Proliferation Of Renal Tubular Epithelial Cells Research Articles

Ablation of Gsa impairs renal tubule proliferation after injury via CDK2/cyclin E.

Acute kidney injury has a high global morbidity associated with an increased risk of death and chronic kidney disease. Renal tubular epithelial cell regeneration following injury may be a decisive factor in renal repair or the progression of acute kidney injury to chronic kidney disease, but the underlying mechanism of abnormal renal tubular repair remains unclear. In the present study, we investigated the role of heterotrimeric G stimulatory protein α-subunit (Gsa) in renal tubular epithelial cell regeneration. We generated renal tubule epithelium-specific Gsa knockout (GsaKspKO) mice to show the essential role of Gsa in renal tubular epithelial cell regeneration in two AKI models: acute aristolochic acid nephropathy (AAN) and unilateral ischemia-reperfusion injury (UIRI). GsaKspKO mice developed more severe renal impairment after AAN and UIRI, higher serum creatinine levels, and more substantial tubular necrosis than wild-type mice. More importantly, Gsa inactivation impaired renal tubular epithelial cell proliferation by reducing bromodeoxyuridine+ cell numbers in the AAN model and inhibiting cyclin-dependent kinase 2/cyclin E1 expression in the UIRI model. This reduced proliferation was further supported in vitro with Gsa-targeting siRNA. Downregulation of Gsa inhibited tubular epithelial cell proliferation in HK-2 and mIMCD-3 cells. Furthermore, Gsa downregulation inhibited cyclin-dependent kinase 2/cyclin E1 expression, which was dependent on the Raf-MEK-ERK signaling pathway. In conclusion, Gsa is required for tubular epithelial cell regeneration during kidney repair after AKI. Loss of Gsa impairs renal tubular epithelial cell regeneration by blocking the Raf-MEK-ERK pathway.

Abstract P144: Integrin β1 in Tubular Epithelium Protects From Acute Injury Induced Fibrosis

Acute kidney injury (AKI) results in the sudden loss of renal function primarily due to renal tubular injury. Repeated injury or maladaptive recovery lead to chronic kidney diseases. All forms of chronic kidney disease are characterized by excessive extracellular matrix (ECM) accumulation. The proximal tubule cells that are injured but not detached from the basement membrane are critical for the restoration of renal functions after the injury incidence. Renal tubular epithelial cell adhesion, migration, and proliferation are required for the kidney to recover from sub-lethal injury. These processes require the ability of cells to interact with the ECM. Integrin, which exists as a complex of α and β subunits, are the principal receptors for ECM proteins. Integrin β1 is the major integrin subunit present in kidney and critically regulates renal epithelial cell functions. We investigated the importance of Integrin β1 in kidney proximal tubule epithelial cells. Integrin β1 was deleted from these cells using the γ-gt cre. These KO mice had no morphological abnormalities but have a higher urine osmolality (2369±881 mΩ) compared to WT (2098±945 mΩ). By using a nephrotoxic injury model, we investigated the role of integrin β1 in AKI. Samples were collected on 3, 5, 7 and 14 days after injury to assess the nature of injury and recovery. Integrin β1 KO mice were more susceptible to injury as evident by protein casts and tubule dilation of tubules. The blood urea nitrogen (BUN) from the plasma showed increased BUN level in KO animals compared to WT controls (26.78±8.23 and 34.05±7.11). After the injury the level was increased to 52.26±18.37 in WT and 60.34±11.58 KO suggesting both were injured to a similar extent. After 14 days the WT recovered better than KO animals (BUN 37.33±10.12 and 48.73±15.92). After 14 KO mice had more collagen deposition compared to WT animals. This data suggests the importance of integrin β1 in the tubular epithelium in promoting the recovery from injury and preventing its progression to chronic kidney disease.

Exogenous biological renal support ameliorates renal pathology after ischemia reperfusion injury in elderly mice.

