After washing, they were fixed in glutaraldehyde/paraformaldehyde, then prepared using standard methods (25). Quantitative PCR Total RNA was purified from whole kidney and other organs using Trizol (5) and from cells using the RNA Easy kit (Qiagen, Valencia, CA, USA). cDNAs or were generated using random hexamers and poly dT primers and iScript (Bio-Rad, Hercules, CA, USA) according to the manufacturers’ instructions. Q-PCR was carried out as described previously using Bio-Rad iQ5 system and SYBR green-based quantification (Bio-Rad) (3). Primer pairs for mGpnmb were the following: 5��-TCATGGAAGTGACTGTCTTT-3��, 5��-CAGACAAGTTCCTGTCATTC-3��; and 5��-GATGTCCTCATTCATGATCC-3��, 5��-TTCAAAGTGTGATTGTTGGA-3�� were tested. Both pairs gave solitary specific bands and comparable results using an annealing temperature of 51��C.

Primer pairs for Gapdh were 5��-CTGAGAAAACCTGCCAAGTA-3��, 5��-AAGAGTGGGAGTTGCTGTTG-3��. Flow cytometry Single-cell suspensions were prepared from blood and kidney as described previously (3, 19). In brief, whole blood was citrated, then PBMCs were separated using Ficoll density gradient (300 g, 20 min), followed by PBS wash 2 times. PBMCs were added to tissue culture wells in DMEM/F12 and washed to remove nonadherent cells after 1 h, or alternatively, were sorted using FACSaria (BD Biosciencesm San Jose, CA, USA), selecting the high-FSC, low-SSC population of monocytes. Whole kidney was minced and then digested using Liberase CI and DNase (Roche, Basel, Switzerland) in HBSS for 30 min at 37��C, then filtered (30 ��m).

Resuspended cells were added to tissue culture wells in complete DMEM/F12, and nonadherent cells washed after 1 h. Alternatively filtered single cells were resuspended in FACS buffer (PBS, 0.1% BSA) (3), labeled with lotus lectin-FITC 1:200 (Dako, Copenhagen, Denmark) and anti-CD45-PE, 1:200 (eBioscience), or anti-Kim-1-biotin-antibodies, 1:200 (RMT1�C4, eBioscience) followed by streptavidin-APC (Jackson ImmunoResearch), or labeled with F4/80-APC antibodies (eBioscience). Cells were sorted by FACSAria for Kim-1+ CD45-cells, LTL+ CD45-cells, or F4/80+ cells. Sorted cells were immediately lysed and RNA purified using the RNA Easy (Qiagen) system.

To determine the presence of cell surface Gpnmb expression, anti-Gpnmb Ecto antibodies or control rabbit IgG, 1:400, were applied to LLCPK1 cells expressing Gpnmb (30 min, 4��C) in FACS buffer, then after washing, anti-rabbit-biotin antibodies were applied (30 min, 4��C), followed by washing and incubation of streptavidin-APC, 1:2000 (20 min, 4��C), followed Carfilzomib by washing and fixation. Fluorescence was compared using FACSCalibur flow cytometry. To detect acid load of M lysosomes, cultured BMMs were treated with LTR as described above, then assessed by flow cytometry in the FL3 channel for fluorescence compared with unlabeled controls.