic nephrotic syndrome in New Zealand children, demographic, clinical features, initial management and outcome after twelve-month follow-up:results of a three-year national surveillance study. J Paediatr Child Health 2007; 43:337-341

11. Knoll GA. Proteinuria in kidney transplant recipients: prevalence, prognosis, and evidence-based management. Am J Kidney Dis 2009; 54: 1131-1144

12. Morozumi K, Takeda A, Uchida K, Mihatsch MJ. Cyclosporine nephrotoxicity: How does it affect renal allograft function and transplant morphology. Transplant Proc 2004; 26 (Suppl 2S): 251S-256S

13. Sahin GM, Sahin S, Kantarci G, Ergin H. Proteinuria after conversion to sirolimus in renal transplant recipients. Transplant Proc 2006; 38: 3473-3475

14. Muller M, Renkawitz R. The glucocorticoid receptor. Biochim Biophys Acta 1991; 1088: 171-182

15. Yan K, Kudo A, Hirano H et al. Subcellular localization of glucocorticoid receptor protein in the human kidney glomerulus. Kidney Int 1999; 56: 65-73

16. Yang CW, Lim SW, Han KW et al. Upregulation of ciliary neurotrophic factor (CNTF) and CNTF receptor alpha in rat kidney with ischemia-reperfusion injury. J Am Soc Nephrol 2001; 12: 749-757

17. Ransom RF, Vega-Warner V, Smoyer WE, Klein J. Differential proteomic analysis of proteins induced by glucocorticoids in cultured murine podocytes. Kidney Int 2005; 67: 1275-1285

18. Miron T, Vancompernolle K, Vandekerckhove J, Wilchek M, Geiger B. A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein. J Cell Biol 1991; 114: 255-261

19. Horwitz J. Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A 1992; 89: 10449-10453

20. Smoyer WE, Ransom RF. Hsp27 regulates podocyte cytoskeletal changes in an in vitro model of podocyte process retraction. Faseb J 2002; 16: 315-326

21. Ransom RF, Lam NG, Hallett MA, Atkinson SJ, Smoyer WE. Glucocorticoids protect and enhance recovery of cultured murine podocytes via actin filament stabilization. Kidney Int 2005; 68: 2473-2483

23. Whaley-Connell A, Habibi J, Wei Y et al. Mineralocorticoid receptor antagonism attenuates glomerular filtration barrier remodeling in the transgenic Ren2 rat. Am J Physiol Renal Physiol 2009; 296: F1013-1022

24. Wada T, Pippin JW, Marshall CB, Griffin SV, Shankland SJ. Dexamethasone prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and Bcl-2- related family proteins. J Am Soc Nephrol 2005; 16: 2615-2625

25. Wada T, Pippin JW, Nangaku M, Shankland SJ. Dexamethasone's prosurvival benefits in podocytes require extracellular signal-regulated kinase phosphorylation. Nephron Exp Nephrol 2008; 109: e8-19

26. Schiffer M, Bitzer M, Roberts IS et al. Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest 2001; 108: 807-816

27. Bailey E, Bottomley MJ, Westwell S et al. Vascular endothelial growth factor mRNA expression in minimal change, membranous, and diabetic nephropathy demonstrated by non-isotopic in situ hybridisation. J Clin Pathol 1999; 52: 735-738

28. Simon M, Grone HJ, Johren O et al. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney. Am J Physiol 1995; 268: F240-250

29. Xing CY, Saleem MA, Coward RJ, Ni L, Witherden IR, Mathieson PW. Direct effects of dexamethasone on human podocytes. Kidney Int 2006; 70: 1038-1045

30. Müller-Deile J, Worthmann K, Saleem M, Tossidou I, Haller H, Schiffer M. The balance of autocrine VEGF-A and VEGF-C determines podocyte survival. Am J Physiol Renal Physiol 2009; 297: F1656-1667

31. Aramburu J, Heitman J, Crabtree GR. Calcineurin: a central controller of signalling in eukaryotes. EMBO Rep 2004; 5: 343-348

32. Faul C, Donnelly M, Merscher-Gomez S et al. The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A. Nat Med 2008; 14: 931-938

33. Mundel P, Reiser J. Proteinuria: an enzymatic disease of the podocyte? Kidney Int 2010; 77: 571-580

34. Mukerji N, Damodaran TV, Winn MP. TRPC6 and FSGS: The latest TRP channelopathy. Biochimica et Biophysica Acta 2007; 1772 859-868