We established an exogenous biological renal support model through the generation of parabiotic mice. At 72 hours after ischemia reperfusion injury (IRI), the aged mice that received exogenous biological renal support showed significantly higher levels of renal cell proliferation and dedifferentiation, lower levels of renal tubular injury, improved renal function, and a lower mortality than those that did not receive exogenous biological renal support. Using the Quantibody Mouse Cytokine Antibody Array, we found that aged IRI mice that received exogenous biological renal support had an up-regulation of multiple inflammatory related cytokines compared to the group that did not receive exogenous biological renal support. We suggest that the exogenous biological renal support might promote renal tubular epithelial cell proliferation and dedifferentiation and improve the prognosis of aged IRI mice. Exogenous biological renal support may play an important role in the amelioration of renal IRI by regulating the expression of multiple cytokines.

Effects of long non-coding RNA LINC00667 on renal tubular epithelial cell proliferation, apoptosis and renal fibrosis via the miR-19b-3p/LINC00667/CTGF signaling pathway in chronic renal failure

The global prevalence of chronic renal failure (CRF) has significantly elevated with various reports indicating there to be a 10% worldwide rate. The functions of long non-coding RNAs (lncRNAs) and their deeper association with CRF at present remain poorly understood. Hence, the aim of the present study was to investigate the altered expressions of lncRNA LINC00667 in CRF and its associated effects on renal tubular epithelial cell proliferation, apoptosis and renal fibrosis through the microRNA-19b-3p (miR-19b-3p)/LINC00667/connective tissue growth factor (CTGF) signaling pathway. Initially, verification of the targeting relationship between LINC00667, CTGF and miR-19b-3p was performed, after which evidence was obtained indicating that miR-19b-3p could negatively regulate LINC00667 and CTGF. The expressions of CTGF in both the CRF and normal renal tissues were determined by immunohistochemistry means, with LINC00667 and CTGF determined to be highly expressed, while poor expression levels of miR-19b-3p were detected among the CRF tissues. The expressions of LINC00667, miR-19b-3p, fibrosis- and epithelial-mesenchymal transition (EMT)-related genes were also examined. The successfully established CRF rat models were treated with varying mimics, inhibitors, and siRNA. ELISA was applied to determine the renal function-related factors. Besides, the renal cell proliferation, migration and apoptosis were detected. In response to LINC00667 silencing, the renal tubular epithelial cells displayed increased proliferation and migration accompanied by reduced apoptosis based on upregulated miR-19b-3p, along with inhibited renal fibrosis and EMT detected. Taken together, the key findings of our study demonstrated that decreased lncRNA LINC00667 could promote renal tubular epithelial cell proliferation and ameliorate renal fibrosis in CRF via the miR-19b-3p/LINC00667/CTGF signaling pathway.

Somatic Mutations in Renal Cyst Epithelium in Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy caused by mutations in PKD1 and PKD2 that is characterized by renal tubular epithelial cell proliferation and progressive CKD. Although the molecular mechanisms involved in cystogenesis are not established, concurrent inactivating constitutional and somatic mutations in ADPKD genes in cyst epithelium have been proposed as a cellular recessive mechanism. We characterized, by whole-exome sequencing (WES) and long-range PCR techniques, the somatic mutations in PKD1 and PKD2 genes in renal epithelial cells from 83 kidney cysts obtained from nine patients with ADPKD, for whom a constitutional mutation in PKD1 or PKD2 was identified. Complete sequencing data by long-range PCR and WES was available for 63 and 65 cysts, respectively. Private somatic mutations of PKD1 or PKD2 were identified in all patients and in 90% of the cysts analyzed; 90% of these mutations were truncating, splice site, or in-frame variations predicted to be pathogenic mutations. No trans-heterozygous mutations of PKD1 or PKD2 genes were identified. Copy number changes of PKD1 ranging from 151 bp to 28 kb were observed in 12% of the cysts. WES also identified significant mutations in 53 non-PKD1/2 genes, including other ciliopathy genes and cancer-related genes. These findings support a cellular recessive mechanism for cyst formation in ADPKD caused primarily by inactivating constitutional and somatic mutations of PKD1 or PKD2 in kidney cyst epithelium. The potential interactions of these genes with other ciliopathy- and cancer-related genes to influence ADPKD severity merits further evaluation.

