Magdalena Woznowski

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.

Protein kinase C alpha (PKC α) 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.

AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

Mice lacking AT(1) angiotensin receptors have an impaired capacity to concentrate the urine, but the underlying mechanism is unknown. To determine whether direct actions of AT(1) receptors in epithelial cells of the collecting duct regulate water reabsorption, we used Cre-Loxp technology to specifically eliminate AT(1A) receptors from the collecting duct in mice (CD-KOs). Although levels of AT(1A) receptor mRNA in the inner medulla of CD-KO mice were significantly reduced, their kidneys appeared structurally normal. Under basal conditions, plasma and urine osmolalities and urine volumes were similar between CD-KO mice and controls. The increase in urine osmolality in response to water deprivation or vasopressin administration, however, was consistently attenuated in CD-KO mice. Similarly, levels of aquaporin-2 protein in inner and outer medulla after water deprivation were significantly lower in CD-KO mice compared with controls, despite its normal localization to the apical membrane. In summary, these results demonstrate that AT(1A) receptors in epithelial cells of the collecting duct directly modulate aquaporin-2 levels and contribute to the concentration of urine.

Age‐Related Penetrance of Hereditary Atypical Hemolytic Uremic Syndrome

Hereditary atypical hemolytic uremic syndrome (aHUS), a dramatic disease frequently leading to dialysis, is associated with germline mutations of the CFH, CD46, or CFI genes. After identification of the mutation in an affected aHUS patient, single‐site gene testing of relatives is the preventive care perspective. However, clinical data for family counselling are scarce.

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.

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.

Hypercalcemia after transplant nephrectomy in a hemodialysis patient: a case report

IntroductionHypercalcemia is a complication often seen in chronic hemodialysis patients. A rare cause of this condition is sarcoidosis. Its highly variable clinical presentation is challenging. Especially in patients suffering chronic kidney graft failure the nonspecific constitutional symptoms of sarcoidosis like fever, weight loss, arthralgia and fatigue may be easily misleading.Case presentationA 51 year old male developed hypercalcemia, arthralgia and B-symptoms after explantation of his kidney graft because of suspected acute rejection. The removed kidney showed vasculopathy and tubulointerstitial nephritis, which had not been overt in the biopsy taken half a year earlier. Despite explantation and withdrawal of the immunosuppression the patients general condition deteriorated progressively. A rapid rise in serum calcium finally provoked us to check for sarcoidosis. CT scans of the lungs, broncho-alveolar-lavage and further lab tests confirmed the diagnosis.ConclusionThis case demonstrates that withdrawal of immunosuppressive drugs sometimes unmasks sarcoidosis. It should be considered as differential diagnosis even in hemodialysis patients, in whom other reasons for hypercalcemia are much more common.

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.

Long-Term Effect of Renal Transplantation and Aging on Hemoglobin A1C Levels: A Case-Control Study in 191 Non-Diabetic Deceased Donor Renal Transplant Recipients

BACKGROUND Reduced renal function in patients with chronic kidney disease is linked to insulin resistance; and impairments in glucose homeostasis, as measured by HbA1c levels, are related to cardiovascular events. Recently, aging has been reported to affect HbA1c levels over time in non-diabetic individuals. The objective of this study was to investigate the association between renal function and aging in non-diabetic deceased-donor renal transplant recipients. MATERIAL AND METHODS A total of 191 patients were analyzed (mean age 50.6±12.2 years, dialysis vintage 6.5±3.1 years, 53.4% male patients). HbA1-c levels were measured on the day of transplantation and on follow-up. The mean follow-up time was 4.9±3.1 years. RESULTS Renal transplantation resulted in an increase in eGFR of 38.6±18.9 mL/min/1.73 m2 as compared to baseline levels on dialysis and the mean eGFR on follow-up was 45.5±18.9 mL/min/1.73 m2. HbA1c levels increased significantly from the day of transplantation to the last follow-up (5.3±0.4% to 5.6±0.4%, p<0.0001). Correlation analysis demonstrated non-significant associations between the change in HbA1c levels and the parameters of age and renal transplant function. CONCLUSIONS In conclusion, we observed a significant increase in HbA1c levels over a 5-year post-transplant follow-up period in non-diabetic deceased-donor renal transplant recipients. In contrast to the non-diabetic general population, the increase in HbA1c observed in this cohort was greater but not associated with aging.