Eva Königshausen

PKCα Mediates β-Arrestin2-dependent Nephrin Endocytosis in Hyperglycemia

Nephrin, the key molecule of the glomerular slit diaphragm, is expressed on the surface of podocytes and is critical in preventing albuminuria. In diabetes, hyperglycemia leads to the loss of surface expression of nephrin and causes albuminuria. Here, we report a mechanism that can explain this phenomenon: hyperglycemia directly enhances the rate of nephrin endocytosis via regulation of the β-arrestin2-nephrin interaction by PKCα. We identified PKCα and protein interacting with c kinase-1 (PICK1) as nephrin-binding proteins. Hyperglycemia induced up-regulation of PKCα and led to the formation of a complex of nephrin, PKCα, PICK1, and β-arrestin2 in vitro and in vivo. Binding of β-arrestin2 to the nephrin intracellular domain depended on phosphorylation of nephrin threonine residues 1120 and 1125 by PKCα. Further, cellular knockdown of PKCα and/or PICK1 attenuated the nephrin–β-arrestin2 interaction and abrogated the amplifying effect of high blood glucose on nephrin endocytosis. In C57BL/6 mice, hyperglycemia over 24 h caused a significant increase in urinary albumin excretion, supporting the concept of the rapid impact of hyperglycemia on glomerular permselectivity. In summary, we have provided a molecular model of hyperglycemia-induced nephrin endocytosis and subsequent proteinuria and highlighted PKCα and PICK1 as promising therapeutic targets for diabetic nephropathy.

Chronic treatment with angiotensin-(1-7) improves renal endothelial dysfunction in apolipoproteinE-deficient mice

BACKGROUND AND PURPOSE ApolipoproteinE‐deficient [apoE (−/−)] mice, a model of human atherosclerosis, develop endothelial dysfunction caused by decreased levels of nitric oxide (NO). The endogenous peptide, angiotensin‐(1‐7) [Ang‐(1‐7)], acting through its specific GPCR, the Mas receptor, has endothelium‐dependent vasodilator properties. Here we have investigated if chronic treatment with Ang‐(1‐7) improved endothelial dysfunction in apoE (−/−) mice.

Circulating Permeability Factors in Primary Focal Segmental Glomerulosclerosis: A Review of Proposed Candidates

Primary focal segmental glomerulosclerosis (FSGS) is a major cause of the nephrotic syndrome and often leads to end-stage renal disease. This review focuses on circulating permeability factors in primary FSGS that have been implicated in the pathogenesis for a long time, partly due to the potential recurrence in renal allografts within hours after transplantation. Recently, three molecules have been proposed as a potential permeability factor by different groups: the soluble urokinase plasminogen activator receptor (suPAR), cardiotrophin-like cytokine factor-1 (CLCF-1), and CD40 antibodies. Both CLCF-1 and CD40 antibodies have not been validated by independent research groups yet. Since the identification of suPAR, different studies have questioned the validity of suPAR as a biomarker to distinguish primary FSGS from other proteinuric kidney diseases as well as suPARs pathogenic role in podocyte damage. Researchers have suggested that cleaved molecules of suPAR have a pathogenic role in FSGS but further studies are needed to determine this role. In future studies, proposed standards for the research of the permeability factor should be carefully followed. The identification of the permeability factor in primary FSGS would be of great clinical relevance as it could influence potential individual treatment regimen.

Three-dimensional electron microscopy reveals the evolution of glomerular barrier injury

Glomeruli are highly sophisticated filters and glomerular disease is the leading cause of kidney failure. Morphological change in glomerular podocytes and the underlying basement membrane are frequently observed in disease, irrespective of the underlying molecular etiology. Standard electron microscopy techniques have enabled the identification and classification of glomerular diseases based on two-dimensional information, however complex three-dimensional ultrastructural relationships between cells and their extracellular matrix cannot be easily resolved with this approach. We employed serial block face-scanning electron microscopy to investigate Alport syndrome, the commonest monogenic glomerular disease, and compared findings to other genetic mouse models of glomerular disease (Myo1e−/−, Ptpro−/−). These analyses revealed the evolution of basement membrane and cellular defects through the progression of glomerular injury. Specifically we identified sub-podocyte expansions of the basement membrane with both cellular and matrix gene defects and found a corresponding reduction in podocyte foot process number. Furthermore, we discovered novel podocyte protrusions invading into the glomerular basement membrane in disease and these occurred frequently in expanded regions of basement membrane. These findings provide new insights into mechanisms of glomerular barrier dysfunction and suggest that common cell-matrix-adhesion pathways are involved in the progression of disease regardless of the primary insult.