Role of JAK3 in the Pathogenesis of Oxidative Stress-Induced Kidney Fibrosis

The Janus kinase (JAK) tyrosine kinase family and JAK/STAT signal transduction pathway may act in kidney fibrogenesis. JAK3 expression was investigated in in vitro and in vivo models of kidney fibrosis involving oxidative stress. There was a marked down-regulation of JAK3 mRNA in rat kidney tubular epithelial cells (NRK52E) and fibroblasts (NRK49F) exposed to 1.0 mM H2O2 for 18–20 h compared with controls, which correlated with increased apoptosis and decreased mitosis in both cell lines. However, JAK3 protein levels were not significantly different in control and H2O2-treated epithelial and fibroblast cultures. JAK3 activation (phospho-tyrosine) increased in NRK52E cells and decreased in NRK49F cells with oxidative stress. STAT3 phosphorylation decreased in both cell lines with oxidative stress compared with controls. JAK3 protein expression and localisation were investigated in kidneys using the unilateral ureteral obstruction (UUO) model (0–7 days, rats) of kidney fibrosis that involves oxidative stress. JAK3 protein expression did not differ between UUO and controls; however, JAK3 localisation increased temporally with UUO, with strong epithelial expression in mitotic cells compared with controls. Apoptotic tubular epithelium showed minimal JAK3. In summary, in vitro, decreased kidney JAK3 mRNA after oxidative stress was not seen translationally. Differences in the activation of the JAK3/STAT3 pathway may have different consequences for renal fibrosis. In vivo, changes in JAK3 protein localisation, and especially its co-localisation with mitotic cells, indicate that JAK3 protein may contribute to renal tubular epithelial cell proliferation after oxidative stress.

Effect of long non-coding RNA TapSAKI on the renal tubular epithelial cells induced by hypoxia

Objective To study the effect of long non-coding(LncRNA) TapSAKI on HK-2 cells of renal tubular epithelial cells induced by hypoxia. Methods The cultured HK-2 cells were divided into the normal oxygen group (group A), hypoxia injury group (group B), hypoxia injury+ control siRNA group (group C), and hypoxia injury+ TapSAKI siRNA group (group D). The real-time quantitative polymerase chain reaction (qPCR) was used to detect the change in TapSAKI expression; proliferation was measured by CCK-8 assay; cell cycle and cell apoptosis were measured by flow cytometry; Western blot was used to detect cycle related CyclinD1 protein, CDK2 and apoptosis related protein Bcl-2 and Bax. Results From the result of qPCR, compared with group A, the expression of TapSAKI gene in group B increased significantly (48.92±0.31 vs.1.00±0.01, P<0.05). Compared with group C, the expression of TapSAKI gene in group D decreased significantly(4.70±0.60 vs.48.31±0.29, P<0.05). The result of CCK-8 showed that the proliferation in group B significantly decreased compared with group A (P<0.05). Compared with group C, the proliferation in group D significantly increased (P<0.05). The flow cytometer test results showed that the apoptosis rate in group B was higher than that in group A [(26.38±1.21)% vs.(6.45±0.46)%, P<0.05]. Comparing with group C, the apoptosis rate in group D decreased significantly [(10.98±0.88)% vs.(21.59±1.30)%, P<0.05]. Compared with group A, Bax expression in group B increased, and Bcl-2 expression decreased (1.304±0.082 vs.0.411±0.002, 0.390±0.007 vs.1.027±0.022, all P<0.05). In group D, the expression of Bax was lower than that in group C, and the expression of Bcl-2 increased, both of which were statistically significant(0.655±0.819 vs.1.419±0.087, 0.819±0.034 vs.0.437±0.014, all P<0.05). Compared with group A, the proportion of G0/G1 phase cells in group B significantly increased (69.82±1.14 vs.34.46±0.82, P<0.05), and the proportion of S phase cells decreased significantly (11.6±0.60 vs.42.23±1.46, P<0.05). Compared with group A, CyclinD1 and CDK2 protein expressions in group B decreased (0.659±0.062 vs.1.723±0.084, 0.414±0.015 vs.0.87±0.031, all P<0.05). Compared with group C, group D G0/G1 phase cells significantly decreased (30.77±0.33 vs.61.81±1.50, P<0.05), the proportion of S phase cells significantly increased(40.32±0.72 vs.17.92±0.71, P<0.05), CyclinD1 and CDK2 protein expressions increased (2.049±0.027 vs.0.626±0.024, 0.89±0.104 vs.0.424±0.012, all P<0.05). Conclusions Under hypoxic conditions, LncRNA TapSAKI in renal tubular epithelial cells was abundant, which may inhibit renal tubular epithelial cell proliferation and accelerate apoptosis.It is suggested that LncRNA TapSAKI may play a role in the deterioration of cell proliferation and apoptosis. Key words: Long non-coding RNA; TapSAKI; Renal tubular epithelial cell; Hypoxia

Transcription Factor Trps1 Promotes Tubular Cell Proliferation after Ischemia-Reperfusion Injury through cAMP-Specific 3',5'-Cyclic Phosphodiesterase 4D and AKT.

