Apoptosis In Proximal Tubules Research Articles

Knockout of Nephron ATP6AP2 Impairs Proximal Tubule Function and Prevents High-Fat Diet-Induced Obesity in Male Mice.

ATP6AP2 expression is increased in the nephron during high-fat diet (HFD) and its knockout (ATP6AP2 KO) reduces body weight (WT) in mice. We evaluated the contribution of ATP6AP2 to urinary glucose (UG) and albumin (Ualb) handling during HFD. We hypothesized that nephron ATP6AP2 KO increases UG and Ualb and minimizes HFD-induced obesity. Eight-week-old male C57BL/6J mice with inducible nephron-specific ATP6AP2 KO and noninduced controls were fed either normal diet (ND, 12% kcal fat) or HFD (45% kcal fat) for 6 months. ATP6AP2 KO mice on ND had 20% (P < 0.01) lower WT compared with controls. HFD-fed mice had 41% (P < 0.05) greater WT than ND-fed control mice. In contrast, ATP6AP2 KO abrogated the increase in WT induced by HFD by 40% (P < 0.05). Mice on HFD had less caloric intake compared with ND controls (P < 0.01). There were no significant differences in metabolic rate between all groups. UG and Ualb was significantly increased in ATP6AP2 KO mice on both ND and HFD. ATP6AP2 KO showed greater levels of proximal tubule apoptosis and histologic evidence of proximal tubule injury. In conclusion, our results demonstrate that nephron-specific ATP6AP2 KO is associated with glucosuria and albuminuria, most likely secondary to renal proximal tubule injury and/or dysfunction. Urinary loss of nutrients may have contributed to the reduced WT of knockout mice on ND and lack of WT gain in response to HFD. Future investigation should elucidate the mechanisms by which loss of renal ATP6AP2 causes proximal tubule injury and dysfunction.

Hyperglycemia-induced oxidative stress in isolated proximal tubules of mouse: the in vitro effects of myricitrin and its solid lipid nanoparticle

Context The hyperglycemia (Hyper) induces oxidative stress in kidney tubular cells. Myricitrin (Myr) has an antioxidant effect along with low bioavailability. Objective The present research investigated the effects of Myr and its solid lipid nanoparticles (SLN) on isolated proximal tubules exposed to the hyperglycemic condition. Materials and Methods In this experimental study, the proximal tubules of mice were dissected by the microdissection method and the tubules were prepared for experimental or Real Time-PCR measurement. Results The malondialdehyde level, transforming growth factor-β, nuclear factor kappa B and Bax genes expression increased in Hyper and decreased in Hyper + Myr and its SLN-treated groups compared to Hyper. Superoxide dismutase, total antioxidant capacity, the viability of proximal tubules and Bcl-2 gene expression decreased in untreated Hyper and increased in all treatment groups compared to Hyper. Conclusion The hyperglycemia-induced oxidative disorder, inflammation and apoptosis in proximal tubules and that administrating Myr and its SLN improved them.

Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development.

The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway.

Protective Effects of Cilastatin against Vancomycin-Induced Nephrotoxicity.

Vancomycin is a very effective antibiotic for treatment of severe infections. However, its use in clinical practice is limited by nephrotoxicity. Cilastatin is a dehydropeptidase I inhibitor that acts on the brush border membrane of the proximal tubule to prevent accumulation of imipenem and toxicity. The aim of this study was to investigate the potential protective effect of cilastatin on vancomycin-induced apoptosis and toxicity in cultured renal proximal tubular epithelial cells (RPTECs). Porcine RPTECs were cultured in the presence of vancomycin with and without cilastatin. Vancomycin induced dose-dependent apoptosis in cultured RPTECs, with DNA fragmentation, cell detachment, and a significant decrease in mitochondrial activity. Cilastatin prevented apoptotic events and diminished the antiproliferative effect and severe morphological changes induced by vancomycin. Cilastatin also improved the long-term recovery and survival of RPTECs exposed to vancomycin and partially attenuated vancomycin uptake by RPTECs. On the other hand, cilastatin had no effects on vancomycin-induced necrosis or the bactericidal effect of the antibiotic. This study indicates that cilastatin protects against vancomycin-induced proximal tubule apoptosis and increases cell viability, without compromising the antimicrobial effect of vancomycin. The beneficial effect could be attributed, at least in part, to decreased accumulation of vancomycin in RPTECs.

Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis

Chronic kidney disease progression can be predicted based on the degree of tubular atrophy, which is the result of proximal tubule apoptosis. The Na+/H+ exchanger NHE1 regulates proximal tubule cell survival through interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], but pathophysiologic triggers for NHE1 inactivation are unknown. Because glomerular injury permits proximal tubule luminal exposure and reabsorption of fatty acid/albumin complexes, we hypothesized that accumulation of amphipathic, long-chain acyl-CoA (LC-CoA) metabolites stimulates lipoapoptosis by competing with the structurally similar PI(4,5)P2 for NHE1 binding. Kidneys from mouse models of progressive, albuminuric kidney disease exhibited increased fatty acids, LC-CoAs, and caspase-2-dependent proximal tubule lipoapoptosis. LC-CoAs and the cytosolic domain of NHE1 directly interacted, with an affinity comparable to that of the PI(4,5)P2-NHE1 interaction, and competing LC-CoAs disrupted binding of the NHE1 cytosolic tail to PI(4,5)P2. Inhibition of LC-CoA catabolism reduced NHE1 activity and enhanced apoptosis, whereas inhibition of proximal tubule LC-CoA generation preserved NHE1 activity and protected against apoptosis. Our data indicate that albuminuria/lipiduria enhances lipotoxin delivery to the proximal tubule and accumulation of LC-CoAs contributes to tubular atrophy by severing the NHE1-PI(4,5)P2 interaction, thereby lowering the apoptotic threshold. Furthermore, these data suggest that NHE1 functions as a metabolic sensor for lipotoxicity.

12. Prevention of cisplatin-induced nephrotoxicity by dichloroacetate

Background There is a continuing need to improve the efficacy of anticancer agents and to reduce the toxicity associated with them. Recently, reversal of the glycolytic phenotype of cancer cells with novel agents such as DCA (dichloroacetate) has been recognised as an important new target for cancer therapy. DCA is currently being assessed as a novel chemotherapeutic in clinical trials. It is anticipated that DCA will be used in combination with other well-established chemotherapeutic drugs. It is therefore critically important to determine the effects of DCA on therapeutic efficacy, and off-target effects of existing frontline anti-cancer drugs. We have recently discovered that DCA induces glutamate cysteine ligase, the rate-limiting step in glutathione (GSH) synthesis, a major cellular antioxidant. We hypothesised that the ability of DCA to stimulate GSH-synthesising capacity could reduce cisplatin-induced nephrotoxicity, which is thought to be mediated in part by oxidative stress. Aims To determine whether DCA can attenuate cisplatin-induced nephrotoxicity, and to determine if DCA influences the anti-cancer properties of cisplatin. Methods 120 Balb/c mice were injected subcutaneously with the syngeneic 4T1 breast cancer cell line and then co-treated with DCA and cisplatin weekly for 1 month. Controls included treatment with cisplatin alone, DCA alone, or normal saline injections. Serum blood urea nitrogen (BUN) and creatinine were measured and kidney damage was assessed histologically. Tumour size was monitored throughout. Results and conclusions DCA prevented increases in serum creatinine, BUN, and renal proximal tubule apoptosis evident in cisplatin-only treated mice. The tumour size in mice in the co-treated group decreased at the same rate as the cisplatin-only treated mice. We have thus concluded that DCA largely prevents the development of cisplatin-induced nephrotoxicity, and does not attenuate its anti-cancer properties.

