Urea ct fs

CKD was induced by surgical 5/6th nephrectomy (SNX) as published,³⁶ (link) with minor adaptations. In short, in a single procedure, surgical removal of the whole left kidney and polectomy (2/3rd) of the right kidney was performed using retroperitoneal incisions. In Sham animals, both kidneys were externalized and the renal capsule was removed. Postoperative buprenorphine (0.03 mg/kg Temgesic) was given subcutaneously for a period of at least 36 h. Rats were weighed 3 times a week, 24-h urines, and blood samples were collected and the SBP was measured (Tail cuff sphygmomanometer–LE 5002 LETICA^®) biweekly. To collect 24-h urines, rats were placed in metabolic cages without food for 24 h, but with free access to water with 2% glucose. Urine was collected in antibiotic/antimycotic solution (A5955; Sigma, St Louis, US) and stored at −20 °C. Blood samples were collected from the tail vein into EDTA Microtainers (BD #365974). Urinary protein levels were measured by Bradford Assay (Bio Rad). Plasma urea was determined by DiaSys Urea CT FS (DiaSys Diagnostic Systems). Plasma creatinine levels were determined by DiaSys Creatinine PAP FS kit (DiaSys Diagnostic Systems,). eGFR was calculated with a plasma creatinine- and urea-based equation specific for rats.³⁷ (link) We determined CKD as established once a threshold of proteinuria exceeding 50 mg/24 h was reached.

Rats were weighed weekly, and at regular intervals 24-hour urine and blood samples were collected and the systolic blood pressure was measured by tail cuff sphygmomanometry at weeks 5, 9 and 11. To collect 24-hour urine, rats were placed in metabolism cages without food for 24 hours, but with free access to water with 2% glucose. For the systemic in vivo pravastatin treatment studies, pravastatin was also supplemented to the drinking water during urine collection. Urine was collected in antibiotic/antimycotic solution (A5955; Sigma) and stored at −80°C. Blood samples were collected from the tail vein. Urinary protein levels were measured with Coomassie blue. Sodium and potassium levels were determined by flame photometry. NO metabolites were measured (Cayman Chemical, Ann Arbor, MI, USA). Plasma urea and plasma and urinary creatinine levels were determined by DiaSys Urea CT FS (DiaSys Diagnostic Systems, Holzheim, Germany). Creatinine clearance was calculated by dividing urine creatinine excretion (μmol/minute/100 g body weight) by plasma creatinine (μmol/μl). Cholesterol and triglycerides were determined by DiaSys Cholesterol FS and DiaSys Triglycerides FS (DiaSys Diagnostic Systems).