Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury

Acute renal failure (ARF) is a common disease with high morbidity and mortality. Recovery from ARF is dependent on the replacement of necrotic tubular cells with functional tubular epithelium. Recent advancement in developmental biology led to the discovery of immature mesenchymal stem cells (MSCs) in bone marrow and several established organs and to the definition of their potential in the recovery from tissue injury. We investigated the effect of MSCs infusion on the recovery from ARF induced by intramuscle injection of glycerol in C57/BL6 mice. In this model, ARF is associated with an extensive necrosis of tubular epithelial cells due to myoglobin- and hemoglobin-induced injury. MSCs were obtained from bone marrow of transgenic mice expressing green fluorescent protein (GFP). MSC GFP-positive cells (MSC-GFP(+)) injected intravenously homed to the kidney of mice with glycerol-induced ARF but not to the kidney of normal mice. MSC-GFP(+) localized in the context of the tubular epithelial lining and expressed cytokeratin, indicating that MSCs engrafted in the damaged kidney, differentiated into tubular epithelial cells and promoted the recovery of morphological and functional alterations. Moreover, MSCs enhanced tubular proliferation as detected by the increased number of proliferating cell nuclear antigen (PCNA) positive cells. A significant contribution of the engrafted MSCs in the regeneration of tubular epithelial cells was shown by the presence of a consistent number of GFP(+) tubular cells 21 days after the induction of injury. In conclusion, these results indicated a tropism of MSCs for the injured kidney and a potential contribution of these cells to tubular regeneration and to the recovery from ARF.     

Precipitable tissue proteins can cause experimental acute renal failure

A previous investigation demonstrated that intravenous infusion of a saline-liver extract into rats causes acute renal failure (ARF), manifested by severe azotemia, extensive cast formation, and patchy tubular necrosis. The purpose of the present study was to explore its pathogenesis. Histologic assessments of rat kidneys made 1.5 hours after liver extract infusion demonstrated eosinophilic material within glomerular capillaries, Bowman's space, and proximal tubular lumina, distal nephron cast formation, and tubular dilation without evidence of tubular necrosis. Renal blood flow at this time was normal but the rats were anuric. Assessments made 24 hours after liver extract infusion demonstrated persisting ARF (blood urea nitrogen, 132 +/- 8; creatinine, 2.54 +/- 0.19 mg/dl), profound cast deposition almost exclusively in the inner medulla/papilla, and the appearance of patchy proximal tubular necrosis. Sephacryl S200 fractionation and 10% polyacrylamide gel electrophoresis of liver extract showed high and low molecular weight proteins (less than 30,000). Proteins in both regions demonstrated prominent acid precipitability (pH 4.5) and autoaggregation (at 37 degrees C). Trace amounts of spontaneously precipitated protein recovered from urine during liver extract infusion demonstrated a predominance of low molecular weight proteins by polyacrylamide gel electrophoresis. Infusing rats with filterable low molecular weight proteins (cytochrome c, ribonuclease, myoglobin) without autoaggregation/acid precipitation characteristics or liver extract made devoid of precipitable proteins failed to induce ARF. However, infusing a kidney extract containing acid precipitating/autoaggregating proteins caused inner medullary/papillary cast formation and ARF. Conclusion: normal parenchymal tissues contain proteins which can undergo glomerular filtration and which can spontaneously aggregate under conditions which exist in the distal nephron. If released into the circulation, or if shed from tubular cells into lumina after nephrotoxic or ischemic renal injury, they could help to induce intratubular obstruction and ARF.     

Acute renal failure — An integrative discussion of morphologic and functional findings

Summary The ultrastructural alterations at the nephron established in animal experiments, were also confirmed, by means of an electron-microscopic examination, in eight cases of human acute renal failure (ARF). Special consideration was given in this study to single cell alterations, particularly in proximal tubular cells, with emphasis being placed on alterations due to single cell damage in the region of the renal fluid compartments. The ultrastructural alterations of the tubular cells in ARF, suggest serious impairment of the cellular capacity for electrolyte transport and metabolic processes. The shunt paths between the tubular fluid compartment and the functional interstitium, arising from necrosis of the tubular cells or dissolution of the gap or tight junctions, were discussed in terms of their significance for the directional, active transport processes of the tubular cells for sodium chloride and the passive water flow. The morphologic findings were reviewed in light of recent findings on cellular membrane processes and electrolyte transport. A reinterpretation of the morphologic and functional findings in ARF is suggested. This takes into consideration single cell function and the integrity of the renal fluid compartments.This study was supported by the Deutsche Forschungsgemeinschaft (Gi 117/2-2 and Bo 216/22-2)     

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated.     