Angiotensin II increases glomerular permeability by β-arrestin mediated nephrin endocytosis

Glomerular permeability and subsequent albuminuria are early clinical markers for glomerular injury in hypertensive nephropathy. Albuminuria predicts mortality and cardiovascular morbidity. AT1 receptor blockers protect from albuminuria, cardiovascular morbidity and mortality. A blood pressure independent, molecular mechanism for angiotensin II (Ang II) dependent albuminuria has long been postulated. Albuminuria results from a defective glomerular filter. Nephrin is a major structural component of the glomerular slit diaphragm and its endocytosis is mediated by β-arrestin2. Ang II stimulation increases nephrin-β-arrestin2 binding, nephrin endocytosis and glomerular permeability in mice. This Ang II effect is mediated by AT1-receptors. AT1-receptor mutants identified G-protein signaling to be essential for this Ang II effect. Gαq knockdown and phospholipase C inhibition block Ang II mediated enhanced nephrin endocytosis. Nephrin Y1217 is the critical residue controlling nephrin binding to β-arrestin under Ang II stimulation. Nephrin Y1217 also mediates cytoskeletal anchoring to actin via nck2. Ang II stimulation decreases nephrin nck2 binding. We conclude that Ang II weakens the structural integrity of the slit diaphragm by increased nephrin endocytosis and decreased nephrin binding to nck2, which leads to increased glomerular permeability. This novel molecular mechanism of Ang II supports the use of AT1-receptor blockers to prevent albuminuria even in normotensives.

Neprilysin is a Mediator of Alternative Renin-Angiotensin-System Activation in the Murine and Human Kidney

Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formation in vivo has not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activation in vivo. Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy.

Chronic p38 mitogen-activated protein kinase inhibition improves vascular function and remodeling in angiotensin II-dependent hypertension.

Introduction: An excess of angiotensin II (Ang II) causes hypertension and vascular injury. Activation of mitogen-activated protein kinase p38 (p38-MAPK) plays a substantial role in Ang II-dependent organ damage. Recently, we showed that p38-MAPK activation regulates the pressor response to Ang II. This study evaluates the effect of chronic p38-MAPK inhibition in Ang II-dependent hypertension. Materials and methods: C57Bl/6J mice were infused with Ang II for 14 days and either treated with the p38-MAPK inhibitor BIRB796 (50 mg/kg/day) or the vehicle as the control. We assessed vascular function in the aorta and isolated perfused kidneys. Results: Chronic p38-MAPK inhibition did not alter blood pressure at the baseline, but attenuated Ang II-induced hypertension significantly (baseline: 122 ± 2 versus 119 ± 4 mmHg; Ang II: 173 ± 3 versus 155 ± 3 mmHg; p < 0.001). In addition, BIRB796 treatment improved vascular remodeling by reducing the aortic media-to-lumen ratio and decreasing the expression of the membrane metalloproteinases (MMP) MMP-1 and MMP-9. Moreover, renal vascular dysfunction induced by chronic Ang II infusion was significantly ameliorated in the BIRP796-treated mice. Acute p38-MAPK inhibition also improved vascular function in the aorta and kidneys of Ang II-treated mice, highlighting the important role of p38-MAPK activation in the pathogenesis of vascular dysfunction. Conclusions: Our findings indicated there is an important role for p38-MAPK in regulating blood pressure and vascular injury, and highlighted its potential as a pharmaceutical target.

