A molecular mechanism for angiotensin II receptor blocker-mediated slit membrane protection: Angiotensin II increases nephrin endocytosis via AT1-receptor-dependent ERK 1/2 activation.

Albuminuria is characterized by a disruption of the glomerular filtration barrier, which is composed of the fenestrated endothelium, the glomerular basement membrane, and the slit diaphragm. Nephrin is a major component of the slit diaphragm. Apart from hemodynamic effects, Ang II enhances albuminuria by ß-Arrestin2-mediated nephrin endocytosis. Blocking the AT1 receptor with candesartan and irbesartan reduces the Ang II-mediated nephrin-ß-Arrestin2 interaction. The inhibition of MAPK ERK 1/2 blocks Ang II-enhanced nephrin-ß-Arrestin2 binding. ERK 1/2 signaling, which follows AT1 receptor activation, is mediated by G-protein signaling, EGFR transactivation, and ß-Arrestin2 recruitment. A mutant AT1 receptor defective in EGFR transactivation and ß-Arrestin2 recruitment reduces the Ang II-mediated increase in nephrin ß-Arrestin2 binding. The mutation of ß-Arrestin2K11,K12, critical for AT1 receptor binding, completely abrogates the interaction with nephrin, independent of Ang II stimulation. ß-Arrestin2K11R,K12R does not influence nephrin cell surface expression. The data presented here deepen our molecular understanding of a blood-pressure-independent molecular mechanism of AT-1 receptor blockers (ARBs) in reducing albuminuria.

Factors Associated With Vaccine-Induced T-Cell Immune Responses Against Severe Acute Respiratory Syndrome Coronavirus 2 in Kidney Transplant Recipients.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important prophylactic measure in kidney transplant recipients (KTRs), but the immune response is often impaired. Here, we examined the T-cell immune response against SARS-CoV-2 in 148 KTRs after 3 or 4 vaccine doses, including 35 KTRs with subsequent SARS-CoV-2 infection. The frequency of spike-specific T cells was lower in KTRs than in immunocompetent controls and was correlated with the level of spike-specific antibodies. Positive predictors for detection of vaccine-induced T cells were detection of spike-specific antibodies, heterologous immunization with messenger RNA and a vector vaccine, and longer time after transplantation. In vaccinated KTRs with subsequent SARS-CoV-2 infection, the T-cell response was greatly enhanced and was significantly higher than in vaccinated KTRs without SARS-CoV-2 infection. Overall, the data show a correlation between impaired humoral and T-cell immunity to SARS-CoV-2 vaccination and provide evidence for greater robustness of hybrid immunity in KTRs.

Immune response to third SARS-CoV-2 vaccination in seronegative kidney transplant recipients: Possible improvement by mycophenolate mofetil reduction.

Modification of vaccination strategies is necessary to improve the immune response to SARS-CoV-2 vaccination in kidney transplant recipients (KTRs). This multicenter observational study analyzed the effects of the third SARS-CoV-2 vaccination in previously seronegative KTRs with the focus on temporary mycophenolate mofetil (MMF) dose reduction within propensity matched KTRs. 56 out of 174 (32%) previously seronegative KTRs became seropositive after the third vaccination with only three KTRs developing neutralizing antibodies against the omicron variant. Multivariate logistic regression revealed that initial antibody levels, graft function, time after transplantation and MMF trough levels had an influence on seroconversion (P < .05). After controlling for confounders, the effect of MMF dose reduction before the third vaccination was calculated using propensity score matching. KTRs with a dose reduction of ≥33% showed a significant decrease in MMF trough levels to 1.8 (1.2-2.5) µg/ml and were more likely to seroconvert than matched controls (P = .02). Therefore, a MMF dose reduction of 33% or more before vaccination is a promising approach to improve success of SARS-CoV-2 vaccination in KTRs.

Inhibition of p38 MAPK decreases hyperglycemia-induced nephrin endocytosis and attenuates albuminuria.

