Freitag, 02.10.2020

14:00 - 15:30

Poster DGfN 2020

Nierenphysiologie (P098 - P102; LA21 - LA23)

Inter-talk between the prostaglandin and purinergic signalling system – direct effect of prostaglandin E2-1-glyceryl ester on human P2Y6 receptor?

J. Wehmöller, M. G. Christensen, H. A. Praetorius; Kiel, Aarhus C/DK, Aarhus/DK

Objective: Prostanoid and purinergic signalling are essential for the proinflammatory component of acute and chronic kidney disease. Nucleotides are released early in response to cell perturbation, whereas the prostaglandin system is activated downstream (1). Interestingly, recent data suggest a direct effect of the COX-2 product prostaglandin E2-1-glyceryl ester (PGE2-G) on the UDP-sensitive P2Y6 receptor (2) expressed in the proximal tubule (3). Thus, we hypothesized that PGE2-G amplifies renal inflammation directly via P2Y6 receptor activation.
Method: To verify the PGE2-G mediated P2Y6 activation, we used 132-1N1 astrocytoma cells devoid of P2 receptors for specific expression of the P2Y6 receptor. We monitored [Ca2+]i signalling by both life-cell microscopy (LCM) and plate reader (PR) by detection of Fluo-4 fluorescence (F) in human P2Y6 expressing 132-1N1 cells (hP2Y6 cells). Data are given as mean F/F0 ± S.E.M. Wild type 132-1N1 cells (WT cells) were used as negative control.
Results: The hP2Y6 cells were activated by UDP in a concentration-dependent manner with an EC50-value of 1.89*10-8 M (95% confidence interval 0.00 to 4.13*10-8 M, PR) to a maximal increase of 1.70 ±0.15 (PR) and 1.81 ±0.23 (LCM). WT cells did not react to UDP (1.07 ±0.01, PR, 1.05 ±0.02, LCM) but to carbachol (3.18 ±0.16, PR, 1.67 ±0.13, LCM). Surprisingly, stimulation of hP2Y6 cells with PGE2-G from several batches in concentrations from 10-11 to 10-6 M did not inflict any change in [Ca2+]i (1.12 ±0.02, PGE2-G 10-6 M, PR). We observed a PGE2-G-induced [Ca2+]i increase compared to vehicle control at concentrations of 10-4 M dissolved in DMSO (4.95 ±0.72, PR). However, we detected a completely similar response in WT cells (4.24 ±1.11, PR). PGE2-G in our preparations was confirmed by HPLC.
Conclusion: Our data cannot confirm a direct effect of PGE2-G on P2Y6 receptors and thus, does not support that this type of signalling is relevant during renal inflammation.
1. Leipziger J, Praetorius H. Renal autocrine and paracrine signaling: A story of self-protection. Physiol Rev. 2020 Jul 1;100(3):1229–89.
2. Brüser A, Zimmermann A, Crews BC, Sliwoski G, Meiler J, König GM, et al. Prostaglandin E2 glyceryl ester is an endogenous agonist of the nucleotide receptor P2Y6. Sci Rep. 2017 May 24;7(1):2380.
3. Bailey MA, Imbert-Teboul M, Turner C, Srai SK, Burnstock G, Unwin RJ. Evidence for basolateral P2Y(6) receptors along the rat proximal tubule: Functional and molecular characterization. J Am Soc Nephrol. 2001 Aug 1;12(8):1640–7.

Angiotensin II receptor blockade stimulates p38 MAPK/NF-kB/COX-2 signaling in kidney cortex to alleviate calcineurin inhibitor nephrotoxicity