35. Schlöndorff J, Del Camino D, Carrasquillo R, Lacey V, Pollak MR. TRPC6 mutationsvassociated with focal segmental glomerulosclerosis cause constitutive activation of NFAT-dependent transcription. Am J Physiol Cell Physiol 2009; 296: C558-569

36. Kuwahara K, Wang Y, McAnally J et al. TRPC6 fulfi lls a calcineurin signaling circuitvduring pathologic cardiac remodeling. J Clin Invest 2006; 116: 3114-3126

37. Sinkins WG, Goel M, Estacion M, Schilling WP Association of immunophilins withvmammalian TRPC channels. J Biol Chem 2004; 279: 34521-34529

38. Hinkes B, Wiggins RC, Gbadegesin R et al. Positional cloning uncovers mutations inPLCE1 r sponsible for a nephrotic syndrome variant that may be reversible.

 

Projektleitung: Schiffer, Mario Prof. (Dr. med.); Förderung: IFB-Tx

 

Weitere Forschungsprojekte der Abteilung:

 

Klinische Forschung: Wirkung von EPO auf chronische Transplantatschädigung

Projektleitung: Kielstein, Jan (PD Dr. med.); Förderung: BMBF/IFB-Tx

 

 

Klinisch-experimentelle Forschung: Rolle von Zelladhäsion und Inflammation bei der Reperfusion

Projektleitung: Güler, Faikah (Prof. Dr. med.), Rong, Song (Dr. med.)

 

Klinische Forschung: Protokollbiopsien in der Nierentransplantation

Projektleitung: Einecke, Gunilla (Dr. med.), Schwarz, Anke (Prof. Dr. med.); Förderung: Roche, Novartis, DFG

 

Klinische Forschung: Proteinurie nach Nierentransplantation

Projektleitung: Schiffer, Mario (Prof. Dr. med.); Förderung: BMBF/IFB-Tx

 

 

Klinische Forschung: Mechanismen der Fibrosierung/Kalzifizierung

Projektleitung: Gwinner, Wilfried (Prof. Dr. med.); Förderung: BMBF/IFB-Tx

 

 

Experimentelle Forschung: Mechanismen der Signaltransduktion des uPA-Rezeptors

Projektleitung: Dumler, Inna (Prof. Dr. rer.nat); Förderung: DFG, German-Israeli-Foundation (GIF)

 

 

Klinische Forschung: AT-Rezeptorblockade und Proteinurie

Projektleitung: Menne, Jan (PD Dr. med.), Haller, Hermann (Prof. Dr. med.); Förderung: Amgen, Novartis, Daiichi-Sankyo

 

 

Klinisch-experimentelle Forschung: PKC und Proteinurie

Projektleitung: Menne, Jan (PD Dr. med.), Haller, Hermann (Prof. Dr. med.); Förderung: BMB

 

 

Experimentelle Forschung: Mechanismen der peritonealen Fibrose

Projektleitung: Haller, Hermann (Prof. Dr. med.), Hiss, Marcus (Dr. med.); Förderung: DFG

 

 

Klinische Forschung: Neue Therapiestrategien bei therapie-resistentem Bluthochdruck

Projektleitung: Menne, Jan (PD Dr. med.), Schmidt, Bernhard (PD Dr. med.)

 

 

Klinische Forschung: Inflammatorische Marker und Bluthochdruck

Projektleitung: Menne, Jan (PD Dr. med.; Förderung: Daiichi Sankyo Japan

 

 

Klinische Forschung: Inflammatorische Marker und Hämodialyse

Projektleitung: Kielstein, Jan PD Dr. med.); Förderung: Fresenius-Stiftung

 

 

Klinische Forschung: Plasmaaustauschverfahren

Projektleitung: Kielstein, Jan (PD Dr. med.)

 

 

Klinisch-experimentelle Forschung: Podozyten und Proteinurie

Projektleitung: Schiffer, Mario (Prof. Dr. med.); Förderung: DFG

 

 

Klinisch-experimentelle Forschung: Endothel und Proteinurie

Projektleitung: Haller, Hermann (Prof. Dr. med.), Ziegler, Wolfgang (Dr. rer. nat), Kirsch, Thorsten (Dr. rer.nat.), Park, Joon-Keun (Dr. rer. nat.), Schuschakowa, Nelly (Dr. rer. nat.); Förderung: DFG