The role of epithelial-to-mesenchymal transition in renal fibrosis

Epithelial-to-mesenchymal transition (EMT) involving injured epithelial cells plays an important role in the progression of fibrosis in the kidney. Tubular epithelial cells can acquire a mesenchymal phenotype, and enhanced migratory capacity enabling them to transit from the renal tubular microenvironment into the interstitial space and escape potential apoptotic cell death. EMT is a major contributor to the pathogenesis of renal fibrosis, as it leads to a substantial increase in the number of myofibroblasts, leading to tubular atrophy. However, recent findings suggest that EMT involving tubular epithelial cell is a reversible process, potentially determined by the surviving cells to facilitate the repopulation of injured tubules with new functional epithelia. Major regulators of renal epithelial cell plasticity in the kidney are two multifunctional growth factors, bone morphogenic protein-7 (BMP-7) and transforming growth factor 1 (TGF-1). While TGF-1 is a well-established inducer of EMT involving renal tubular epithelial cells, BMP-7 reverses EMT by directly counteracting TGF--induced Smad-dependent cell signaling in renal tubular epithelial cells. Such antagonism results in the repair of injured kidneys, suggesting that modulation of epithelial cell plasticity has therapeutic advantages.Abbreviations ALK Activin-like kinase - bFGF Basic fibroblast growth factor - BMP Bone morphogenic protein - ECM Extracellular matrix - EGF Epithelial growth factor - EMT Epithelial-to-mesenchymal transition - FSP1 Fibroblast specific protein 1 - IL-1 Interleukin 1 - LAP Latency-associated polypeptide - MET Mesenchymal-to-epithelial transition - MMP Matrix metalloproteinase - TBM Tubular basement membrane - TGF Transforming growth factor     

樟柳碱、东莨菪碱、山莨菪碱对急性肾衰大鼠肾微血管损伤的保护作用

目的 :研究急性肾衰时肾微血管的损伤及樟柳碱、东莨菪碱、山菪莨碱对这些损伤的影响。方法 :采用左肾动脉注射油酸的方法建立大鼠急性肾衰模型 ,采用电镜、光镜及TTC显色观察大鼠急性肾衰不同时期肾微血管超微结构、肾组织结构及TTC显色的改变。结果 :左肾动脉注射油酸后 1 0min ,肾小球毛细血管内皮细胞坏死 ;肾小球毛细血管不同程度充血肿胀 ,在皮质与髓质交界区 ,可见小血管内充满大量红细胞 ;TTC显色呈鲜红色。注射油酸后 6h和 2 4h上述损伤加重 ,TTC显然出现典型的不显色苍白区。三种莨菪药物不同程度减轻了急性肾衰时肾微血管损伤 ,改善了肾组织缺血 ,减少了肾小管上皮细胞坏死数。结论 :急性肾衰时很早即出现肾微血管的损伤 ,三种莨菪药对急性肾衰时肾微血管和组织结构的损伤均有保护作用。     

Tubular ultrastructure in acute renal failure in man: Epithelial necrosis and regeneration

Summary It is not clear whether tubular cell necrosis is present or not in acute renal failure (ARF) of ischaemic type (acute tubular necrosis). In order to get quantitative data, using precisely defined criteria for tubular cell necrosis, 25 renal biopsies from 24 patients with ARF (11 obtained in the active phase, 14 in the early recovery period) were compared with 12 control biopsies. In all 1959 proximal cells and 1603 distal cells were analysed by electron microscopy. Cellular disintegration was very rare in all groups. Shrinkage necrosis (apoptosis) was not present in the proximal tubules of the controls and was rare in ARF (1.6–2.1%). In the distal tubules of controls 2.7% of all cells showed shrinkage necrosis. The incidence in ARF was not significantly increased. Non-replacement sites in distal tubules (probablyloci where cells have recently been desquamated) were significantly increased in number (5.2%) in the active phase in ARF compared to controls and recovery. The relative number of regenerating cells was not increased.These data show that there is no widespread necrosis of tubular cells in ARF. The increased incidence in distal tubules of focal, denuded areas of the basement membrane in the active phase of ARF indicates a slightly increased desquamation of cells and/or a failure to cover such sites by adjacent cells. This process is not restricted to the brief induction phase of ARF but continues during the whole active phase.     