J. Hu, Y. Xu, S. Bachmann, K. Mutig; Berlin

Objective: Calcineurin inhibitors (CNI) such as cyclosporine A (CsA) are routinely used in patients undergoing organ transplantation to achieve optimal immunosuppression. Nephrotoxic side effects of CNI include stimulation of the renin-angiotensin system (RAS), suppression of renal cortical cyclooxygenase 2 (COX-2), and pathophysiological alterations of glomerular filtration rate and sodium balance. Regulation of COX-2 depends on local, as well as systemic effects of CNI. We hypothesized that COX-2 suppression is related to RAS activity and studied possible signaling pathways in CNI-treated animal and cell models.
Method: Wistar rats receiced CNI (CsA; 25 mg/kg*d), RAS antagonist (candesartan; 5mg/kg*d), or COX-2 inhibitor (celecoxib; 50 mg/kg*d) alone or in combinations at short (3 days) or long term (3 weeks) to study COX-2 and RAS parameters as well as renal function, metabolism, and cell biology. Cultured macula densa (MD) cells were treated with CsA, angiotensin II (Ang II), p38 MAPK inhibitor, and NF-kB inhibitor in various combinations.
Results: Calcineurin inhibition using CsA or siRNA significantly upregulated COX-2 activity in cultured MD cells via activation of p38 MAPK and NF-kB. Concomitant application of Ang II abrogated these effects, demonstrating a dominant role for the RAS. In rats, both 3 days and 3 weeks CsA treatments stimulated renin biosynthesis, reduced cortical COX-2 expression, decreased creatinine clearance, and induced sodium retention due to activation of major distal salt transporters, NKCC2 and NCC. Pathophysiology was partially (3 days) or completely (3 weeks) restored by simultaneous administration of candesartan. Celecoxib largely recapitulated effects of CsA and substantially reduced the beneficial effects of RAS antagonism thus unmasking the COX-2-dependent nephrotoxic CsA effects.
Conclusion: In sum, we have identified calcineurin as an endogenous COX-2 inhibitor, acting via suppression of p38 MAPK and NF-kB activity in MD cells. CNI-induced RAS activation critically reduces COX-2 activity in the cortex likely by overriding local, stimulatory effects of calcineurin inhibition. Our data support the use of RAS antagonism to alleviate the CNI nephrotoxicity.

Die Ödementstehung beim nephrotischen Syndrom der Maus ist unabhängig von der Enzym- und Gerüstfunktion der membranständigen Serinprotease Prostasin.

D. Essigke, B. Bohnert, M. Xiao, R. Szabo, T. H. Bugge, F. Artunc; Tübingen, Bethesda/USA

Hintergrund: Die membranständige Serinprotease Prostasin (Prss8) wird im distalen Tubulus exprimiert und reguliert den epithelialen Natriumkanal (ENaC) sowohl durch seine Enzym- als auch durch seine Gerüstfunktion. Beim nephrotischen Syndrom (NS) kommt es zu einer proteolytischen Aktivierung des ENaC, die wiederum zu einer Natriumretention und Ödementstehung führt. Die Bedeutung von Prostasin bei der proteolytischen Aktivierung des ENaC im NS in vivo ist bisher nicht bekannt.
Methode: Zur Untersuchung dieser Fragestellung wurden zwei verschiedene knock in-Mauslinien verwendet, die entweder eine fehlende Proteaseaktivität (Prss8-S238A) oder eine fehlende Aktivierbarkeit (Prss8-R44Q) aufwiesen. Nach Induktion mittels einmaliger i.v.-Injektion von Doxorubicin (14,5 μg/g KG) wurde der Verlauf des NS über 10 Tage untersucht. Die Bestimmung der ENaC Aktivität erfolgte bei gesunden und nephrotischen Mäusen durch Gabe von Vehikel (5µl/g KG) bzw. Triamteren (10µg/g KG) i.p. über die 6h-Natriumausscheidung.
Ergebnisse: Nach Doxorubicin-Injektion entwickelten die Mäuse ein vergleichbares NS mit einer Proteinurie von 155±8 mg/mg Kreatinin (Prss8-wt, n=9), 154±9 mg/mg Krea (Prss8-S238A, n=10) und 166±10 mg/mg Krea (Prss8-R44Q, n=9). Das Ansprechen auf Triamteren stieg bei allen Genotypen im Vergleich zum uninduzierten Zustand als Korrelat einer ENAC-Aktivierung signifikant an. Im Folgenden kam es bei allen Genotypen zu einer Natriumretention mit Abfall der Urin-Natriumausscheidung von im Mittel 275±22 µmol/mg Krea (alle Genotypen) auf minimal 13±4 µmol/mg Krea bei Prss8-wt, 14±3 µmol/mg Krea bei Prss8-S238A und 14±4 µmol/mg Krea bei Prss8-R44Q (p=0,44). Dies führte zu einer vergleichbaren Gewichtszunahme als Ausdruck der Ödementstehung (+25±3% bei Prss8-wt, +20±2% bei Prss8-S238A und +28±3% bei Prss8-R44Q, p=0,16). Die im NS mittels ELISA bestimmte Prostasin-Ausscheidung im Urin unterschied sich ebenfalls nicht signifikant zwischen den Mauslinien.
Zusammenfassung: Weder die Enzym- noch die Gerüstfunktion von Prostasin haben einen signifikanten Einfluss auf Natriumretention und Ödementstehung im NS. Prostasin ist für die proteolytische ENaC-Aktivierung im NS nicht essentiell, was auf eine entscheidende Rolle von löslichen Serinproteasen hinweist.