Trichorhinophalangeal 1 (Trps1) is a transcription factor essential for epithelial cell morphogenesis during kidney development, but the role of Trps1 in AKI induced by ischemia-reperfusion (I/R) remains unclear. Our study investigated Trps1 expression during kidney repair after acute I/R in rats and explored the molecular mechanisms by which Trps1 promotes renal tubular epithelial cell proliferation. Trps1 expression positively associated with the extent of renal repair after I/R injury. Compared with wild-type rats, rats with knockdown of Trps1 exhibited significantly delayed renal repair in the moderate I/R model, with lower GFR levels and more severe morphologic injury, whereas rats overexpressing Trps1 exhibited significantly accelerated renal repair after severe I/R injury. Additionally, knockdown of Trps1 inhibited and overexpression of Trps1 enhanced the proliferation of renal tubular epithelial cells in rats. Chromatin immunoprecipitation sequencing assays and RT-PCR revealed that Trps1 regulated cAMP-specific 3',5'-cyclic phosphodiesterase 4D (Pde4d) expression. Knockdown of Trps1 decreased the renal protein expression of Pde4d and phosphorylated Akt in rats, and dual luciferase analysis showed that Trps1 directly activated Pde4d transcription. Furthermore, knockdown of Pde4d or treatment with the phosphatidylinositol 3 kinase inhibitor wortmannin significantly inhibited Trps1-induced tubular cell proliferation in vitro Trps1 may promote tubular cell proliferation through the Pde4d/phosphatidylinositol 3 kinase/AKT signaling pathway, suggesting Trps1 as a potential therapeutic target for kidney repair after I/R injury.

CD47 regulates renal tubular epithelial cell self-renewal and proliferation following renal ischemia reperfusion

Defects in renal tubular epithelial cell repair contribute to renal ischemia reperfusion injury, cause acute kidney damage, and promote chronic renal disease. The matricellular protein thrombospondin-1 and its receptor CD47 are involved in experimental renal ischemia reperfusion injury, although the role of this interaction in renal recovery is unknown. We found upregulation of self-renewal genes (transcription factors Oct4, Sox2, Klf4 and cMyc) in the kidney of CD47(-/-) mice after ischemia reperfusion injury. Wild-type animals had minimal self-renewal gene expression, both before and after injury. Suggestive of cell autonomy, CD47(-/-) renal tubular epithelial cells were found to increase expression of the self-renewal genes. This correlated with enhanced proliferative capacity compared with cells from wild-type mice. Exogenous thrombospondin-1 inhibited self-renewal gene expression in renal tubular epithelial cells from wild-type but not CD47(-/-) mice, and this was associated with decreased proliferation. Treatment of renal tubular epithelial cells with a CD47 blocking antibody or CD47-targeting small interfering RNA increased expression of some self-renewal transcription factors and promoted cell proliferation. In a syngeneic kidney transplant model, treatment with a CD47 blocking antibody increased self-renewal transcription factor expression, decreased tissue damage, and improved renal function compared with that in control mice. Thus, thrombospondin-1 via CD47 inhibits renal tubular epithelial cell recovery after ischemia reperfusion injury through inhibition of proliferation/self-renewal.

Alternatively activated macrophages as therapeutic agents for kidney disease: in vivo stability is a key factor

Infusing ex vivo-generated alternatively activated macrophages (AAM) has shown promise in experimental systems as a therapeutic strategy for inflammatory kidney disease. In the mouse Adriamycin nephropathy model, however, Cao et al. report that AAM derived from bone marrow precursors fail to ameliorate disease severity. Absence of the anticipated protective effect resulted from a loss of macrophage anti-inflammatory (M2) phenotype following trafficking to injured kidney-an effect that was mediated by localized colony-stimulating factor-1-dependent macrophage proliferation.

