Renal Impairment with Sublethal Tubular Cell Injury in a Chronic Liver Disease Mouse Model

The pathogenesis of renal impairment in chronic liver diseases (CLDs) has been primarily studied in the advanced stages of hepatic injury. Meanwhile, the pathology of renal impairment in the early phase of CLDs is poorly understood, and animal models to elucidate its mechanisms are needed. Thus, we investigated whether an existing mouse model of CLD induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) shows renal impairment in the early phase. Renal injury markers, renal histology (including immunohistochemistry for tubular injury markers and transmission electron microscopy), autophagy, and oxidative stress were studied longitudinally in DDC- and standard diet–fed BALB/c mice. Slight but significant renal dysfunction was evident in DDC-fed mice from the early phase. Meanwhile, histological examinations of the kidneys with routine light microscopy did not show definitive morphological findings, and electron microscopic analyses were required to detect limited injuries such as loss of brush border microvilli and mitochondrial deformities. Limited injuries have been recently designated as sublethal tubular cell injury. As humans with renal impairment, either with or without CLD, often show almost normal tubules, sublethal injury has been of particular interest. In this study, the injuries were associated with mitochondrial aberrations and oxidative stress, a possible mechanism for sublethal injury. Intriguingly, two defense mechanisms were associated with this injury that prevent it from progressing to apparent cell death: autophagy and single-cell extrusion with regeneration. Furthermore, the renal impairment of this model progressed to chronic kidney disease with interstitial fibrosis after long-term DDC feeding. These findings indicated that DDC induces renal impairment with sublethal tubular cell injury from the early phase, leading to chronic kidney disease. Importantly, this CLD mouse model could be useful for studying the pathophysiological mechanisms of sublethal tubular cell injury.     

Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.     

NLRP3 Inflammasome Mediates Albumin-induced Renal Tubular Injury through Impaired Mitochondrial Function

Proteinuria serves as a direct causative factor of renal tubular cell injury and is highly associated with the progression of chronic kidney disease via uncertain mechanisms. Recently, evidence demonstrated that both NLRP3 inflammasome and mitochondria are involved in the chronic kidney disease progression. The present study was undertaken to examine the role of NLRP3 inflammasome/mitochondria axis in albumin-induced renal tubular injury. In patients with proteinuria, NLRP3 was significantly up-regulated in tubular epithelial cells and was positively correlated with the severity of proteinuria. In agreement with these results, albumin remarkably activated NLRP3 inflammasome in both in vitro renal tubular cells and in vivo kidneys in parallel with significant epithelial cell phenotypic alteration and cell apoptosis. Genetic disruption of NLRP3 inflammasome remarkably attenuated albumin-induced cell apoptosis and phenotypic changes under both in vitro and in vivo conditions. In addition, albumin treatment resulted in a significant mitochondrial abnormality as evidenced by the impaired function and morphology, which was markedly reversed by invalidation of NLRP3/caspase-1 signaling pathway. Interestingly, protection of mitochondria function by Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) or cyclosporin A (CsA) robustly attenuated albumin-induced injury in mouse proximal tubular cells. Collectively, these findings demonstrated a pathogenic role of NLRP3 inflammasome/caspase-1/mitochondria axis in mediating albumin-induced renal tubular injury. The discovery of this novel axis provides some potential targets for the treatment of proteinuria-associated renal injury.     

A novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in rat vascular smooth muscle and renal proximal tubular cells

Angiotensin II stimulates cellular hypertrophy in cultured vascular smooth muscle and renal proximal tubular cells. This effect is believed to be one of earliest morphological changes of heart and renal failure. However, the precise molecular mechanism involved in angiotensin II-induced hypertrophy is poorly understood. In the present study we report the isolation of a novel angiotensin II type 1 receptor-associated protein. It encodes a 531-amino acid protein. Its mRNA is detected in all human tissues examined but highly expressed in the human kidney, pancreas, heart, and human embryonic kidney cells as well as rat vascular smooth muscle and renal proximal tubular cells. Protein synthesis and relative cell size analyzed by flow cytometry studies indicate that overexpression of the novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in cultured rat vascular smooth muscle and renal proximal tubular cells. In contrast, the hypertrophic effects was reversed in renal proximal tubular cell lines expressing the novel gene in the antisense orientation and its dominant negative mutant, which lacks the last 101 amino acids in its carboxyl-terminal tail. The hypertrophic effects are at least in part mediated via protein kinase B activation or cyclin-dependent kinase inhibitor, p27(kip1) protein expression level in vascular smooth muscle, and renal proximal tubular cells. Moreover, angiotensin II could not stimulate cellular hypertrophy in renal proximal tubular cells expressing the novel gene in the antisense orientation and its mutant. These findings may provide new molecular mechanisms to understand hypertrophic agents such as angiotensin II-induced cellular hypertrophy.     