Pharmacological activation of the soluble guanylyl cyclase affects renal afferent and efferent arterioles differentially

M. Z. Xu, I. Wennysia, L. Zhao, T. Schomber, D. Braun, S. Golz, H. Summer, A. Benardeau, E. Y. Lai, F. B. Lichtenberger, R. Schubert, P. B. Persson, A. Patzak; Berlin, Wuppertal, Hangzhou/CN, Augsburg

Objective: Renal arteriolar tone depends considerably on the dilatory action of nitric oxide (NO) via activation of the soluble guanylyl cyclase (sGC) and cGMP action. NO deficiency contributes to impaired renal perfusion and the development of acute kidney injury (AKI). Here, the hypotheses of a differential potency of sGC-activation and PDE inhibition, respectively, in renal afferent (AA) and efferent arterioles (EA) resulting in differential dilation of AA and EA upon pharmacological sGC-activation after hypoxia/re-oxygenation is tested.
Method: Experiments were performed in isolated, perfused mouse glomerular arterioles under isobaric conditions. Hypoxia (0.1% oxygen) was achieved by using a hypoxia chamber.
Results: Phosphodiesterase 5 (PDE5) and sGC subunits were considerably expressed on the mRNA level in AA. PDE5 inhibition with sildenafil diminished the responses to Ang II bolus application in AA, but not EA. Sildenafil induced a vasodilatation in Ang II-pre-constricted vessels, which was stronger in EA than AA. The sGC-activator BAY 58-2667 dilated EA stronger than AA after pre-constriction with Ang II. In L-NAME pre-treated and Ang II pre-constricted vessels, BAY 58-2667 dilated arterioles similar to controls without L-NAME treatment. Further, BAY 58-2667 induced dilatation in iodinated contrast medium treated AA and dilated EA, but not in AA, after hypoxia/re-oxygenation.
Conclusion: The results reveal an important role of the NO-sGC-cGMP system for renal arteriolar dilatation. The study suggests beneficial effects of sGC-activation in situations of renal vasoconstriction with NO-deficiency. The stronger effect of NO-independent sGC-activation in EA during NO-deficiency and after hypoxia/re-oxygenation indicates an influence of sGC activators on the glomerular filtration in this context.

Tolvaptan treatment abolishes the corticomedullary osmolality gradient in the kidney

F. Boivin, K. M. Schmidt-Ott; Berlin

Objective: The reabsorption of electrolyte-free water in the renal collecting duct is dependent on aquaporin-mediated transcellular water permeability and a high interstitial osmolality. Vasopressin, the key hormone responsive to water deprivation, activates renal Vasopressin 2 Receptors (V2R), which induces apical trafficking and membrane incorporation of Aquaporin 2. In addition, V2R has been reported to increase urea reabsorption in the inner medullary collecting duct and NaCl reabsorption in the thick ascending limb, raising the possibility that V2R contributes to the establishment of medullary hypertonicity required for water reabsorption. Here, we evaluated the involvement of V2R in establishing the corticomedullary osmolality gradient of the kidney.
Method: C57Bl/6N mice were fed a Tolvaptan-enriched diet (V2R antagonist, Tol) for 48 hours. Control (Ctrl) animals were fed the same diet without Tolvaptan. All groups were given ad libitum access to food and water. Water intake and urinary osmolality were measured. To evaluate the intrarenal osmolality gradient, we measured tissue osmolality in the different compartments of the kidney across the cortico-medullary axis.
Results: Daily water intake was significantly increased in Tolvaptan-treated mice compared to controls (Day 1; Ctrl: 11.96ml vs Tol: 38.03, p<0.05, Day 2; Ctrl: 13.22ml vs Tol: 35.27, p<0.05) and urinary osmolality was significantly decreased after 48 hours of Tolvaptan treatment (1755 mOsm/kgH2O Ctrl vs 790.6 mOsm/kgH2O Tol, p<0.001). Renal tissue osmolality was significantly decreased in the inner stripe of the outer medulla (401.8 mOsm/kgH2O Tol vs. 713.8 mOsm/kgH2O Ctrl, p<0.001) and in the inner medulla (602.2 mOsm/kgH2O Tol vs. 1360 mOsm/kgH2O Ctrl, p<0.001). However, no significant changes were observed in the renal cortex (378.5 mOsm/kgH2O Tol vs. 432.1 mOsm/kgH2O Ctrl).
Conclusion: Together, our data demonstrate that Tolvaptan reduces tissue osmolality across the cortico-medullary axis in the kidney, leading to a near-complete loss of the intrarenal osmolality gradient.  Tolvaptan treatment in mice therefore provides an experimental model to evaluate the cellular and pathophysiological effects of interstitial hypertonicity.