Resistance to hypertension mediated by intercalated cells of the collecting duct

The renal collecting duct (CD), as the terminal segment of the nephron, is responsible for the final adjustments to the amount of sodium excreted in urine. While angiotensin II modulates reabsorptive functions of the CD, the contribution of these actions to physiological homeostasis is not clear. To examine this question, we generated mice with cell-specific deletion of AT1A receptors from the CD. Elimination of AT1A receptors from both principal and intercalated cells (CDKO mice) had no effect on blood pressures at baseline or during successive feeding of low- or high-salt diets. In contrast, the severity of hypertension caused by chronic infusion of angiotensin II was paradoxically exaggerated in CDKO mice compared with controls. In wild-type mice, angiotensin II induced robust expression of cyclooxygenase-2 (COX-2) in renal medulla, primarily localized to intercalated cells. Upregulation of COX-2 was diminished in CDKO mice, resulting in reduced generation of vasodilator prostanoids. This impaired expression of COX-2 has physiological consequences, since administration of a specific COX-2 inhibitor to CDKO and control mice during angiotensin II infusion equalized their blood pressures. Stimulation of COX-2 was also triggered by exposure of isolated preparations of medullary CDs to angiotensin II. Deletion of AT1A receptors from principal cells alone did not affect angiotensin II-dependent COX2 stimulation, implicating intercalated cells as the main source of COX2 in this setting. These findings suggest a novel paracrine role for the intercalated cell to attenuate the severity of hypertension. Strategies for preserving or augmenting this pathway may have value for improving the management of hypertension.

A novel in vivo method to quantify slit diaphragm protein abundance in murine proteinuric kidney disease

Injury of the glomerular filter causes proteinuria by disrupting the sensitive interplay of the glomerular protein network. To date, studies of the expression and trafficking of glomerular proteins have been mostly limited to in vitro or histologic studies. Here, we report a novel in vivo biotinylation assay that allows the quantification of surface expression of glomerular proteins in mice. Kidneys were perfused in situ with biotin before harvest. Afterwards glomeruli were isolated and lyzed. The protein of interest was separated by immunoprecipitation and the amount of surface-expressed protein was quantified by Western blot analysis with streptavidin staining. As proof-of-concept, we examined the presence of nephrin in the slit diaphragm in two well-established murine models of proteinuric kidney disease: nephrotoxic nephritis and adriamycin nephropathy. In proteinuric animals, significantly less nephrin was detected in the slit diaphragm. When proteinuria decreased once again during the course of disease, the amount of surface nephrin returned to the baseline. Our present results suggest that our assay is a valuable tool to study the glomerular filter in proteinuric kidney diseases. Note that the assay is not limited to proteins expressed in the slit diaphragm, and all surface proteins that are accessible to biotin perfusion and immunoprecipitation qualify for this analysis.

Pulmonary Cytomegalovirus Replication in Renal Transplant Patients with Late Onset Pneumonitis

BACKGROUND In renal transplant patients, pneumonitis may be caused by cytomegalovirus (CMV pneumonitis). This condition is usually accompanied by CMV viremia. However, CMV may also reactivate locally in the lungs of renal transplant patients with pneumonitis due to other pathogens. The impact of local CMV replication/reactivation in the lungs is unknown. MATERIAL AND METHODS All renal transplant patients at the Duesseldorf transplant center in the time from 01/2004 to 1/2008 were analyzed concerning pulmonary CMV replication in the setting of pneumonitis. RESULTS Of 434 renal transplant recipients, 25 patients were diagnosed with pneumonitis. From these 25 patients with pneumonitis, 7 presented with isolated pulmonary CMV replication but without relevant CMV viremia 8±4.2 months after renal transplantation. Three of the 7 patients needed long-term respiratory support by invasive mechanical ventilation. Pulmonary opportunistic infections were diagnosed in 6 of the 7 patients. Therapy consisted of a reduction in immunosuppression, ganciclovir, and antibiotics. CONCLUSIONS Our findings suggest that pulmonary CMV replication occurs in renal transplant patients with pneumonitis even at later phases after renal transplantation. This finding seems to be a frequent complication (7/25 patients). Clinicians should be aware of this condition because blood-based screening assays for CMV will remain negative.