Heat shock prevents simulated ischemia-induced apoptosis in renal tubular cells via a PKC-dependent mechanism

Heat shock produces cellular tolerance to a variety of adverse conditions; however, the protective effect of heat shock on renal cell ischemic injury remains unclear. Protein kinase C (PKC) has been implicated in the signaling mechanisms of acute preconditioning, yet it remains unknown whether PKC mediates heat shock-induced delayed preconditioning in renal cells. To study this, renal tubular cells (LLC-PK1) were exposed to thermal stress (43 degrees C) for 1 h and heat shock protein (HSP) 72 induction was confirmed by Western blot analysis. Cells were subjected to simulated ischemia 24 h after thermal stress, and the effect of heat shock (delayed preconditioning) on ischemia-induced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) and B cell lymphoma 2 (Bcl(2)) expression (Western) was determined. Subsequently, the effect of PKC inhibition on HSP72 induction and heat stress-induced ischemic tolerance was evaluated. Thermal stress induced HSP72 production, increased Bcl(2) expression, and prevented simulated ischemia-induced renal tubular cell apoptosis. PKC inhibition abolished thermal induction of HSP72 and prevented heat stress-induced ischemic tolerance. These data demonstrate that thermal stress protects renal tubular cells from simulated ischemia-induced apoptosis through a PKC-dependent mechanism.     

Renal tubular acidosis: developments in our understanding of the molecular basis

Renal tubular acidosis is a metabolic acidosis due to impaired acid excretion by the kidney. Hyperchloraemic acidosis with a normal anion gap and normal (or near normal) glomerular filtration rate, and in the absence of diarrhoea, defines this disorder. However, systemic acidosis is not always evident and renal tubular acidosis can present with hypokalaemia, medullary nephrocalcinosis and recurrent calcium phosphate stone disease, as well as growth retardation and rickets in children, or short stature and osteomalacia in adults. Renal dysfunction in renal tubular acidosis is not always confined to acid excretion and can be part of a more generalised renal tubule defect, as in the renal Fanconi syndrome. Isolated renal tubular acidosis is more usually acquired, due to drugs, autoimmune disease, post-obstructive uropathy or any cause of medullary nephrocalcinosis. Less commonly, it is inherited and may be associated with deafness, osteopetrosis or ocular abnormalities. The clinical classification of renal tubular acidosis has been correlated with our current physiological model of how the nephron excretes acid, and this has facilitated genetic studies that have identified mutations in several genes encoding acid and base ion transporters. In vitro functional studies of these mutant proteins in cell expression systems have helped to elucidate the molecular mechanisms underlying renal tubular acidosis, which ultimately may lead to new therapeutic options in what is still treatment only by giving an oral alkali.     

Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

Kidney is one of the most important organs in maintaining the normal life activities. With the high abundance of mitochondria, renal tubular cell plays the vital role in functioning in the reabsorption and secretion of kidney. Reports have shown that mitochondrial dysfunction is of great importance to renal tubular cell senescence and subsequent kidney ageing. However, the underlying mechanisms are not elucidated. Cannabinoid receptor 2 is one of the two receptors responsible for the activation of endocannabinoid system. CB2 is primarily upregulated in renal tubular cells in chronic kidney diseases and mediates fibrogenesis. However, the role of CB2 in tubular mitochondrial dysfunction and kidney ageing has not been clarified. In this study, we found that CB2 was upregulated in kidneys in 24-month-old mice and d -galactose (d -gal)-induced accelerated ageing mice, accompanied by the decrease in mitochondrial mass. Furthermore, gene deletion of CB2 in d -gal-treated mice could greatly inhibit the activation of β-catenin signalling and restore the mitochondrial integrity and Adenosine triphosphate (ATP) production. In CB2 knockout mice, renal tubular cell senescence and kidney fibrosis were also significantly inhibited. CB2 overexpression or activation by the agonist AM1241 could sufficiently induce the decrease in PGC-1α and a variety of mitochondria-related proteins and trigger cellular senescence in cultured human renal proximal tubular cells. CB2-activated mitochondrial dysfunction and cellular senescence could be blocked by ICG-001, a blocker for β-catenin signalling. These results show CB2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing. The intrinsic mechanism may be related to its activation in β-catenin signalling.     

Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury

Several studies demonstrated that treatment with mesenchymal stem cells (MSCs) reduces cisplatin mortality in mice. Microvesicles (MVs) released from MSCs were previously shown to favor renal repair in non lethal toxic and ischemic acute renal injury (AKI). In the present study we investigated the effects of MSC-derived MVs in SCID mice survival in lethal cisplatin-induced AKI. Moreover, we evaluated in vitro the effect of MVs on cisplatin-induced apoptosis of human renal tubular epithelial cells and the molecular mechanisms involved. Two different regimens of MV injection were used. The single administration of MVs ameliorated renal function and morphology, and improved survival but did not prevent chronic tubular injury and persistent increase in BUN and creatinine. Multiple injections of MVs further decreased mortality and at day 21 surviving mice showed normal histology and renal function. The mechanism of protection was mainly ascribed to an anti-apoptotic effect of MVs. In vitro studies demonstrated that MVs up-regulated in cisplatin-treated human tubular epithelial cells anti-apoptotic genes, such as Bcl-xL, Bcl2 and BIRC8 and down-regulated genes that have a central role in the execution-phase of cell apoptosis such as Casp1, Casp8 and LTA. In conclusion, MVs released from MSCs were found to exert a pro-survival effect on renal cells in vitro and in vivo, suggesting that MVs may contribute to renal protection conferred by MSCs.     

高糖背景下白蛋白抑制肾小管上皮细胞的自噬表达

目的:探讨高糖背景下白蛋白造成肾小管间质损伤的作用及其机制。方法:体外培养大鼠近端肾小管上皮细胞系NRK-52E细胞,观察高糖培养环境下细胞自噬表达的改变;同时观察低浓度牛血清白蛋白(BSA)单独刺激,对肾小管上皮细胞自噬蛋白表达的影响以及细胞凋亡蛋白的表达改变;接着在高糖培养环境下加入低浓度的白蛋白刺激,观察肾小管上皮细胞的损伤效应及自噬表达情况。结果:高糖培养条件下肾小管上皮细胞自噬蛋白Beclin-1表达增加(P0.05),低浓度白蛋白也诱导肾小管上皮细胞自噬蛋白Beclin-1、Atg12表达增加(P0.05),以及细胞凋亡蛋白cleaved caspase3的轻度增加,乳酸脱氢酶活性增加(P0.05);但高糖培养下,少量白蛋白却抑制了肾小管上皮细胞自噬蛋白Beclin-1、Atg12的表达,并且显著增加了肾小管上皮细胞的凋亡蛋白cleaved caspase3的表达(P0.05)。结论:自噬是细胞自身的一种保护机制。在高糖背景下,白蛋白通过影响自噬的自身调节机制,促进了肾小管间质的损害作用。     

Kidney Specific Protein-Positive Cells Derived from Embryonic Stem Cells Reproduce Tubular Structures In Vitro and Differentiate into Renal Tubular Cells

Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.     

Reactive oxygen species-mediated endoplasmic reticulum stress contributes to aldosterone-induced apoptosis in tubular epithelial cells

Apoptosis contributes to tubular epithelial cell death and atrophy in aldosterone (Aldo)-induced renal injury. This study aimed to determine mechanisms underlying Aldo-induced reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress in tubular epithelial cells. Intracellular ROS generation was evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. Apoptosis was detected by annexin V/propidium iodide staining and flow cytometry. ER stress induced protein and mRNA were evaluated by Western blot and real-time PCR, respectively. Aldo promoted tubular epithelial cell apoptosis, increased intracellular ROS production and induced ER stress, as evidenced by increased expression of glucose-regulated protein 78 (GRP78) and CAAT/enhancer-binding protein homologous protein (CHOP) in a dose- and time-dependent manner. Additionally, siRNA knockdown of CHOP and antioxidant N-acetyl-l-cysteine (NAC) attenuated ER stress-mediated apoptosis. NAC also could inhibit Aldo-induced expression of GRP78 and CHOP. Altogether, these observations suggest that Aldo induces apoptosis via ROS-mediated, CHOP-dependent activation in renal tubular epithelial cells.     