Notch in the kidney: development and disease

Notch signalling is a highly conserved cell-cell communication mechanism that regulates development, tissue homeostasis, and repair. Within the kidney, Notch has an important function in orchestrating kidney development. Recent studies indicate that Notch plays a key role in establishing proximal epithelial fate during nephron segmentation as well as the differentiation of principal cells in the renal collecting system. Notch signalling is markedly reduced in the adult kidney; however, increased Notch signalling has been noted in both acute and chronic kidney injury. Increased glomerular epithelial Notch signalling has been associated with albuminuria and glomerulosclerosis, while tubular epithelial Notch activation caused fibrosis development most likely inducing an improper epithelial repair pathway. Recent studies thereby indicate that Notch is a key regulator of kidney development, repair, and injury.     

Failure of BCL-2 up-regulation in proximal tubular epithelial cells of donor kidney biopsy specimens is associated with apoptosis and delayed graft function

SUMMARY: In renal transplantation, postischemic acute renal failure (ARF) develops in more than 20% of patients. We investigated whether tubular epithelial cells obtained from donor kidneys without subsequent ARF express a different pattern of survival genes, compared with cells from kidneys exhibiting ARF. Donor kidney biopsy specimens were obtained before transplantation from eight recipients of cadaveric kidneys with primary graft function (CAD-PF), eight patients with biopsy-proven ARF without rejection (CAD-ARF), and eight recipients of living donor kidneys with primary graft function (LIV). One thousand proximal tubular epithelial cells per biopsy specimen were isolated by laser capture microdissection. Quantitative analysis of apoptosis and the apoptosis regulatory genes Bcl-2, Bcl-xL, and Bax were performed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling staining and real-time PCR, respectively. Primary cultures of human proximal tubular epithelial cells served as calibrator. The number of apoptotic cells was significantly higher in CAD-ARF compared with LIV and CAD-PF (1.5 +/- 1.1% [p < 0.05] vs. 0.3 +/- 0.2% vs. 0.4 +/- 0.2%; mean +/- SD). The apoptosis inhibitors Bcl-2 and Bcl-xL were significantly up-regulated in renal tubular cells of recipients without ARF compared with CAD-ARF. The ratios of Bcl-2/GAPDH normalized to calibrator were as follows: LIV 48 +/- 30, CAD-PF 38 +/- 55, and CAD-ARF 5 +/- 7 (p < 0.05). The corresponding ratios for Bcl-xL were as follows: LIV 6 +/- 6, CAD-PF 5 +/- 3, and CAD-ARF 1 +/- 1 (p < 0.05). No difference in the expression of the proapoptotic Bax could be observed. These data suggest that failure of proximal tubular cells to respond to injury by up-regulation of survival factors from the Bcl-2 family contributes to postischemic ARF in patients after cadaveric renal transplantation.     

Renal regeneration following d-serine induced acute tubular necrosis.

Regeneration of the ray kidney was observed for six days after inducing acute tubular necrosis of the proximal pars recta with d-serine (80 mg/100 g body weight. Regenerating cells appear by two days post-treatment, and re-epithelialization of the nephron is completed within six days, with the most mature cells approaching normal morphology. Regeneration originates from viable cells adjacent to the necrotic zone which divide and follow a template provided by the intact basement membrane. Transient, cytoplasmic regenerative activity among developing tubular cells is characterized by the presence of large, irregularly shaped nuclei, prominent nucleoli, abundant ribosomes and lysosomes, and abnormal mitochondrial configurations. Microfilaments appear to be involved in the formation of apical microvilli and the basal labyrinth of plasmalemmal convolutions. These data suggest that d-serine induced acute tubular necrosis of the proximal pars recta may be followed by rapid, patterned regeneration along an intact basement membrane, and that microfilaments are involved in differentiation of cellular morphology.     

