Sixteen to 30 whole glomerular profiles were evaluated (corresponding to 205�C471 order inhibitor reference points). The total volume of mesangium or capillaries per glomerulus (V(Mes,Glom) and V(Cap,Glom), respectively) was obtained by multiplying the respective volume fraction by mean v(Glom). All results were corrected for embedding shrinkage (34). Immunofluorescence staining of kidney sections. Kidneys were frozen in OCT compound and sectioned at 6 ��m (Leica Kryostat). The sections were fixed with 4% paraformaldehyde, blocked in PBS containing 5% BSA, and incubated for 1 hour with primary antibodies as indicated. After PBS rinse for several times, fluorophore-conjugated secondary antibodies (Invitrogen) were applied for 30 minutes.

Images were taken using a Zeiss laser scan microscope equipped with a ��63 water immersion objective or a Zeiss fluorescence microscope equipped with ��20 and ��40 oil immersion objectives. Quantification of immunofluorescence results. Images were acquired by a Zeiss Axioscope 40FL microscope, equipped with AxioCam MRc5 digital video camera and immunofluorescence apparatus (Carl Zeiss SpA). Images were recorded using AxioVision software 4.3 and analyzed by the AxioVision analysis module (Carl Zeiss SpA). Glomeruli were selected as region of interest (ROI), and a macro (consisting of a color threshold procedure, followed by filtering and Danielsson��s algorithm) was applied to select stained areas and to calculate their relative quantity as a percentage of the ROI area. Isolation and characterization of adult mouse glomeruli.

Glomeruli were isolated using Dynabead perfusion and were glass-glass�Chomogenized in lysis buffer (containing 20 mM CHAPS and 1% Triton X-100) (55). After centrifugation (15,000 g, 15 minutes, 4��C) protein concentration was determined by Dc Protein-Assay (Bio-Rad). Equal amounts of protein were separated on SDS page. Western blots were densitometrically analyzed using LabImage software. Ratios of protein band intensity to loading control protein band intensity are shown (Figures (Figures1C,1C, 3C, 6F, and 7C). Patients and microarray analysis. Human renal biopsies from patients and controls were collected with informed consent within the framework of the European Renal cDNA Bank �� Kr?ner-Fresenius Biopsy Bank (35) and the Pima Indian Diabetes Study (56), respectively. The former biopsy bank has been approved by the cantonal ethics committee of Zurich, Switzerland, the latter by the National Institute of Diabetes and Digestive and Kidney Diseases in Bethesda, Batimastat Maryland, USA. The approval allows exclusively the report of aggregate data; no individual data are reported or deposited. Glomeruli were microdissected, total RNA isolated, linearly amplified, and hybridized to Affymetrix HG-U 133 Plus 2.