The role of long-term label-retaining cells in the regeneration of adult mouse kidney after ischemia/reperfusion injury

Abstract

Background: Label-retaining cells (LRCs) have been recognized as rare stem and progenitor-like cells, but their complex biological features in renal repair at the cellular level have never been reported. This study was conducted to evaluate whether LRCs in kidney are indeed renal stem/progenitor cells and to delineate their potential role in kidney regeneration.

Methods: We utilized a long-term pulse chase of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in C57BL/6J mice to identify renal LRCs. We tracked the precise morphological characteristics and locations of BrdU(+)LRCs by both immunohistochemistry and immunofluorescence. To examine whether these BrdU(+)LRCs contribute to the repair of acute kidney injury, we analyzed biological characteristics of BrdU(+)LRCs in mice after ischemia/reperfusion (I/R) injury.

Results: The findings revealed that the nuclei of BrdU(+) LRCs exhibited different morphological characteristics in normal adult kidneys, including nuclei in pairs or scattered, fragmented or intact, strongly or weakly positive. Only 24.3 ± 1.5 % of BrdU(+) LRCs co-expressed with Ki67 and 9.1 ± 1.4 % of BrdU(+) LRCs were positive for TUNEL following renal I/R injury. Interestingly, we found that newly regenerated cells formed a niche-like structure and LRCs in pairs tended to locate in this structure, but the number of those LRCs was very low. We found a few scattered LRCs co-expressed Lotus tetragonolobus agglutinin (LTA) in the early phase of injury, suggesting differentiation of those LRCs in mouse kidney.

Conclusions: Our findings suggest that LRCs are not a simple type of slow-cycling cells in adult kidneys, indicating a limited role of these cells in the regeneration of I/R injured kidney. Thus, LRCs cannot reliably be considered stem/progenitor cells in the regeneration of adult mouse kidney. When researchers use this technique to study the cellular basis of renal repair, these complex features of renal LRCs and the purity of real stem cells among renal LRCs should be considered.

Keywords: AKI; Ischemia/reperfusion injury; Kidney regeneration; Label-retaining cells; Renal progenitor cells; Renal stem cells.

Fig. 1

Restoration of renal function in C57BL/6J mice after I/R injury (n = 4 in each group). a Serum creatinine (Scr) level significantly increased one day after injury and was restored to normal gradually over time. b The peak value of blood urea nitrogen (BUN) was found at the end of the first day after injury, followed by return to normal levels (P < 0.05, I/R injured mice vs sham group). I/R ischemia/reperfusion

Fig. 2

Renal histology in I/R injury. Kidney tissues from C57BL/6J mice with 22 min of bilateral renal ischemia or sham operation were collected on 1, 2, 3, 4, 5, 6, 9, 14, and 28 days and stained with H&E for histological examination. During the first 3 days of injury, kidney tissues showed severe tubular dilatation, loss of brush border, and sloughed debris in tubular lumen space. On day 4, the newly generated tubular cells increased rapidly. Nine days after I/R injury, the structure of damaged tissues showed normal histological architecture in cortex, medulla, and papilla (arrows, on day 4 and 6, indicate the special niche-like structure of newly restored tubular cells; magnification × 400). I/R ischemia/reperfusion, H&E hematoxylin and eosin

Fig. 3

The newly regenerated cells formed a niche-like structure on day 4 during the repair process of I/R injury. a Kidney tissues stained with H&E for histological examination by magnification × 200; b Ki67-positive cells in medulla and papilla by immunohistochemistry (magnification × 400); c Ki67-positive cells in medulla and papilla by immunofluorescence (magnification × 400). I/R ischemia/reperfusion, H&E hematoxylin and eosin

Fig. 4

Localization of BrdU-retaining cells in 8-week-old mouse kidneys using immunofluorescence. a BrdU⁺ LRCs (red) scattered among normal adult kidneys; b BrdU-retaining cells in cortex, medulla, and papilla. BrdU-retaining cells were quantified by counting the number of positive nuclei in five randomly selected fields of sections under a fluorescence microscope (magnification × 200). BrdU 5-bromo-2'-deoxyuridine, LRCs label-retaining cells

Fig. 5

Localization of BrdU-retaining cells by staining with the DAB lectin in 8-week-old mouse kidneys. a In the cortex, most of the BrdU-retaining cells were located among the tubular cells, and a few of them were scattered in renal interstitium; b Localization of BrdU-retaining cells in the medulla; c BrdU-retaining cells on the renal papilla; d Quantitative analysis of BrdU+ LRCs located in pairs (solid arrows) and BrdU+ LRCs revealed nuclear fragmentation and disassembly (dotted arrows, magnification × 400). BrdU 5-bromo-2'-deoxyuridine, DAB diaminobenzidine, LRCs label-retaining cells

Fig. 6

BrdU-retaining cells in mouse kidneys after I/R injury. After an 8-week chase period, the BrdU-labeled C57BL/6J mice were subjected to renal bilateral I/R injury. Kidney tissues were harvested 1, 2, 3, 4, 5, 6, 9, 14, and 28 days after I/R injury. BrdU-retaining cells were stained a visible brown color without using the hematoxylin. a Quantification of BrdU-retaining cells in I/R injured kidneys from the cortex, medulla, and papilla; LRCs diminished over time with renal functional recovery; four days after injury, the number of LRCs decreased dramatically. b Kidney sections from BrdU-stained cortex, medulla, and papilla showed the disappearance of LRCs after I/R injury. Upon restoration and recovery of the structure of the I/R injured kidney to normal within 1 month, LRCs were difficult to detect (magnification × 200). BrdU 5-bromo-2'-deoxyuridine, I/R ischemia/reperfusion, LRCs label-retaining cells

Fig. 7

Analysis of the expression of Ki67-positive cells (a, c) and TUNEL-positive cells (b, d) in mouse kidneys after IRI (magnification × 400)

Fig. 8

Contribution of BrdU⁺ LRCs in the regeneration of I/R injured mouse kidneys. a Double-immunofluorescence staining for BrdU (red) and Ki67 (green) to visualize the role of BrdU⁺ LRCs in cell proliferation 4 days after IRI. Newly generated cells formed a special niche-like structure (green), and only some of the BrdU⁺ LRCs co-expressed Ki-67 in this area (arrows). b Double immunofluorescence was used to determine BrdU⁺ LRCs undergoing cellular apoptosis on day 4. Scattered BrdU⁺ LRCs co-expressed TUNEL (arrows) (arrows indicate the double-stained location; magnification × 400). BrdU 5-bromo-2'-deoxyuridine, LRCs label-retaining cells, I/R ischemia/reperfusion

Fig. 9

BrdU⁺ LRCs (red) co-expressed LTA (green) both in normal and I/R injured kidney (The arrows indicate LRCs in pairs, the arrowheads indicate isolated LRCs; magnification × 400). BrdU 5-bromo-2'-deoxyuridine, LRCs label-retaining cells, LTA Lotus tetragonolobus agglutinin, I/R ischemia/reperfusion