Targeting of sodium–glucose cotransporters with phlorizin inhibits polycystic kidney disease progression in Han:SPRD rats

Renal tubular epithelial cell proliferation and transepithelial cyst fluid secretion are key features in the progression of polycystic kidney disease (PKD). As the role of the apical renal sodium-glucose cotransporters in these processes is not known, we tested whether phlorizin inhibits cyst growth and delays renal disease progression in a rat model of PKD. Glycosuria was induced by subcutaneous injection of phlorizin in male heterozygous (Cy/+) and wild-type Han:SPRD rats. Phlorizin induced immediate and sustained glycosuria and osmotic diuresis in these rats. Cy/+ rats treated with phlorizin for 5 weeks showed a significant increase in creatinine clearance, a lower 2-kidneys/body weight ratio, a lower renal cyst index, and reduced urinary albumin excretion as compared with vehicle-treated Cy/+ rats. Measurement of Ki67 staining found significantly lower cell proliferation in dilated tubules and cysts of Cy/+ rats treated with phlorizin, as well as a marked inhibition of the activated MAP kinase pathway. In contrast, the mTOR pathway remained unaltered. Phlorizin dose dependently inhibited MAP kinase in cultured tubular epithelial cells from Cy/+ rats. Thus, long-term treatment with phlorizin significantly inhibits cystic disease progression in a rat model of PKD. Hence, induction of glycosuria and osmotic diuresis (glycuresis) by renal sodium-glucose cotransporters inhibition could have a therapeutic effect in polycystic kidney disease.

CD98 Increases Renal Epithelial Cell Proliferation by Activating MAPKs

CD98 heavy chain (CD98hc) is a multifunctional transmembrane spanning scaffolding protein whose extracellular domain binds with light chain amino acid transporters (Lats) to form the heterodimeric amino acid transporters (HATs). It also interacts with β1 and β3 integrins by its transmembrane and cytoplasmic domains. This interaction is proposed to be the mechanism whereby CD98 mediates cell survival and growth via currently undefined signaling pathways. In this study, we determined whether the critical function of CD98-dependent amino acid transport also plays a role in cell proliferation and defined the signaling pathways that mediate CD98-dependent proliferation of murine renal inner medullary collecting duct (IMCD) cells. We demonstrate that downregulating CD98hc expression resulted in IMCD cell death. Utilizing overexpression studies of CD98hc mutants that either lacked a cytoplasmic tail or were unable to bind to Lats we showed that CD98 increases serum-dependent cell proliferation by a mechanism that requires the CD98hc cytoplasmic tail. We further demonstrated that CD98-dependent amino acid transport increased renal tubular epithelial cell proliferation by a mechanism that does not require the CD98hc cytoplasmic tail. Both these mechanisms of increased renal tubular epithelial cell proliferation are mediated by Erk and p38 MAPK signaling. Although increased amino transport markedly activated mTor signaling, this pathway did not alter cell proliferation. Thus, these studies demonstrate that in IMCD cells, the cytoplasmic and extracellular domains of CD98hc regulate cell proliferation by distinct mechanisms that are mediated by common MAPK signaling pathways.

Loss of activator of G‐protein signaling 3 impairs renal tubular regeneration following acute kidney injury in rodents

The intracellular mechanisms underlying renal tubular epithelial cell proliferation and tubular repair following ischemia-reperfusion injury (IRI) remain poorly understood. In this report, we demonstrate that activator of G-protein signaling 3 (AGS3), an unconventional receptor-independent regulator of heterotrimeric G-protein function, influences renal tubular regeneration following IRI. In rat kidneys exposed to IRI, there was a temporal induction in renal AGS3 protein expression that peaked 72 h after reperfusion and corresponded to the repair and recovery phase following ischemic injury. Renal AGS3 expression was localized predominantly to the recovering outer medullary proximal tubular cells and was highly coexpressed with Ki-67, a marker of cell proliferation. Kidneys from mice deficient in the expression of AGS3 exhibited impaired renal tubular recovery 7 d following IRI compared to wild-type AGS3-expressing mice. Mechanistically, genetic knockdown of endogenous AGS3 mRNA and protein in renal tubular epithelial cells reduced cell proliferation in vitro. Similar reductions in renal tubular epithelial cell proliferation were observed following incubation with gallein, a selective inhibitor of Gβγ subunit activity, and lentiviral overexpression of the carboxyl-terminus of G-protein-coupled receptor kinase 2 (GRK2ct), a scavenger of Gβγ subunits. In summary, these data suggest that AGS3 acts through a novel receptor-independent mechanism to facilitate renal tubular epithelial cell proliferation and renal tubular regeneration.