PI3K/Akt pathway mediates high glucose-induced lipogenesis and extracellular matrix accumulation in HKC cells through regulation of SREBP-1 and TGF-β1

Previous studies have shown that high glucose stimulates renal SREBP-1 gene expression and increases renal tubular cells lipid metabolism, however, the mechanisms remain elusive. In the present study we demonstrated that PI3K/Akt pathway was activated in human renal proximal tubular cell line (HKC) exposed to high glucose accompanied with up-regulation of SREBP-1, TGF-β1, lipid droplets deposits and extracellular matrix production. Inhibition of PI3K/Akt pathway by chemical LY294002 or specific short hairpin RNA (shRNA) vector prevented SREBP-1 and TGF-β1 up-regulation, as well as ameliorated HKC cells lipogenesis and extracellular matrix accumulation. These findings indicate that PI3K/Akt pathway potentially mediates high glucose-induced lipogenesis and extracellular matrix accumulation in HKC cells.     

In search of adult renal stem cells

The therapeutic potential of adult stem cells in the treatment of chronic degenerative diseases has becoming increasingly evident over the last few years. Significant attention is currently being paid to the development of novel treatments for acute and chronic kidney diseases too. To date, promising sources of stem cells for renal therapies include adult bone marrow stem cells and the kidney precursors present in the early embryo. Both cells have clearly demonstrated their ability to differentiate into the kidney's specialized structures. Adult renal stem cells have yet to be identified, but the papilla is where the stem cell niche is probably located. Now we need to isolate and characterize the fraction of papillary cells that constitute the putative renal stem cells. Our growing understanding of the cellular and molecular mechanisms behind kidney regeneration and repair processes - together with a knowledge of the embryonic origin of renal cells - should induce us, however, to bear in mind that in the kidney, as in other mesenchymal tissues, the need for a real stem cell compartment might be less important than the phenotypic flexibility of tubular cells. Thus, by displaying their plasticity during kidney maintenance and repair, terminally differentiated cells may well function as multipotent stem cells despite being at a later stage of maturation than adult stem cells. One of the major tasks of Regenerative Medicine will be to disclose the molecular mechanisms underlying renal tubular plasticity and to exploit its biological and therapeutic potential.     

Mesenchymal-like progenitors derived from human embryonic stem cells promote recovery from acute kidney injury via paracrine actions

Background aimsThe engraftment of mesenchymal stem cells (MSCs) is reported to promote recovery of renal function in animal models of acute kidney injury (AKI). However, it is unknown whether mesenchymal-like progenitors (MPs) derived from human embryonic stem cells (hESCs) can mediate similar therapeutic effects. We investigated the responses of recipient renal tissue to engraftment of hESC-MPs and underlying mechanisms of these effects.MethodsWe measured blood urea nitrogen and creatinine levels of AKI mice with hESC-MPs transplantation and control mice. We performed renal morphology analysis by immunohistochemistry and electron microscopy to confirm the renoprotective effects of engrafted hESC-MPs. Proliferation, apoptosis and gene expression of tubular cells were also monitored by immunohistochemistry and real-time quantitative polymerase chain reaction to investigate the mechanisms that occurred.ResultsAfter transplantation of hESC-MPs into mice with cisplatin-induced AKI, improvements in renal function and recovery from tubular epithelial cell injury were observed. Engrafted hESC-MPs were localized to areas of injured kidney 5 days after cisplatin induction, where they promoted tubular cell proliferation and decreased kidney cell apoptosis. The beneficial effect was further confirmed by the capability of the engrafted cells to up-regulate renal gene expression of anti-inflammatory cytokines and pro-survival cytokines. Meanwhile, infusion of these cells reduced renal gene expression of pro-inflammatory cytokines and monocyte chemotactic protein-1, a chemokine that stimulates monocyte and macrophage infiltration.ConclusionsOur results show that infused hESC-MPs may promote recovery from AKI by regulating related cytokines.     