Die Herabregulation von Nephronektin durch TGFß in Podozyten wird sowohl über den kanonischen als auch über den nicht-kanonischen Weg vermittelt

N. Sopel, A. Ohs, M. Schiffer, J. Müller-Deile; Erlangen


Hintergrund: The extracellular matrix protein nephronectin (NPNT) is essential for kidney development and has recently been shown to be differentially expressed in various kidney diseases. In e. g. diabetic glomerulosclerosis NPNT has been observed to be upregulated, while it was down regulated in membranous glomerulonephropathy. A knockdown of npnt in zebrafish embryos caused proteinuria, podocyte foot process effacement and a thickening of the lamia rara interna of the glomerular basement membrane. Furthermore a down regulation of NPNT by transforming growth factor beta (TGFβ), as well as microRNA 378a (miR-378a) has been described.
To further elucidate the mechanisms by which TGFβ is regulating NPNT expression in podocytes, we are currently studying the blockade of the different parts of the canonical as well as the non-canonical TGFβ pathway.

Methode: In order to investigate the mechanisms by which TGFβ down regulates NPNT in podocytes we seeded immortalized human podocytes. After 10 to 12 days of differentiation, the cells were serum starved overnight, then pre-incubated for 30 minutes with inhibitors for different parts of the canonical and the non-canonical TGFβ signaling pathway before culture in complete media with or without TGFβ. Cells were either harvested for protein or RNA isolation and analyzed with Western Blot or qPCR or fixed for immunofluorescence.

Ergebnisse: Our results indicate that TGFβ-mediated down regulation of NPNT is dependent on both arms of the TGFβ pathway. Blockade of the canonical pathway leads to an increase in NPNT expression on protein level, indicating an important role in signal transduction. Targeting of single molecules of the non-canonical pathways leads to more ambiguous results, as inhibition of e. g. p38, AKT or ROCK results in either up or down regulation of NPNT protein expression.
Interestingly, addition of TGFβ after pre-incubation with the different inhibitors reduces NPNT expression regardless of the effect observed with the inhibitor alone.

Zusammenfassung: Inhibition of single signaling molecules in the canonical and non-canonical TGFβ pathway results in different outcomes regarding NPNT expression under physiological conditions. While blockade of the canonical pathway leads to increased NPNT levels, non-canonical pathway compounds might have opposing roles in NPNT regulation.
In summary, we propose an intricate regulation of NPNT by TGFβ via both the canonical and the non-canonical pathway.


High chloride and the aldosterone-mineralocorticoid-receptor-pathway

H. Pham, H. Vitzthum, N. Hauswald, S. Shibata, C. Meyer-Schwesinger, H. Ehmke; Hamburg, Tokyo/J

Objective: Aldosterone plays an integral role in maintaining electrolyte and volume homeostasis. During physiological conditions, aldosterone is secreted by the adrenal glands in response to decreased blood pressure or increased plasma potassium. As a steroid hormone, it binds to the intracellular mineralocorticoid receptor (MR). After activation, the MR is translocated to the nucleus and the transcription of specific genes is activated. However, we recently observed in vivo that despite high aldosterone levels no MR translocation to the nucleus was observable in renal principal cells (PCs) when extracellular chloride was increased. The cellular mechanisms behind this effect remain unclear. In this study, we question whether phosphorylation of MR (MR-S843P) is present in PCs. MR phosphorylation is known to prevent MR translocation in renal intercalated cells (ICs) (S. Shibata et al., Cell Metab 18, 660, 2013).
Method: Murine cortical connecting duct (mCCD) cells were used as a cellular model of principal cells (H. P. Gaeggeler et al., J. Am. Soc. Nephrol. 16, 878, 2005). Cells were stimulated with aldosterone in the presence of normal (112 mM) or high (127 mM) extracellular chloride ([Cl-]e). The intracellular localization of MR and MR-S843P was determined by immunohistochemistry. In addition, the protein abundance of ULK1 (unc-51-like kinase 1), the kinase responsible for phosphorylation of MR in ICs, and its phosphorylated, inactive form ULK1-S757P was analyzed by immunoblotting.
Results: Immunofluorescence revealed that MR is not translocated to the nucleus of mCCD cells when the extracellular [Cl-] is elevated despite aldosterone stimulation. Furthermore, cytoplasmic MR-S843P signal was enhanced in the cytoplasm of mCCD cells treated with high [Cl-]e. In line with these results, immunoblot studies showed a decreased ULK1-S757P/ULK1 protein ratio, indicating a higher ULK1 activity after treatment with high [Cl-]e.
Conclusion: These findings show that high extracellular chloride is sufficient to suppress the activation of the aldosterone-MR-pathway in vitro. Furthermore, the results suggest that high extracellular chloride might prevent MR translocation to the nucleus by enhanced phosphorylation of the MR in PCs.