Renal regeneration following D-serine induced acute tubular necrosis

Regeneration of the rat kidney was observed for six days after inducing acute tubular necrosis of the proximal pars recta with d-serine (80 mg/100g body weight). Regenerating cells appear by two days post-treatment, and re-epithelialization of the nephron is completed within six days, with the most mature cells approaching normal morphology. Regeneration originates from viable cells adjacent to the necrotic zone which divide and follow a template provided by the intact basement membrane. Transient, cytoplasmic regenerative activity among developing tubular cells is characterized by the presence of large, irregularly shaped nuclei, prominent nucleoli, abundant ribosomes and lysosomes, and abnormal mitochondrial configurations. Microfilaments appear to be involved in the formation of apical microvilli and the basal labyrinth of plasmalemmal convolutions. These data suggest that d-serine. inducedacute tubular necrosis of the proximal pars recta may be followed by rapid, patterned regeneration along an intact basement membrane, and that microfilaments are involved in differentiation of cellular morphology.     

Human amniotic fluid stem cell preconditioning improves their regenerative potential

Human amniotic fluid stem (hAFS) cells, a novel class of broadly multipotent stem cells that share characteristics of both embryonic and adult stem cells, have been regarded as promising candidate for cell therapy. Taking advantage by the well-established murine model of acute kidney injury (AKI), we studied the proregenerative effect of hAFS cells in immunodeficient mice injected with the nephrotoxic drug cisplatin. Infusion of hAFS cells in cisplatin mice improved renal function and limited tubular damage, although not to control level, and prolonged animal survival. Human AFS cells engrafted injured kidney predominantly in peritubular region without acquiring tubular epithelial markers. Human AFS cells exerted antiapoptotic effect, activated Akt, and stimulated proliferation of tubular cells possibly via local release of factors, including interleukin-6, vascular endothelial growth factor, and stromal cell-derived factor-1, which we documented in vitro to be produced by hAFS cells. The therapeutic potential of hAFS cells was enhanced by cell pretreatment with glial cell line-derived neurotrophic factor (GDNF), which markedly ameliorated renal function and tubular injury by increasing stem cell homing to the tubulointerstitial compartment. By in vitro studies, GDNF increased hAFS cell production of growth factors, motility, and expression of receptors involved in cell homing and survival. These findings indicate that hAFS cells can promote functional recovery and contribute to renal regeneration in AKI mice via local production of mitogenic and prosurvival factors. The effects of hAFS cells can be remarkably enhanced by GDNF preconditioning.     

The progression of renal diseases: On the pathogenesis of renal interstitial fibrosis

Summary Renal interstitial fibrosis (RIF) frequently occurs in inflammatory and non-inflammatory kidney diseases and is associated with a decline in renal excretory function. Fibroblasts which occupy the renal interstitium are involved mainly in the formation of RIF not only by the production of extracellular matrix, but also by regulatory processes. They respond to a variety of cytokines released by different cell types. To investigate mechanisms leading to RIF, immunohistochemical analysis and cell cultures of renal biopsies in various renal diseases have been performed. T lymphocytes are the major cells infiltrating the renal interstitium, and their number correlates with the impairment of renal function. In most forms of glomerulonephritis accompanied by interstitial inflammation, an abnormal expression of HLA-DQ/-DP molecules, frequently associated with an aberrant expression of the intercellular adhesion molecule 1 (ICAM-1), was observed on proximal tubular epithelial cells, indicating that these cells may play a role in antigen presentation. The cell biological experiments revealed the presence of the three mitotic fibroblast types (MFI-MFIII) and the three postmitotic types (PMFIV-PMFVI) in the cell culture. The number of fibroblasts in primary and passage-1 culture was increased sevenfold in cultures derived from kidneys with RIF (FKIF cells) in comparison to normal kidneys (NKF cells). FKIF cells show hyperproliferative growth and synthesize an increased amount of total collagen, especially types III and V. These cells express a protein, named FIBROSIN, which seems to be specific for FKIF cells. Further extended cell biological analyses are currently being performed to investigate interactions of tubular cells, lymphocytes, macrophages, and fibroblasts in order to shed more light on the pathomechanisms involved in fibrogenesis leading to renal interstitial fibrosis.Abbreviations AG antigen - DMEM Dulbecco's modified Eagle's medium - ECM extracellular matrix - FACS fluorescence activated cell sorter - FGF fibroblast growth factor - FKIF fibroblast derived from kidneys with interstitial fibrosis - FSGS focal segmental glomerulosclerosis - GBM glomerular basement membrane - GM-CSF granulocyte/macrophage-colony stimulating factor - GN glomerulonephritis - HLA human leukocyte antigen - ICAM-1 intercellular adhesion molecule 1 - ICI interstitial cellular infiltrate - IgA N immunoglobulin A nephropathy - IL interleukin - INF- interferon gamma - LFA leukocyte function antigen - M-CSF macrophage-colony stimulating factor - MF mitotic fibroblast - MGN membranous glomerulonephritis - MesPrGN mesangioproliferative glomerulonephritis - MHC major histocompatibility complex - Min Ch minimal changes - Min Les minor lesions - MMC mitomycin C - MPGN membranoproliferative glomerulonephritis - NKF normal kidney fibroblast - NSF normal skin fibroblast - PDGF platelet-derived growth factor - PMF postmitotic fibroblast - PTEC proximal tubular epithelial cells - TGF transforming growth factor - TNF tumor necrosis factor - RIF renal interstitial fibrosis - RPGN rapidly progressive glomerulonephritis - TBM tubular basement membrane - TCR T-cell receptor Preprint of a lecture to be read at the 22nd Congress of the Gesellschaft für Nephrologie, Heidelberg, September 15–18, 1991 (Editor: Prof. Dr. E. Ritz, Heidelberg)Supported by the Deutsche Forschungsgemeinschaft DFG Mu 523/3-5, SFB 223, Project B5/Ro 527/2-2 and by the Alexander von Humboldt-Stiftung     

