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).

Rationale and design of a large registry on renal denervation: the Global SYMPLICITY registry.

AIMS: Hypertension is a global healthcare concern associated with a wide range of comorbidities. The recognition that elevated sympathetic drive plays an important role in the pathogenesis of hypertension led to the use of renal artery denervation to interrupt the efferent and afferent sympathetic nerves between the brain and kidneys to lower blood pressure. Clinical trials of the Symplicity™ renal denervation system have demonstrated that radiofrequency ablation of renal artery nerves is safe and significantly lowers blood pressure in patients with severe resistant (systolic BP >160 mmHg) hypertension. Smaller ancillary studies in hypertensive patients suggest a benefit from renal denervation in a variety of conditions such as chronic kidney disease, glucose intolerance, sleep apnoea and heart failure. METHODS AND RESULTS: The Global SYMPLICITY registry, which incorporates the GREAT SYMPLICITY registry initiated in Germany, is being conducted worldwide to evaluate the safety and efficacy of treatment with the Symplicity renal denervation system in real-world uncontrolled hypertensive patients, looking first at subjects with severe resistant hypertension to confirm the results of prior clinical trials, but then also subjects with a wider range of baseline blood pressure and coexisting comorbidities. CONCLUSIONS: The rationale, design and first baseline data from the Global SYMPLICITY registry are presented.

Implementation and first results of a German chronic kidney disease registry.

Advanced chronic kidney disease (CKD) is gaining increasing medical and economical importance, but little information exists about treatment variation and the impact of routine clinical treatments on survival, quality of life, and cost. We demonstrate the first results of a national electronic registry of nephrology clinic data that will serve as a resource for the prospective observation of CKD patients in Germany. A large network of German nephrologist practices is currently joining the project. Routinely obtained clinical data for non-dialysis dependent CKD patients are documented in health records electronically, and elements from these data are extracted using QuaNT (Qualitätssicherung Nephrologie und Transplantation) to create a centralized database. Here, we report cross-sectional data from 59 participating nephrology clinics and 6,187 patients with CKD Stage 3 - 5 in 2011. Mean age ± standard deviation (SD) was 72 ± 12 years. The distribution of CKD 3, 4, and 5 (non-dialysis) was 60%, 33%, and 8%, respectively. The major renal diseases were hypertension/vascular nephropathy (47%) and diabetic nephropathy (26%). Reninangiotensin-system inhibitor prescription was 78%. Vitamin D prescription was 50%, phosphate binders 6%, iron (oral or i.v.) 19%, and erythropoietin-stimulating agents 14%. This electronic registry follows clinical nephrology care and outcomes for CKD patients in Germany, and increased participation is anticipated. As a component of the initiative, variation in patient care will be studied to identify best treatment practices in analyses integrated into the international CKD Outcomes and Practice Patterns Study (CKDopps).

A phase 1, single-dose study of fresolimumab, an anti-TGF-ß antibody, in treatment-resistant primary focal segmental glomerulosclerosis.

Primary focal segmental glomerulosclerosis (FSGS) is a disease with poor prognosis and high unmet therapeutic need. Here, we evaluated the safety and pharmacokinetics of single-dose infusions of fresolimumab, a human monoclonal antibody that inactivates all forms of transforming growth factor-ß (TGF-ß), in a phase I open-label, dose-ranging study. Patients with biopsy-confirmed, treatment-resistant, primary FSGS with a minimum estimated glomerular filtration rate (eGFR) of 25 ml/min per 1.73 m(2), and a urine protein to creatinine ratio over 1.8 mg/mg were eligible. All 16 patients completed the study in which each received one of four single-dose levels of fresolimumab (up to 4 mg/kg) and was followed for 112 days. Fresolimumab was well tolerated with pustular rash the only adverse event in two patients. One patient was diagnosed with a histologically confirmed primitive neuroectodermal tumor 2 years after fresolimumab treatment. Consistent with treatment-resistant FSGS, there was a slight decline in eGFR (median decline baseline to final of 5.85 ml/min per 1.73 m(2)). Proteinuria fluctuated during the study with the median decline from baseline to final in urine protein to creatinine ratio of 1.2 mg/mg with all three Black patients having a mean decline of 3.6 mg/mg. The half-life of fresolimumab was ∼14 days, and the mean dose-normalized Cmax and area under the curve were independent of dose. Thus, single-dose fresolimumab was well tolerated in patients with primary resistant FSGS. Additional evaluation in a larger dose-ranging study is necessary.