The renoprotective effect of cGMP phosphodiesterase inhibitor and nitroprusside in a rat model of cyclosporin A-induced nephrotoxicity

The cyclosporin A (CsA) nephrotoxicity limits its usefulness as an immunosuppression. We studied the administration of both nitroprusside and phosphodiesterase-5 inhibitor (udenafil) in order to determine whether these agents could ameliorate the renal injury in CsA nephrotoxicity. 30 8-week-old SD rats were divided into 5 groups: the control (1), SQ with 15 mg/kg CsA (Group 2), CsA along with 5 mg/kg IP nitroprusside (Group 3), CsA with 10 mg/kg PO udenafil (Group 4), and CsA with udenafil and nitroprusside (Group 5). Group showed an increase in creatinine compared o the control group. Group 5 showed a decrease in creatinine compared to Group 2. In TUNEL, Group 2 increased apoptosis in proximal tubules compared to control. Group 5 showed a decrease in apoptosis compared to Groups 2, 3, and 4. In IHC, the eNOS in Group 2 was stronger than in the controls. Groups 3, 4, and 5 showed decreased staining intensity compared to Group 2. In IHC, the VEGF in Groups 2, 3, and 4 increased compared to the controls. The eNOS protein expression was increased in both Groups 3 and 5 compared to the controls. The VEGF protein expression was increased in Groups 3 and 5 compared to Group 2. The eNOS mRNA was decreased in Group 2 compared to the control group and tended to increase in Groups 3, 4, and 5 compared to Group 2. The VEGF mRNA was increased in Group 2 and tended to increase more in Groups 3 and 5. The udenafil and nitroprusside ameliorated renal injury in rat model of CsA nephrotoxicity. The mechanism appears to be associated with decreasing tubular apoptosis by decreasing eNOS and increasing VEGF.

Role of caveolin and heat shock protein 70 interaction in the antioxidative effects of an angiotensin II type 1 receptor blocker in spontaneously hypertensive rats proximal tubules

Role of caveolin and heat shock protein 70 interaction in the antioxidative effects of an angiotensin II type 1 receptor blocker in spontaneously hypertensive rats proximal tubules

Cisplatin-induced macroautophagy occurs prior to apoptosis in proximal tubules in vivo

Autophagy is an intracellular bulk degradation process induced by cell starvation. Autophagy was recently reported to be induced by various stresses such as hypoxia, ischemia/reperfusion, toxins, and denatured proteins, and to affect cell survival and death. Light chain 3-II (LC3-II) is specifically located on double membrane-bound autophagosomes that envelop disused proteins or organelles. Transgenic mice in which green fluorescent protein (GFP) was fused to LC3 (LC3-GFP) were administered cisplatin (20 mg/kg). After euthanasia at times between 0 and 72 h, kidneys were excised for immunohistochemical analyses. Microscopic examinations of the generated NRK-52E cell lines stably transfected with LC3-GFP, and Western blot analyses of NRK-52E cells, were undertaken after cisplatin treatment with or without autophagy inhibitors and beclin 1 small interfering RNA (siRNA). Autophagosomes increased in the proximal tubular cells of transgenic mice from 12 h after cisplatin injection (20 mg/kg). The time course for this was faster than those for tubular necrosis and apoptosis. Autophagosomes also increased in NRK-52E cells after cisplatin treatment, with the time course for this being faster than that for apoptosis. When autophagy was suppressed by autophagy inhibitors or beclin 1 siRNA, the level of apoptosis was also suppressed. Autophagy occurs in proximal tubular cells after cisplatin treatment and is involved in cell death in renal tubular injury. Our data suggest that autophagy is a kind of cell damage index and that cells with activated autophagy will be scavenged by apoptosis.