Adult stem cell-like tubular cells reside in the corticomedullary junction of the kidney

The existence of adult renal stem cells has long been suspected because the kidney is capable of regeneration in response to injury, such as acute tubular necrosis (ATN), but their location, or niche, has not been fully defined yet. The aim of this study was to identify the niche of adult renal stem cells responsible for the tubular regeneration. The location of label-retaining cells (LRCs) was studied in adult mouse kidneys after administration of a pulse of bromodeoxyuridine (BrdU) during embryonic period. To study regional participation in renal tubular regeneration, the expression of the proliferation marker Ki-67 was examined after induction of unilateral ATN in mouse kidneys. Regional colony-forming capacity was examined using cultured cells derived from normal mouse and human kidneys and their multipotency was examined in human kidneys. LRCs in adult mouse kidneys were mostly tubular epithelial cells and concentrated constantly in the outer stripe of the corticomedullary junction (CMJ). In the ATN model, Ki-67 positive cells were concentrated in the tubular epithelial cells of the outer stripe, not only in the ATN kidneys but also in the contralateral non-ATN kidneys. High colony-forming capacity was noted in the CMJ of mouse and human kidneys. Cultured cells derived from a single human CMJ cell revealed multipotency, differentiating not only into tubular cells but also into glomerular podocytes. These results demonstrate that the CMJ of the kidney contains label-retaining, renal-repairing, highly colony-forming multipotent stem cell-like tubular cells, suggesting the CMJ as the niche of adult renal stem cells.     

Molecular mechanisms of acute renal failure following ischemia/reperfusion

Acute renal failure (ARF) necessitating renal replacement therapy is a common problem associated with high morbidity and mortality in the critically ill. Hypotension, followed by resuscitation, is the most common etiologic factor, mimicked by ischemia/reperfusion (I/R) in animal models. Although knowledge of the pathophysiology of ARF in the course of this condition is increasingly detailed, the intracellular and molecular mechanisms leading to ARF are still incompletely understood. This review aims at describing the role of cellular events and signals, including collapse of the cytoskeleton, mitochondrial and nuclear changes, in mediating cell dysfunction, programmed cell death (apoptosis), necrosis and others. Insight into the molecular pathways in the various elements of the kidney, such as vascular endothelium and smooth muscle and tubular epithelium leading to cell damage upon I/R will, hopefully, open new therapeutic modalities, to mitigate the development of ARF after hypotensive episodes and to promote repair and resumption of renal function once ARF has developed.     