Ascofuranone: A Possible Therapeutic Tool for Autosomal Dominant Polycystic Kidney Disease? – Letter

We read with interest the article by Jeong et al. (1) showing how ascofuranone induces G1 arrest through a p53-independent activation of p21WAF1/CIP1 due to the disruption of c-Myc in human cancer cell lines. Interestingly, defects in cell cycle arrest occur in autosomal dominant polycystic kidney disease (ADPKD) and are correlated with polycystin-1 (PC-1) malfunctioning and with secondary failure to upregulate p21 in renal and biliary epithelial cells (2), in addition to increased expression of c-Myc. Renal and liver cysts are key features of ADPKD, in which they develop through a cellular recessive mechanism involving loss-of-heterozygosity and somatic mutations (3), as in the case of onco-suppressor gene–related cancers. Recently, it has been shown how p21 is decreased in cysts of ADPKD patients, in which p21 levels are inversely correlated with renal tubular epithelial cell proliferation (4). A high PKD1 mutation rate has been considered to explain the number of cysts observed in ADPKD. Furthermore, lowering the PKD1 expression is sufficient to cause polycystic disease, leading to the hypothesis that, in patients heterozygous for PKD1 (ADPKD patients), reduction of the normal allele products below a critical level, due to genetic, environmental, and stochastic factors, can determine the formation of cysts and other ADPKD manifestations (5). As PC-1 upregulates p21 levels, the latter can vary following the genetic and/or acquired PC-1 modifications. The p21 impairment observed in ADPKD can be considered even pivotal in determining the loss of heterozygosity, as the premature transition from G1 phase to S phase and the increased proliferation of the epithelial cells could enhance the probability of second mutations. A cyclin-dependent kinase inhibitor, roscovitine, has been already shown to arrest progression in a murine model of ADPKD, to increase p21 levels, and to decrease renal tubular epithelial cell proliferation, with no effect on apoptosis (4). Therapies acting on pivotal molecules of the early phase of ADPKD pathogenesis could not only slow the growth of the formed cysts but also prevent the development of new cysts, reducing the probability of somatic mutations acting on cell cycle and proliferation, as conceived for the cyclin-dependent kinase inhibitor roscovitine (4) or the mTOR inhibitor rapamycin. Another therapeutic with these features is ascofuranone; recent discovery shows that it induces G1 arrest through p21WAF1/CIP1 activation and c-Myc disruption (1). The recent findings by Jeong et al. lead us to consider the possibility to evaluate ascofuranone as a therapeutic tool for ADPKD.See the Response, p. 3101.No potential conflicts of interest were disclosed.

HIV-1 viral protein r induces ERK and caspase-8-dependent apoptosis in renal tubular epithelial cells

HIV-associated nephropathy (HIVAN) is the most common cause of end-stage renal disease in persons with HIV/AIDS and is characterized by focal glomerulosclerosis and dysregulated renal tubular epithelial cell (RTEC) proliferation and apoptosis. HIV-1 viral protein r (Vpr) has been implicated in HIV-induced RTEC apoptosis but the mechanisms of Vpr-induced RTEC apoptosis are unknown. The aim of this study was therefore to determine the mechanisms of Vpr-induced apoptosis in RTEC. Apoptosis and caspase activation were analyzed in human RTEC (HK2) after transduction with Vpr-expressing and control lentiviral vectors. Bax and BID were inhibited with lentiviral shRNA, and ERK activation was blocked with the MEK1,2 inhibitor, U0126. Vpr induced apoptosis as indicated by caspase 3/7 activation, PARP-1 cleavage and mitochondrial injury. Vpr activated both caspases-8 and 9. Inhibition of Bax reduced Vpr-induced apoptosis, as reported in other cell types. Additionally, Vpr-induced cleavage of BID to tBID and suppression of BID expression prevented Vpr-induced apoptosis. Since sustained ERK activation can activate caspase-8 in some cell types, we studied the role of ERK in Vpr-induced caspase-8 activation. Vpr induced sustained ERK activation in HK2 cells and incubation with U0126 reduced Vpr-induced caspase-8 activation, BID cleavage and apoptosis. We detected phosphorylated ERK in RTEC in HIVAN biopsy specimens by immunohistochemistry. These studies delineate a novel pathway of Vpr-induced apoptosis in RTEC, which is mediated by sustained ERK activation, resulting in caspase 8-mediated cleavage of BID to tBID, thereby facilitating Bax-mediated mitochondrial injury and apoptosis.

