IMMUNODISSECTION OF THE HUMAN PROXIMAL NEPHRON - FLOW SORTING OF S1S2S3, S1S2 AND S3 PROXIMAL TUBULAR CELLS

We report on the use of several proximal tubular cell (PTC) surface ma rkers and corresponding antibodies in fluorescence-activated cell sort ing (FACS), and their ability to identify and flow sort cells of defin ed proximal tubular origin (S1S2S3) or of defined proximal subsegmenta l origin (S1S2 only/S3 only). We tested monoclonal/polyclonal antibodi es directed against five different surface peptidases [leucine aminope ptidase (LAP), neutral endopeptidase 24.11 (NEP), dipeptidyl peptidase IV (DPPIV), aminopeptidase A (APA) and gamma-glutamyl transferase (ga mma-GT)], the S3 segment-specific marker intestinal type alkaline phos phatase (LAP) and as S1S2 marker (TN20-antigen), originally proposed a s a surface marker for interstitial fibroblasts. Segment (proximal tub ular vs. distal tubular) and proximal subsegmental (S1S2 vs. S3) expre ssion of all five surface peptidases and TN20 antigen were first asses sed by comparing immunohistochemical staining on normal human kidney t issue with staining for well-known segment-specific differentiation ma rkers (intestinal type alkaline phosphatase, Tamm-Horsfall protein) on adjacent sections. All five peptidases were found to be expressed to a certain degree in all subsegments (S1 S2 and S3) of the proximal nep hron, whereas expression was never seen int he more distal parts of th e nephron. Flow cytometry was performed on cells obtained following gr adient purification of collagenase-digested human renal tissue. Labeli ng cells for expression of LAP, NEP or DPPIV resulted in high yields o f specifically labeled PTC (S1S2S3 origin). Labeling with anti-LAP res ulted in the clearest distinction between positive and negative cell s ubpoulations, and therefore LAP was considered the best PTC marker for the use in FACS. LAP histochemical staining on sorted cells showed th at flow sorting with monoclonal antibody (moAb) 250 (anti-intestinal t ype alkaline phosphatase) allowed sorting of S3 cells with > 90% purit y. Likewise, moAb TN20 enabled us to obtain a highly purified S1S2 pop ulation as confirmed by the absence of LAP on sorted cells.