Effect of an acute insulin administration on tubular transport in mice with and without insulin resistance

N. Emektar, B. Bohnert, F. Artunc; Tübingen

Objective: Obwohl die Niere nicht zu den klassischen Zielorganen von Insulin zählt, wird der Insulinrezeptor in fast allen Zellen einschließlich des Tubulussystems exprimiert. Die Beeinflussung des tubulären Transports durch eine akute Insulingabe ist beschrieben. Es ist jedoch nicht klar, inwieweit diese Prozesse durch eine Insulinresistenz beeinflusst werden.
Method: Zur Untersuchung dieser Fragestellung wurden adipöse Leptin-defiziente BTBR-Mäuse (Lepob/ob) im Alter von 35-50 Tagen mit schlanken Wildtyp-Kontrollen (Lepwt/wt) verglichen. Den Mäusen wurde Vehikel (NaCl 0,9%, 15µl/gKG) und im Abstand von zwei Tagen Insulin i.p. (0,1mU/gKG) verabreicht und der Urin jeweils über 2h gesammelt. Aus den Urinproben wurde die Ausscheidung von Na+,K+,Ca2+,Mg2+ und PO4³- gemessen und auf Urinvolumen (UV), Kreatinin (Krea) und Körpergewicht (KG) normiert.
Results: Das KG der Lepob/ob-Mäuse (n=14-20) war mit 49,9±1g im Alter von 56 Tagen höher als das der Wildtyp-Geschwister mit 31,3±1g (n=8-14,p<0,0001). Der Blutzucker (BZ) lag nach 6h Fasten bei den Lepob/ob-Mäusen bei 171±6mg/dl und bei den Lepwt/wt-Mäusen bei 173±4mg/dl (p=0,3). 30min nach Gabe von NaCl zeigte sich der BZ mit 174±4mg/dl (Lepob/ob) bzw.179±6mg/dl (Lepwt/wt) unverändert. 30min nach Insulingabe fiel der BZ bei den Lepwt/wt-Mäusen auf 90±7mg/dl ab, während der BZ der Lepob/ob-Mäuse aufgrund ihrer Insulinresistenz weniger stark auf 156±7mg/dl abfiel. Das ausgeschiedene UV bzw. die ausgeschiedene Krea-Menge waren zwischen den Genotypen und Behandlungen vergleichbar. Die Lepob/ob-Mäuse zeigten jedoch unter Vehikelgabe ein signifikant niedrigeres UV und schieden eine geringere Menge Krea aus. Nach Insulingabe fand sich keine Änderung der absoluten und Krea-normierten Urinausscheidung von Na+,K+,Ca2+,Mg2+ und PO43- bei Lepwt/wt-Mäusen im Vergleich zur Vehikelgabe. Bei den Lepob/ob-Mäusen fand sich hingegen nach Insulingabe eine erhöhte absolute Ausscheidung von Na+,K+,Ca2+ und Mg2+, die PO43--Ausscheidung veränderte sich nicht. Für Na+ und Ca2+ persistierte diese Änderung nach Normierung auf die ausgeschiedene Krea-Menge.
Conclusion: In diesem Modell findet sich keine Beeinflussung des tubulären Transports durch eine akute Insulingabe bei den insulinsensitiven Lepwt/wt-Mäusen. Hingegen wurden die Urin-Na+- und Ca2+-Ausscheidung bei den insulinresistenten Lepob/ob-Mäusen gesteigert, was unerwartet erscheint und weiter untersucht werden muss.


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