Efficacy and tolerability of the fixed lercanidipine-enalapril combination in the treatment of patients with essential hypertension.

The purpose of this first prospective observational cohort study after launch was to document the efficacy and tolerability of therapy with the fixed combination of lercanidipine HCl (CAS 132866-11-6), a third-generation calcium antagonist, and the angiotensin converting enzyme (ACE) inhibitor enalapril maleate (CAS 76095-16-4) in patients with essential hypertension in daily practice. Both parts of the observational study were conducted with the fixed combination of enalapril maleate 10 respectively 20 mg and lercanidipine HCl 10 mg, one part with Zaneril and the other with Zanipress. The fixed combination is marketed under these product names in Germany. The data of 8,440 patients with a mean age of 62 years and a mean body mass index (BMI) of 28 kg/m2 were evaluated. 84% of the patients had already received pre-treatment for essential hypertension. 26% of all the patients were known to have had hypertension for at least 10 years. Around 70% had further concomitant diseases. The mean blood pressure before the beginning of the study was 162.5/ 94.5 mmHg, the mean reduction after around three months was 28.4/ 13.5 mmHg. Just under 80% of the patients reached the desired target blood pressure after the observation period. The global assessment of the physicians shows that the efficacy of the drugs was "very good" to "good" for 94% of the patients. Global tolerability was assessed as "very good" and "good" for 97% of the patients. Compliance was stated as "very good" and "good" for 97% of the patients. Adverse drug reactions were documented for 2% of all patients.

Sympathetic overactivity--the Cinderella of cardiovascular risk factors in dialysis patients.

Cardiovascular morbidity and mortality is exceedingly high in patients with chronic renal failure. Sympathetic overactivity is an important pathomechanism contributing to progression of renal disease as well as cardiovascular complications. For more than 30 years it has been known that plasma levels of norepinephrine are elevated in chronic renal failure pointing to increased sympathetic nerve activity. The kidneys are richly innervated by efferent sympathetic and afferent sensory nerves. They participate in many reflex adjustments of renal function. Initially, this finding had not been attributed to increased efferent sympathetic drive, but rather to reduced renal clearance and defective neuronal reuptake of norepinephrine. At this time, however, the evidence for increased sympathetic drive is solid. Interventions to reduce sympathetic overactivity will provide new therapeutic approaches. The available experimental and clinical evidence to suggest such a pathophysiological role of sympathetic overactivity is summarized in this current review.

beta-Arrestin2 mediates nephrin endocytosis and impairs slit diaphragm integrity.

beta-Arrestins mediate internalization of plasma membrane receptors. Nephrin, a structural component of the glomerular slit diaphragm, is a single transmembrane spanning receptor and belongs to the family of adhesion molecules. Its mutation causes a hereditary nephrotic syndrome. We report the previously undescribed interaction of beta-arrestin2 with the nephrin C terminus. The phosphorylation status of nephrin Y1193 regulates inversely the binding of beta-arrestin2 and podocin. The Src-family member Yes, known to enhance podocin-nephrin interaction by nephrin phosphorylation, diminishes beta-arrestin2-nephrin interaction. beta-Arrestin2 induces nephrin endocytosis and attenuates nephrin signaling. This finding suggests that nephrin Y1193 serves as a molecular switch that determines the integrity of the slit diaphragm by functional competition between beta-arrestin2 and podocin. This concept offers a molecular pathomechanism of slit diaphragm distortion and opens therapeutic avenues for glomerular diseases.