Reactive oxygen species differently regulate renal tubular epithelial and interstitial cell proliferation after ischemia and reperfusion injury

Reactive oxygen species (ROS) function as an inducer of cell death and survival or proliferative factor, in a cell-type-specific and concentration-dependent manner. All of these roles are critical to ischemia-induced renal functional impairment and progressive fibrotic changes in the kidney. In an effort to define the role of ROS in the proliferation of tubular epithelial cells and of interstitial cells in kidneys recovering after ischemia and reperfusion (I/R) injury, experimental mice were subjected to 30 min of bilateral kidney ischemia and administered with manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP), a superoxide dismutase mimetic, from 2 to 15 days after I/R for 14 days daily (earlier and longer) and from 8 to 15 days after I/R for 8 days daily (later and shorter). Cell proliferation was assessed via 5'-bromo-2'-deoxyuridine (BrdU) incorporation assays for 20 h before the harvest of kidneys. After I/R, the numbers of BrdU-incorporating cells increased both in the tubules and interstitium. MnTMPyP administration was shown to accelerate the proliferation of tubular epithelial cells, presenting tubule-specific marker proteins along tubular segments, whereas it attenuated the proliferation of interstitial cells, evidencing α-smooth muscle actin, fibroblast-specific protein-1, F4/80, and NADPH oxidase-2 proteins; these results indicated that ROS attenuates tubular cell regeneration, but accelerates interstitial cell proliferation. Earlier and longer MnTMPyP treatment more effectively inhibited tissue superoxide formation, the increment of interstitial cells, and the decrement of epithelial cells compared with later and shorter treatment. After I/R, apoptotic cells appeared principally in the tubular epithelial cells, but not in the interstitial cells, thereby indicating that ROS is harmful in tubule cells, but is not in interstitial cells. In conclusion, ROS generated after I/R injury in cell proliferation and death performs a cell-type-specific and concentration-dependent role, even within the same tissues, and timely intervention of ROS is crucial for effective therapies.

PXII-12 Leflunomide inhibits BK virus replication but simultaneously compromises renal tubular epithelial cell proliferation

PXII-12 Leflunomide inhibits BK virus replication but simultaneously compromises renal tubular epithelial cell proliferation

Netrin-1 increases proliferation and migration of renal proximal tubular epithelial cells via the UNC5B receptor

The cellular hallmark of kidney repair is a rapid proliferation of renal tubular epithelial cells ultimately leading to the restoration of nephron structure and function. Netrin-1 was discovered as a neural guidance cue and found to be expressed outside the nervous system, including in kidney. Previous work showed that netrin-1 is upregulated in response to ischemic injury and ameliorates ischemic injury. The objectives of this study were to determine the role of netrin-1 in renal tubular epithelial cell proliferation and migration in vitro. Real-time RT-PCR analysis showed that netrin-1 and its receptors UNC5B and neogenin are highly expressed in cultured mouse renal epithelial cells (TKPTS), whereas the expression of the Deleted in Colon Cancer (DCC), UNC5A, UNC5C, and UNC5D receptors is negligible or undetectable. Netrin-1 protein was induced in the edges of mechanical wounds in vitro. Netrin-1 increased TKPTS cell proliferation in a dose-dependent manner. The netrin-1-induced increase in TKPTS cell proliferation was completely prevented by small interfering RNA (siRNA) inhibition of UNC5B receptor but not UNC5C receptor expression. Netrin-1 also increased TKPTS cell migration in vitro, and this was also mediated through the UNC5B receptor. Netrin-1 increased the phosphorylation of Akt and ERK. Inhibition of phosphatidylinositol 3-kinase and MEK1/2 completely inhibited netrin-1-induced cell proliferation but not migration. These results indicate that netrin-1 increases renal tubular epithelial cell proliferation and migration through the UNC5B receptor. Moreover, the increase in cell proliferation, but not migration, was mediated via activation of Akt and ERK pathways.