Chronic hyperglycemia, as in diabetes mellitus, may cause glomerular damage with microalbuminuria as an early sign. Noteworthy, even acute hyperglycemia can increase glomerular permeability before structural damage of the glomerular filter can be detected. Despite intensive research, specific antiproteinuric therapy is not available so far. Thus, a deeper understanding of the molecular mechanisms of albuminuria is desirable. P38 MAPK signaling is involved in the development of hyperglycemia-induced albuminuria. However, the mechanism of increased p38 MAPK activity leading to increased permeability and albuminuria remained unclear. Recently, we demonstrated that acute hyperglycemia triggers endocytosis of nephrin, the key molecule of the slit diaphragm, and induces albuminuria. Here, we identify p38 MAPK as a pivotal regulator of hyperglycemia-induced nephrin endocytosis. Activated p38 MAPK phosphorylates the nephrin c-terminus at serine 1146, facilitating the interaction of PKCα with nephrin. PKCα phosphorylates nephrin at threonine residues 1120 and 1125, mediating the binding of ß-arrestin2 to nephrin. ß-arrestin2 triggers endocytosis of nephrin by coupling it to the endocytic machinery, leading to increased glomerular permeability. Pharmacological inhibition of p38 MAPK preserves nephrin surface expression and significantly attenuates albuminuria. KEY MESSAGES: Acute hyperglycemia triggers endocytosis of nephrin. Activated p38 MAPK phosphorylates the nephrin c-terminus at serine 1146, facilitating the interaction of PKCα with nephrin. PKCα phosphorylates nephrin at threonine residues 1120 and 1125, mediating the binding of ß-arrestin2 to nephrin. ß-arrestin2 triggers endocytosis of nephrin by coupling it to the endocytic machinery, leading to a leaky glomerular filter. Pharmacological inhibition of p38 MAPK preserves nephrin surface expression and significantly attenuates albuminuria under hyperglycemic conditions.

Intensity of mycophenolate mofetil treatment is associated with an impaired immune response to SARS-CoV-2 vaccination in kidney transplant recipients.

Kidney transplant recipients (KTRs) are extremely vulnerable to SARS-CoV-2 infection and show an impaired immune response to SARS-CoV-2 vaccination. We analyzed factors related to vaccination efficiency in KTRs. In a multicenter prospective observational study (NCT04743947), IgG antibodies levels against SARS-CoV-2 spike S1 subunit and their neutralization capacity after SARS-CoV-2 vaccination were analyzed in 225 KTRs and compared to 176 controls. After the vaccination, 56 (24.9%) KTRs became seropositive of whom 68% had neutralizing antibodies. This immune response was significantly lower compared to controls (239 [78-519] BAU/ml versus 1826 [560-3180] BAU/ml for KTRs and controls, p < .0001). The strongest predictor for an impaired response was mycophenolate mofetil (MMF) treatment. Multivariate regression analysis revealed that MMF-free regimen was highly associated with seroconversion (OR 13.25, 95% CI 3.22-54.6; p < .001). In contrast, other immunosuppressive drugs had no significant influence. 187 out of 225 KTRs were treated with MMF of whom 26 (13.9%) developed antibodies. 23 of these seropositive KTRs had a daily MMF dose ≤1 g. Furthermore, higher trough MMF concentrations correlated with lower antibody titers (R -0.354, p < .001) supporting a dose-dependent unfavorable effect of MMF. Our data indicate that MMF dose modification could lead to an improved immune response.

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy.

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte foot processes contain actin bundles that bind to cytoskeletal adaptor proteins such as podocin. Those adaptor proteins, such as podocin, link the backbone of the glomerular slit diaphragm, such as nephrin, to the actin cytoskeleton. Studying the localization and function of these and other podocytic proteins is essential for the understanding of the glomerular filter's role in health and disease. The presented protocol enables the user to visualize actin, podocin, and nephrin in cells with super resolution imaging on a conventional microscope. First, cells are stained with a conventional immunofluorescence technique. All proteins within the sample are then covalently anchored to a swellable hydrogel. Through digestion with proteinase K, structural proteins are cleaved allowing isotropical swelling of the gel in the last step. Dialysis of the sample in water results in a 4-4.5-fold expansion of the sample and the sample can be imaged via a conventional fluorescence microscope, rendering a potential resolution of 70 nm.

Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70.

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 µL). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

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.

Recent Treatment Advances and New Trials in Adult Nephrotic Syndrome.

The etiology of nephrotic syndrome is complex and ranges from primary glomerulonephritis to secondary forms. Patients with nephrotic syndrome often need immunosuppressive treatment with its side effects and may progress to end stage renal disease. This review focuses on recent advances in the treatment of primary causes of nephrotic syndrome (idiopathic membranous nephropathy (iMN), minimal change disease (MCD), and focal segmental glomerulosclerosis (FSGS)) since the publication of the KDIGO guidelines in 2012. Current treatment recommendations are mostly based on randomized controlled trials (RCTs) in children, small RCTs, or case series in adults. Recently, only a few new RCTs have been published, such as the Gemritux trial evaluating rituximab treatment versus supportive antiproteinuric and antihypertensive therapy in iMN. Many RCTs are ongoing for iMN, MCD, and FSGS that will provide further information on the effectiveness of different treatment options for the causative disease. In addition to reviewing recent clinical studies, we provide insight into potential new targets for the treatment of nephrotic syndrome from recent basic science publications.

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.

Angiotensin II increases glomerular permeability by ß-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 ß-arrestin2. Ang II stimulation increases nephrin-ß-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 ß-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.

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.

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.

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 suPAR's 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.

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.

PKC alpha mediates beta-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 ß-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 ß-arrestin2 in vitro and in vivo. Binding of ß-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-ß-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.