rtTA;tetO.Cre mice and were provided by Nicola Wanner (Renal Division, University Hospital Freiburg). All animal studies were approved by the Committee on Research Animal Care, Regierungspr?sidium, Freiburg. Protein overload and subsequent analysis. Rictor��podocyte kinase inhibitor Nutlin-3a female mice (n = received endotoxin-free BSA (Sigma-Aldrich A9430) (250 mg/ml, dissolved in PBS) intraperitoneally for 5 consecutive days (10 mg/g body weight) (31, 32). Urinary albumin excretion rates were analyzed before injections and at days 1 to 8 after the first injection. STZ-induced diabetes mellitus. Heterozygous podocyte-specific Raptor-knockout mice RaptorHet podocyte mice (n = 5) and control littermates (n = 11) received 2 doses of intraperitoneal STZ (Sigma-Aldrich) (125 ��g/g body weight) in 50 mM sodium citrate buffer on days 1 and 4 (50).

Urinary albumin excretion rates were analyzed before injection and 4, 8, 12, and 16 weeks after injection. Kidneys were harvested and processed for histological and ultrastructural analyses after the 16-week follow-up. Urine and serum analyses. Urinary albumin and urinary or serum creatinine, respectively, were measured using mouse albumin�Cspecific ELISA (Bethyl) and creatinine kits (Labor-Technik). Proteinuria was expressed as mg albumin/mg creatinine. Blood glucose was measured using Accu-Check Sensor (51). Blood pressure analysis. Blood pressure analysis was performed using a Panlab LE5001 control unit using a single animal heating unit and a LE5160MM transducer and cuff. Animals were accommodated to the equipment for 3 days before measurements were taken.

Blood pressure values represent the mean systolic arterial pressure of 3 consecutive measurements for each mouse. Morphological analysis. Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin, GMA/MMA, Epon (34), or in Lowicryl K4M resin (Electron Microscopy Sciences) and further processed for PAS staining, transmission electron microscopy, or scanning electron microscopy, respectively. Sclerosis index was done as described by el Nahas et al. (24). Histological analyses. Quantitative stereological analyses of kidney sections were performed as described previously (34). Briefly, the mean glomerular volume (mean v(Glom)) was determined from the mean glomerular profile area (mean A(Glom)) (52), which was obtained by measuring 100 systematically sampled glomerular profiles per animal.

The physical dissector principle was applied for counting podocytes (Q�C) as described, using semithin sections (53, 54). The numerical density of podocytes in glomeruli (NV(P/Glom)) was calculated as the quotient of the sum of Q�C divided by the dissector volume. Brefeldin_A The number of podocytes per glomerulus (N(P,Glom)) was calculated multiplying NV(P/Glom) and mean v(Glom). The volume fraction of podocytes per glomerulus (Vv(P/Glom)) was determined by point counting method. The mean podocyte volume (mean v(P)) was calculated dividing Vv(P/Glom) by NV(P/Glom).