Ablation of developing podocytes disrupts cellular interactions and nephrogenesis both inside and outside the glomerulus

Podocyte loss in adults leads to glomerulosclerosis. However, the impact of podocyte loss on glomerulogenesis and the development of the kidney as a whole has not been directly studied. Here, we used a podocyte-specific Cre transgene to direct the production of diphtheria toxin (DTA) inside podocytes during nephrogenesis. Affected podocytes underwent translational arrest and apoptosis, leading to oliguria, proteinuria, hematuria, interstitial hemorrhage, and perinatal death. Glomerular cell-cell interactions were disrupted, even before overt podocyte apoptosis. VEGF production by podocytes was greatly decreased, and this was associated with reduced endothelial fenestration and altered glomerular vascular architecture. In addition to these glomerular anomalies, embryonic podocyte ablation also led to structural changes and increased apoptosis in proximal tubules. The collecting ducts, however, only showed molecular changes that are likely an indirect effect of the greatly reduced urine flow. Although podocyte loss significantly impacted the development and maintenance of the vasculature both inside and outside the glomerulus, our results suggest that there is a lack of long-range signaling from deep-seated, mature glomeruli to the differentiating cells in the outer nephrogenic zone. This study illustrates the tight integration of various cell types in the developing kidney and shows that the impact of podocyte loss during development is much greater than that in adults. This study also shows the specificity and effectiveness of a genetically controlled podocyte ablation system in mice where the additional readily available tools can further expand its applications.

Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation

Aristolochic acid contamination in herbal remedies leads to interstitial fibrosis, tubular atrophy, and renal failure in humans. To study the cellular mechanisms contributing to the pathophysiology of this renal disease, we studied Wistar rats treated with aristolochic acid and measured tubular and interstitial cell proliferation, epithelial/mesenchymal cell marker expression, tubular membrane integrity, myofibroblast accumulation, oxidative stress, mitochondrial damage, tubular apoptosis, and fibrosis. Oxidative stress, a loss of cadherin concomitant with vimentin expression, basement membrane denudation with active caspase-3 expression, and mitochondrial injury within tubular cells were evident within 5 days of administration of the toxin. During the chronic phase, interstitial mesenchymal cells accumulated in areas of collagen deposits. Impaired regeneration and apoptosis of proximal tubular cells resulted in tubule atrophy with a near absence of dedifferentiated cell transmembrane migration. We suggest that resident fibroblast activation plays a critical role in the process of renal fibrosis during aristolochic acid toxicity.

Glomerulotubular disconnection in neonatal mice after relief of partial ureteral obstruction

Ureteropelvic junction obstruction is a common cause of congenital obstructive nephropathy. To study the pathogenesis of nephropathy, a variable-partial, complete or a sham unilateral ureteral obstruction (UUO) was produced in mice within 2 days of birth. The obstruction was released in some animals at 7 days and kidneys harvested at 7-42 days of age for histologic and morphometric study. Renal parenchymal growth was stunted by partial UUO with the impairment proportional to the duration and severity of obstruction. Proximal tubule apoptosis and glomerulotubular disconnection led to nephron loss. Relief of partial UUO arrested glomerulotubular disconnection, resolved tubule atrophy, and interstitial fibrosis with remodeling of the renal architecture. Relief of severe UUO did not result in recovery. Compensatory growth of the contralateral kidney depended on the severity of obstruction. Our studies indicate that relief of moderate UUO will minimize nephron loss. Application of this technique to mutant mice will help develop future therapies to enhance nephron recovery.

Cadmium-induced ceramide formation triggers calpain-dependent apoptosis in cultured kidney proximal tubule cells