Cellular events in experimental unilateral ischemic renal atrophy and in regeneration after contralateral nephrectomy

Two related morphological studies were undertaken in rats. In the first, cellular events involved in the development of ischemic renal atrophy induced by renal artery stenosis were recorded. One primary objective was to document the pathogenetic role that a distinct form of cell death, termed apoptosis, played in the development of renal tubular atrophy. A small, partially closed ligating clip was used to produce stenosis of the left renal artery, or a sham operation was performed. Animals were killed 2-28 days after operation. The ensuing ischemic renal atrophy was studied histologically and ultrastructurally, and apoptosis was counted in paraffin sections, using established criteria for its identification. Nuclear [3H]thymidine uptake was used as an indicator of cell proliferation. Morphometric studies recorded changes in area of transected tubular profiles. Correlation was sought between the morphological changes, data gained by the above quantitations, and the progressive reduction in renal mass that occurred during the experiment. Our results showed that during the acute phase (2-8 days), cell death was effected by both apoptosis and necrosis and increased tubular epithelial cell labeling and mitoses provided evidence of epithelial repair. During the chronic phase (10-28 days), when the mass of the ischemic kidney underwent significant reduction, cell death was effected by apoptosis alone, and the level of tubular epithelial cell labeling and mitosis returned to near normal. Intraepithelial macrophages were significant in removing the apoptotic bodies. Area of tubular epithelium was reduced in atrophic tubules, and it is proposed that this reduction may be explained by apoptotic cell deletion, as well as cell shrinkage. In the second study, evidence of regeneration was sought in atrophic kidneys after surgical reversal of renal artery stenosis and, in other animals, after unilateral nephrectomy of the contralateral kidney. Our results showed that regeneration, involving both hypertrophy and hyperplasia, was stimulated only by removal of the hypertrophied contralateral kidney and occurred whether or not stenosis of the renal artery was reversed.     

Plasma protein extravasation and vascular endothelial growth factor expression with endothelial nitric oxide synthase induction in gentamicin-induced acute renal failure in rats

Microvascular hyperpermeability to plasma proteins via vascular endothelial growth factor (VEGF) with endothelial nitric oxide synthase (eNOS) induction may contribute to wound healing through matrix remodeling. However, vascular hyperpermeability is not examined in acute renal failure (ARF), a unique form of wound healing. Subcutaneous injection of gentamicin (400 mg/kg per day for 2 days in divided doses every 8 h) in rats increased serum creatinine levels and induced tubular damage, which peaked at day 6, after the last gentamicin injection. Ki67-positive regenerating proximal tubules (PTs) peaked in number at day 6 and almost covered the bare tubular basement membrane (TBM) by day 10. Staining of fibrinogen and plasma fibronectin began to increase in the peritubular regions as early as day 0, steadily increased in TBM and tubular lumen until day 6 and then decreased. Hyperpermeable peritubular capillaries were identified by extravasation of perfused-fluoresceinated dextran (both 70 kDa and 250 kDa) into peritubular regions as early as day 0 and prominently into TBM and tubular lumen at day 6. Electron microscopy further suggested the intraendothelial pathway of dextran. Immunoreactive VEGF increased in the damaged and regenerating PTs. Immunoreactive VEGF receptors-1 and -2 did not change, but immunoreactive eNOS increased in the peritubular capillaries after induction of ARF. Western blotting for VEGF and eNOS supported the immunostaining findings. In addition, we assessed the effects of NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) on vascular hyperpermeability during the recovery phase of this model. Treatment with l-NAME (s.c. at a dose of 100 mg/kg/day from day 3 to day 6) decreased extravasation of perfused-250-kDa dextran and significantly inhibited the regenerative repair of PTs at day 6 when compared with vehicle-treated rats. In conclusion, plasma protein extravasation occurred, leading to matrix remodeling, such as the process of wound healing during the tubular repair in gentamicin-induced ARF. Since VEGF-induced vascular hyperpermeability may depend on NO production, VEGF/VEGF receptor system with eNOS induction might be responsible for this process.     

