| BackgroundIn recent years, the number of patients with acute or chronic kidney disease (CKD) has been increased with the extension of human life and the change of modern life rhythm. The current treatment for patients who progress to ESRD only rely on dialysis or a kidney transplant, but dialysis treatment still has many serious complications and transplantation treatment cannot be carried out effectively due to the shortage of donor kidney. Futher more, the medical expenses for these patients are very expensive. Resently, the data from U.S. health organization showed that costs for patients with CKD and uremia accounted for 27.6% of the total health budget, although the number of them only accounted for 7% of the totle medical people. The cost of each ESRD patien is about $61800 per year (RMB 410000), and the cost of a CKD for stage 4 is about$12700 (RMB 80000). In our country, the cost for these treatments is lower than the United States, but it is also a heavy burden for our society.Given the grim situation of the treatment on renal failure at present, we need to find new effective treatments. An important characteristic of ESRD is the obvious reduction in residual functional-nephron, so the ideal treatment is to make the injured kidney to form new nephrons. Adult kidney regeneration has been confirmed in fishes, although this phenomenon is rarely observed in adult mammals. Interestingly, after acute injury, the adult kidney undergoes a quickly regenerative response to recover from renal functional failure by generating new cells to replace damaged ones either in human or in animal. In recent years more and more evidences existed for the potential regenerative capacity of human and animal adult kidneys. Thus, to develop ideal treatments for kidney diseases, a basic prerequisite is a clear understanding of which cells contribute to repair injured kidney.The cellular and molecular basis of adult acute renal repair is unclear at present. That cells responsible for kidney regeneration are terminally differentiated tubular cells or multipotent progenitor cells or adult renal stem cells is still controversial. The evidence that adult stem/progenitor cells contribute to the restoration of renal tubules after ischemic injury emerged form about the last ten years. In 2003, Maeshima et al. firstly demonstrated the existence of renal progenitor-like tubular cells and the participation of that cells in tubular regeneration by a 2-wk chase period of LRC technique. After this research, LRC technique improved by a long chase period to search for stem/progenitor cells in the adult mouse kidney. Using this technique, more studies have reported that renal stem/progenitor cells participate in the regeneration process of the ischemic injured kidney and are labeled in specific locations, such as renal papilla, S3 segment of proximal tubules, and the junction of cortex and outer medulla. But based on the technique of stem-cell specific surface markers such as CD133 and CD24, studies have shown that renal progenitor cells are located at urinary pole of the Bowman's capsule that contribute to regenerate podocytes and tubular cells. In addition, using a lineage-tracing technique, recent studies have been demonstrated that surviving tubular epithelial cells repair the kidney after injury without specialized progenitors. There is no consensus about the presence of renal stem/progenitor cells in adult kidney by different approaches.Therefore, further research is required to evaluate whether these label-retaining cells in kidney are in fact renal stem/progenitor cells and their roles in kidney regeneration in order to clarify the cellular basis of renal repair.Objectives1. to observe the potential regenerative capability of adult mouse kidneys after I/R injury2. to examine the distribution of long-term LRCs in adult mouse kidneys3. to investigate the real role of long-term LRCs in the recovery process of I/R injured mouse kidneys, and to identify whether these cells can be considered as the real adult renal stem cells/progenitor cells or notMethods1. Long-term BrdU labeled mouse model. To investigate the long-term LRCs distribution in mouse kidneys, postnatal mice were injected intraperitoneally with BrdU at dose of 50?g/g twice daily from 12 hours after birth for 3 days. Then labeled pups were allowed to grow without a medical operation until 8-week-old mice.2. Renal ischemia/reperfusion injury model.8-week-old BrdU-labeling male C57BL/6J mice were subjected to renal bilateral I/R injury. Briefly, mice were anesthetized with chloral hydrate (125 mg/kg, intraperitoneally), and kidneys were exposed through a midline incision. Then the renal artery and vein were isolated from surrounding tissues and kidneys were subjected to ischemia by clamping bilateral renal pedicles with non-traumatic mirovascular clamps for 22 min. In the sham group, the renal pedicles were isolated without using clamps. After removing of the clamps, verification of reperfusion, the incision were sutured in two layers and 1 ml of 37? saline was injected into the abdomen to supplement fluid loss. Mouse body temperature maintained at 37? using a warming pad before awake. Mice were sacrificed at 1d,2d,3d,4d,5d,6d,9d,14d,28d after I/R injury to collect blood and tissues samples.3. Assessment of renal function. Serum was isolated from the blood samples by centrifugation at 3000 rpm,5 minutes. Scr and BUN were measured at the clinical laboratory.4. Morphological studies. Renal tissues were fixed, embedded in paraffin and cut into 5 u m sections. For general histology, sections were stained with H&E. BrdU immunohistochemistry was performed to determine the LRCs staining. The quantification of BrdU-labeled cells was determined by counting the number of positive nuclei on 5 selected fields of