Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney

Abstract

We searched for evidence for a contribution of stem cells in growth of the proximal S3 segments of healthy rats. According to the stem cell model, stem cells are undifferentiated and slow cycling; the bulk of cycling cells are transit amplifying, rapidly cycling cells. We show the following. 1) By continuous application of a thymidine analog (ThA) for 7 days, S3 proximal epithelial cells in healthy kidneys display a high-cycling rate. 2) Slow-cycling cells, identified by lack of ThA uptake during 14 days of continuous ThA application up to death and by expression of the cell cycle protein Ki67 at death, have the same degree of differentiation as quiescent cells. 3) To detect rapidly cycling cells, rats were killed at various time points after injection of a ThA. Double immunofluorescence for ThA and a cell cycle marker was performed, with colocalization indicating successive divisions. During one week after division, daughter cells display a very low proliferation rate, indicating the absence of rapidly cycling cells. 4) Labeling with cyclin D1 showed that this low proliferation rate is due to cycle arrest. 5) More than 50% of the S3 cells entered the cell cycle 36 h after a potent proliferative stimulus (lead acetate injection). We conclude that generation of new cells in the proximal tubule relies on division of differentiated, normally slow-cycling cells. These may rapidly enter the cycle under an adequate stimulus.

Origin of regenerating tubular cells after acute kidney injury

“…This is consistent with findings of Le Hir and coworkers, who showed that in growing adolescent normal rats, tubular cells undergo cellular divisions while remaining fully differentiated (19,20). In contrast, in human kidneys, we noted that STCs showed a dedifferentiated phenotype with loss of brush border and of the basolateral labyrinth (12).…”

Repair of injured proximal tubule does not involve specialized progenitors

“…Such slow clearance would bias our results toward a small increase in double-labeled cells, as has been previously reported (21). The observation that proximal tubule cells undergo cell cycle arrest after division in growing rats is consistent with our low mea- sured frequency of CldU-and Ki67-copositive cells after IRI (18). Taken together, our observations do not support even a minor contribution of specialized intratubular progenitors to epithelial repair.…”

“…To accurately quantify two sequential rounds of DNA synthesis in the same cell, we used the thymidine analogs, 5-chloro-2-deoxyuridine (CldU) and 5-iodo-2-deoxyuridine (IdU), in a strategy validated in solid organs, including pancreas (16), heart (17), and adult, uninjured kidney (18). To assess the specificity of CldU and IdU in our renal IRI model, we performed unilateral IRI and killed mice 48 h after injury.…”

“…Detection of CldU and IdU was accomplished using modifications of published protocols (16,18,36). Paraffin sections (4 μM) were rehydrated, nuclear membranes permeabilized with 0.2% Triton X-100 in PBS for 5 min, and antigens retrieved by treatment with heated citrate (Antigen Unmasking Solution; VectorLabs).…”

Isolation and Characterization of Progenitor-Like Cells from Human Renal Proximal Tubules

“…Tubular regeneration may be a stochastic process in which the least-injured cells dedifferentiate and repopulate the denuded basal membranes. 4,5 This view has been challenged by the concept of renal and/or bone marrow-derived stem cells participating in the regenerative process. 6 Recent data, however, indicate a minor role for the latter cell type, 7,8 and the search for the defining features of adult tubular stem or progenitor cells has, to date, been inconclusive, with discrepant results regarding their proper histologic markers and their histologic location.…”