Expression of silencer of death domains and death-receptor-3 in normal human kidney and in rejecting renal transplants

We have previously reported the pattern of cellular expression of tumor necrosis factor receptors (TNFR) in human kidney and their altered expression in transplant rejection. We have extended our studies to examine the expression of Silencer of Death Domains (SODD), a protein that binds to the cytoplasmic portion of TNFR1 to inhibit signaling in the absence of ligand. In normal human kidney SODD is expressed in glomerular endothelial cells where it colocalizes with TNFR1. During acute rejection both SODD and TNFR1 are lost from glomeruli, but we found strong expression of SODD on the luminal surface of tubular epithelial cells. This occurs in the absence of detectable TNFR1 expression, suggesting that SODD could interact with other proteins at these sites. Several other members of the TNF superfamily, including Fas and death receptors (DR)-3, -4, and -5, also contain intracellular death domains, but SODD only interacts with the death domain of DR3. We therefore studied the expression of DR3 in human kidney, and report that this death receptor is up-regulated in renal tubular epithelial cells and endothelial cells of some interlobular arteries, in parallel with SODD, during acute transplant rejection. In less severe rejection episodes, DR3 and SODD were more focally induced, generally at sites of mononuclear cell infiltrates. In ischemic allografts, eg, with acute tubular necrosis but no cellular rejection, DR3 was induced on tubular epithelial cells and on glomerular endothelial cells. These data confirm that TNF receptor family members are expressed in a regulated manner during renal transplant rejection, and identify DR3 as a potential inducible mediator of tubular inflammation and injury.     

黄芪甲苷孵育脂肪源性干细胞对顺铂诱导肾小管上皮细胞凋亡的影响

背景：干细胞移植用于治疗急性肾损伤的有效性已经被多个研究证实,但其对肾小管上皮细胞损伤的修复机制尚不明确。 目的：观察黄芪甲苷孵育后的脂肪源性干细胞对顺铂诱导的肾小管上皮细胞凋亡的保护作用及机制。 方法：实验分为4组。2.5 μmol/L顺铂诱导肾小管上皮细胞 24 h,建立肾小管细胞损伤模型(顺铂损伤组);将脂肪源性干细胞与损伤肾小管上皮细胞共培养(脂肪源性干细胞+损伤肾小管上皮细胞组);利用Transwell小室将20 mg/L黄芪甲苷孵育脂肪源性干细胞48 h后与损伤肾小管上皮细胞共培养(黄芪甲苷孵育脂肪源性干细胞+损伤肾小管上皮细胞组);以正常肾小管上皮细胞做对照(正常对照组)。 结果与结论：与肾小管上皮细胞损伤组相比,AV/PI和TUNEL结果均显示脂肪源性干细胞+肾小管上皮细胞组和20 mg/L 黄芪甲苷脂肪源性干细胞+肾小管上皮细胞组肾小管上皮细胞发生凋亡的比例和数量明显减少;ELISA结果表明20 mg/L黄芪甲苷脂肪源性干细胞+肾小管上皮细胞组胰岛素样生长因子1分泌显著提高(P < 0.05);Western blot进一步显示20 mg/L 黄芪甲苷脂肪源性干细胞+肾小管上皮细胞组caspase-3蛋白水平明显下降,而Bcl-2的表达量明显增加(P < 0.05)。表明黄芪甲苷孵育的人脂肪源性干细胞对顺铂诱导的肾小管上皮细胞凋亡具有抑制作用,从而有利于肾小管损伤的早期恢复,其保护机制可能与增加胰岛素样生长因子1分泌,抑制caspase-3表达、上调Bcl-2水平有关。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Bone marrow mesenchymal stem cells ameliorate rat acute renal failure by differentiation into renal tubular epithelial-like cells

In the present study, we investigated the therapeutic potential of mesenchymal stem cells (MSCs) in a rat acute renal failure (ARF) model and explored the possible in vivo and in vitro mechanisms of action. Rat and human MSCs were isolated from bone marrow. After being co-cultured with injured kidney tissues in trans-well dishes in vitro, the rat MSCs became rounded renal tubular epithelial-like cells, and highly expressed renal markers such as cytokeratin 18 (CK18) and aquaporin-1 (AQP1). Human MSCs were infused into rats with ARF, and techniques of microscopy, histology, PCR, RT-PCR and fluorescence in situ hybridization were used to characterize the MSCs after transplantation. We found that there were more exogenous human MSCs localized to injured kidney tissues. The kidney recovery rate in the transplanted MSC group was higher than in the control group. Genes associated with human renal tubular epithelial cells such as AQP1 and parathyroid hormone receptor 1 were detected. These findings suggest that the injured kidney tissue induced rat and human MSCs to differentiate into renal tubular epithelial-like cells in vitro and in vivo, and exogenous human MSCs can home specifically to injured regions and efficiently cure rat ARF. These results demonstrate that cell therapy has potential as a novel intervention in ARF.