P21 is decreased in polycystic kidney disease and leads to increased epithelial cell cycle progression: roscovitine augments p21 levels

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease with few treatment options other than renal replacement therapy. p21, a cyclin kinase inhibitor which has pleiotropic effects on the cell cycle, in many cases acts to suppress cell cycle progression and to prevent apoptosis. Because defects in cell cycle arrest and apoptosis of renal tubular epithelial cells occur in PKD, and in light of earlier reports that polycystin-1 upregulates p21 and that the cyclin-dependent kinase inhibitor roscovitine arrests progression in a mouse model, we asked whether (1) p21 deficiency might underlie ADPKD and (2) the mechanism of the salutary roscovitine effect on PKD involves p21.Methodsp21 levels in human and animal tissue samples as well as cell lines were examined by immunoblotting and/or immunohistochemisty. Apoptosis was assessed by PARP cleavage. p21 expression was attenuated in a renal tubular epithelial cell line by antisense methods, and proliferation in response to p21 attenuation and to roscovitine was assessed by the MTT assay.ResultsWe show that p21 is decreased in human as well as a non-transgenic rat model of ADPKD. In addition, hepatocyte growth factor, which induces transition from a cystic to a tubular phenotype, increases p21 levels. Furthermore, attenuation of p21 results in augmentation of cell cycle transit in vitro. Thus, levels of p21 are inversely correlated with renal tubular epithelial cell proliferation. Roscovitine, which has been shown to arrest progression in a murine model of PKD, increases p21 levels and decreases renal tubular epithelial cell proliferation, with no affect on apoptosis.ConclusionThe novelty of our study is the demonstration in vivo in humans and rat models of a decrement of p21 in cystic kidneys as compared to non-cystic kidneys. Validation of a potential pathogenetic model of increased cyst formation due to enhanced epithelial proliferation and apoptosis mediated by p21 suggests a mechanism for the salutary effect of roscovitine in ADPKD and supports further investigation of p21 as a target for future therapy.

Increased Cell Proliferation is Attenuated by Ca2+ Chelation in a Mouse Model of ARPKD

Increased renal tubular epithelial cell proliferation has been demonstrated to precede cyst development in various models of polycystic kidney disease. Our lab has previously shown, in cilia-deficient cortical collecting duct cells from the Oak Ridge Polycystic Kidney mouse (orpk cilia(−)), that there is increased apical Ca2+ permeability which causes elevated sub-apical Ca2+ levels compared with cilia-competent counterparts (orpk cilia(+)). We hypothesize that elevated Ca2+ levels in orpk cilia(−) cells may lead to increased cell proliferation. In the present studies, cell proliferation rates were measured in orpk cilia(+) and cilia(−) cells by cell counting using a hemacytometer. Cells from both groups were seeded at equal density, and allowed to attach for 24 hours. Treatments to lower [Ca2+]i using BAPTA-AM were initiated 24 hours post-seeding, and repeated at 24 hour intervals. We found that untreated orpk cilia(−) cells grew at a 36% faster rate than cilia(+) cells (2.1*10^5 vs. 1.5*10^5 cells/(plate*day)). 10 μM BAPTA-AM attenuated the growth rate by nearly 57% in cilia(−) cells (2.1*10^5 vs. 0.9*10^5 cells/(plate*day)). The effect of BAPTA-AM on the proliferation rate of orpk cilia(+) cells was less pronounced, as Ca2+ chelation inhibited growth by approximately 22% (1.5*10^5 vs. 1.2*10^5 cells/(plate*day)). Furthermore, proliferation rates of BAPTA-AM treated orpk cilia(−) and cilia(+) cells were comparable (0.9*10^5 vs. 1.2*10^5 cells/(plate*day)). Thus, renal cystogenesis may result from increased proliferation due to elevated sub-apical Ca2+ levels in the orpk cilia(−) model.