sections in a blinded manner by a light microscope at X 200. The average number of the 5 fields was recorded as the number of BrdU-retaining nuclei per field.5. Double-color immunofluorescence. Renal tissues were embedded in OTC freezing medium.5 ?m sections were cut to detect the expression of BrdU and Ki-67, BrdU and TUNEL, BrdU and LAT by indirect immunofluorescence.6. Statistical analysis. Data are presented as means+SEM. Differences between groups were analyzed by one-way ANOVA using SPSS 16.0. P<0.05 was considered statistically significant.Results1. The restoration of adult renal tissues in mice after I/R injury1.1 The recovery of renal functionScr and BUN were measured as markers of renal function at 0 (baseline),1,2, 3,4,5,6,9,14,28 days after ischemia.Scr was significantly increased at the end of 1 day after injury. The peak value of Scr was 9-fold greater than the mean baseline value (Scr in umol/1,1 day after injury vs baseline:175.93±36.61 vs 19.53±5.03, p<0.05), then tended to a normal level within 9 days. The trend of BUN after I/R injury in mice was consistent with Scr.1.2 The recovery of renal morphologyTo verify and localize the renal injury and regeneration directly, we examined the histological changes of kidney tissues with ischemic AKI by H&E staining. Twenty-four hours after I/R injury, we observed the typical renal tubular damage such as severe tubular dilatation, loss of brush border, sloughed debris in tubular lumen space, and denuded basement membrane. These typical renal tubular damage lasted for 3 days in renal cortex, medulla and papilla. But on the fourth day after I/R injury, the newly generated tubular cells increased rapidly and formed a special niche-like structure for new cells. The renal structure tended to restore rapidly within the next 3-5 days.9 days after I/R injury, kidney tissues showed apparently normal histological architecture of renal cortex, medulla and papilla.2. The distribution of long-term LRCs in mouse kidney after I/R injury2.1 The location and morphology of long-term LRCs in adult mouse kidneyAfter a 8-wk chase period, we found that BrdU-retaining cells scattered among adult kidneys. Most of the BrdU-retaining cells located on the tubular cells, the few of them scattered on renal interstitium. Some of BrdU+LRCs located in pairs, nd the number of these BrdU+LRCs was 21.7 ± 1.5%. And we firstly observed that the nucleus of long-term BrdU+LRCs exhibited differently morphological characteristics in normal adult kidneys, and about 10.1±0.9%of their nuclei were fragmentation and dissolution.2.2 The number of long-term LRCs in mouse kidney after I/R injuryThe distribution of BrdU+LRCs faded out over time with renal functional recovery. Compared with the baseline (0 day after injury), on the end of 4th day the number of BrdU+LRCs in the cortex, medulla and papilla were dramatically decreased by 85±2%,81 ±5%and 95±5%, respectively. And when the structure of I/R injured kidney restored to normal within 1 month, LRCs were difficult to be detected3. Not all of long-term LRCs play a positive role in the regeneration process of I/R injured kidneysOn the 4th day after injury, a period of the renal recovery with much tubule proliferation and few cell death, we observed that not all of BrdU+LRCs were positive for Ki-67, which specifically recognizes cellular proliferation. Some of BrdU+LRCs were not located in the special niche-like structure formed by Ki-67 positive cells. In addition, we examined the expression of TUNEL(cell apoptosis maker) on BrdU+LRCs, and found that part of scattered BrdU-retaining cells co-expressed with TUNEL on the 4th day after injury.4. The phenotype of long-term LRCs in mouse kidneys after I/R injuryTo investigate the BrdU+LRCs cells are immature or dedifferentiated in the early period of I/R injury, we use the double-color immunofluorescence. We found that some isolated scattered BrdU+LRCs co-expressed LTA both in normal and in the first 3 days after injury. These results indicated a few LRCs were in a differentiated state in the early phase of renal injury. Interestingly, BrdU+LRCs located in pairs did not co-express LTA in the regeneration process of I/R injured mouse kidney.Conclusion1. Our results showed that adult kidneys had a spontaneous capability to restore from AKI in mice. And we found that the newly generated cells formed a special niche-like structure on the fourth day after I/R injury.2. Our study demonstrated that long-term LRCs scattered among adult kidneys. And we speculate that long-term LRCs which contribute to the regeneration process of renal I/R injury are those that located in pairs and in the special structure formed by new proliferating cells. Only this part of LRCs may reenter the cell cycle in response to acute injury in mouse kidneys.3. We firstly found that the nucleus of long-term BrdU+LRCs exhibited differently morphological characteristics in normal adult kidneys. And our results showed that LRCs were not one simple type of cells in adult kidneys, suggesting putative renal stem/progenitor cell niches located by the LRC technique in previous studies, such as renal papilla, need to be further revaluated.4. Our results showed some LRCs were in a differentiated state during the early damaged time, indicating these LRCs may be not the real stem-cell pool, and new regenerated cells may not derive from renal long-term LRCs completely.5. Our findings on the characteristics of renal LRCs may provide a rational explanation to the inconsistent results by different techniques in researches of which cells contribute to repair injured kidney. And if researchers search for real stem/progenitor cells in adult kidneys by the LRC technique, these complex phenomena of LRCs need to be considered. |
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