Mitochondrial superoxide production contributes to vancomycin-induced renal tubular cell apoptosis

Vancomycin chloride (VCM), a glycopeptide antibiotic, is widely used for the therapy of infections caused by methicillin-resistant Staphylococcus aureus. However, nephrotoxicity is a major adverse effect in VCM therapy. In this study, we investigated the cellular mechanisms underlying VCM-induced renal tubular cell injury in cultured LLC-PK1 cells. VCM induced a concentration- and time-dependent cell injury in LLC-PK1 cells. VCM caused increases in the numbers of annexin V-positive/PI-negative cells and TUNEL-positive cells, indicating the involvement of apoptotic cell death in VCM-induced renal cell injury. The VCM-induced apoptosis was accompanied by the activation of caspase-9 and caspase-3/7 and reversed by inhibitors of these caspases. Moreover, VCM caused an increase in intracellular reactive oxygen species production and mitochondrial membrane depolarization, which were reversed by vitamin E. In addition, mitochondrial complex I activity was inhibited by VCM as well as by the complex I inhibitor rotenone, and rotenone mimicked the VCM-induced LLC-PK1 cell injury. These findings suggest that VCM causes apoptotic cell death in LLC-PK1 cells by enhancing mitochondrial superoxide production leading to mitochondrial membrane depolarization followed by the caspase activities. Moreover, mitochondrial complex I may play an important role in superoxide production and renal tubular cell apoptosis induced by VCM.     

Neural regulation of renal tubular sodium reabsorption and renin secretion

The entire mammalian nephron, including the juxtaglomerular apparatus, receives an exclusive noradrenergic innervation. Renal tubular alpha 1 adrenoceptors mediate the alterations in tubular segmental sodium, chloride, and water reabsorption that occur in response to direct or reflex changes in efferent renal sympathetic nerve activity. Specific tubular segments so identified are the proximal convoluted tubule, the loop of Henle (thick ascending limb), and the collecting duct. Alterations in efferent renal sympathetic nerve activity represent an important physiological contribution to the overall role of the kidney in the regulation of external sodium balance in conscious animals during both dietary sodium restriction and acute and chronic increases in total-body sodium. Progressively more intense activation of the renal nerves recruits a series of adrenergically mediated influences on renin secretion that are additive, ranging from subtle (modulation of nonneural mechanisms without directly causing renin secretion) to marked (renal vasoconstriction, antinatriuresis, high renin secretion rates). Juxtaglomerular granular cell beta 1 adrenoceptors mediate renin secretion responses to frequencies of renal nerve stimulation that do not cause renal vasoconstriction; at higher frequencies of renal nerve stimulation where renal vasoconstriction is present, renal vascular alpha 1 adrenoceptors mediate a portion of the renin secretion response.     

Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell.

The excretory cell extends a tubular process, or canal, along the basolateral surface of the epidermis to form the nematode renal epithelium. This cell can undergo normal tubulogenesis in isolated cell culture. Mutations in 12 genes cause excretory canal cysts in Caenorhabditis elegans. Genetic interactions, and their similar phenotypes, suggest these genes may encode functionally related proteins. Depending upon genotype and individual canal, defects range from focal cysts, flanked by normal width segments, to regional cysts involving the entire tubule. Oftentimes the enlarged regions are convoluted or partially septated. In mutants with very large cysts, renal function is measurably impaired. Based on histology and ultrastructure, canal cysts likely result from defects of the apical membrane domain. These mutants provide a model of tubulocystic disease without hyperplasia or basement membrane abnormalities. Similar apical mechanisms could regulate tubular morphology of vertebrate nephrons.     

Role of TGF-β in chronic kidney disease: an integration of tubular,glomerular and vascular effects

Transforming growth factor beta (TGF-β) has been recognized as an important mediator in the genesis of chronic kidney diseases (CKD), which are characterized by the accumulation of extracellular matrix (ECM) components in the glomeruli (glomerular fibrosis, glomerulosclerosis) and the tubular interstitium (tubulointerstitial fibrosis). Glomerulosclerosis is a major cause of glomerular filtration rate reduction in CKD and all three major glomerular cell types (podocytes or visceral epithelial cells, mesangial cells and endothelial cells) participate in the fibrotic process. TGF-β induces (1) podocytopenia caused by podocyte apoptosis and detachment from the glomerular basement membrane; (2) mesangial expansion caused by mesangial cell hypertrophy, proliferation (and eventually apoptosis) and ECM synthesis; (3) endothelial to mesenchymal transition giving rise to glomerular myofibroblasts, a major source of ECM. TGF-β has been shown to mediate several key tubular pathological events during CKD progression, namely fibroblast proliferation, epithelial to mesenchymal transition, tubular and fibroblast ECM production and epithelial cell death leading to tubular cell deletion and interstitial fibrosis. In this review, we re-examine the mechanisms involved in glomerulosclerosis and tubulointerstitial fibrosis and the way that TGF-β participates in renal fibrosis, renal parenchyma degeneration and loss of function associated with CKD.