A major target of cadmium (Cd(2+)) toxicity is the kidney proximal tubule (PT) cell. Cd(2+)-induced apoptosis of PT cells is mediated by sequential activation of calpains at 3-6 h and caspases-9 and -3 after 24-h exposure. Calpains also partly contribute to caspase activation, which emphasizes the importance of calpains for PT apoptosis by Cd(2+). Upstream processes underlying Cd(2+)-induced calpain activation remain unclear. We describe for the first time that 10-50 microM Cd(2+) causes a significant increase in ceramide formation by approximately 22% (3 h) and approximately 72% (24 h), as measured by diacylglycerol kinase assay. Inhibition of ceramide synthase with fumonisin B(1) (3 microM) prevents ceramide formation at 3 h and abolishes calpain activation at 6 h, which is associated with significant attenuation of apoptosis at 3-6 h with Hoechst 33342 nuclear staining and/or 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) death assays. This indicates that Cd(2+) enhances de novo ceramide synthesis and that calpains are a downstream target of ceramides in apoptosis execution. Moreover, addition of C(6)-ceramide to PT cells increases cytosolic Ca(2+) and activates calpains. Apoptosis mediated by C(6)-ceramide at 24 h is significantly reduced by caspase-3 inhibition, which supports cross talk between calpain- and caspase-dependent apoptotic pathways. We conclude that Cd(2+)-induced apoptosis of PT cells entails endogenous ceramide elevation and subsequent Ca(2+)-dependent calpain activation, which propagates kidney damage by Cd(2+).

Apoptosis induction is associated with decreased NHE1 expression in neonatal unilateral ureteric obstruction

OBJECTIVE To examine the participation of NHE1 in the regulation of the apoptotic response in neonatal obstruction, as the ubiquitously expressed NHE1 isoform is an important component of regulatory volume increase. MATERIALS AND METHODS Rats had a unilateral ureteric obstruction (UUO) or a sham operation, and the kidneys were harvested 5 and 14 days afterward. Cellular apoptosis in proximal tubules (PT) and collecting ducts (CD) was assessed using a standard assay, and NHE1 expression in the renal cortex assessed using reverse transcription-polymerase chain reaction and Western blots. Mitochondrial apoptosis was evaluated by Bax/BcL2 expression, and caspase-3 expression and activity. In addition, we evaluated the in vivo administration of increasing doses of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on NHE1 inhibition associated with the induction of apoptosis. RESULTS After 14 days there were consistently more apoptotic cells in CD than in PT, associated with a lower expression of NHE1 at the mRNA and protein levels. There was increased expression of the Bax/BcL2 ratio, linked to decreased pro-caspase-3 protein levels and with increased caspase-3 activation. NHE1 inhibition by increasing doses of EIPA induced epithelial cell apoptosis and increased caspase-3 activity in a dose-dependent manner. After in vitro incubation with amiloride (100 mm) there was less NHE1 expression associated with reduced 32 kDa pro-caspase-3 protein levels. Kidneys obstructed for 5 days showed no changes in NHE1 expression or induction of apoptosis. CONCLUSION In neonatal obstruction, we suggest that the decreased NHE1 expression could be a signal-transduction event participating in the induction of epithelial tubular cell apoptosis, through the regulation of the BcL-2 gene family and activation of caspase-3.

Inducible Nitric Oxide Synthase and Apoptosis in Murine Proximal Tubule Epithelial Cells

Since inducible nitric oxide synthase (iNOS) and proximal tubule injury are known to be critical determinants of lipopolysaccharide (LPS)-induced renal failure, the role of nitric oxide (NO) in proximal tubule cell apoptosis was examined. An 18-h treatment with a combination of LPS (5 microg/ml) and interferon-gamma (IFN-gamma, 100 units/ml) synergistically induced iNOS and produced a 20-fold increase in NO generation in the TKPTS murine proximal tubule cell line. NO generation by LPS + IFN-gamma was blocked by a specific iNOS blocker, L-N6-(1-iminoethyl)-lysine (L-NIL, 1 mM). To assess the role of iNOS-derived NO in proximal tubule cell apoptosis, annexin V- and propidium iodide-labeled cells were analyzed by flow cytometry. Neither the induction of iNOS nor its inhibition produced significant apoptotic cell death in TKPTS cells. Two exogenous NO donors were used to examine the role of NO more directly in proximal tubule apoptosis. Although both sodium nitroprusside (SNP), an iron-containing, nitrosonium cation donor, and S-nitroso-N-acetylpenicillamine (SNAP), a noniron-containing, NO generator, produced a concentration-dependent increase in NO generation, only SNP increased apoptotic cell death in TKPTS cells (5.9 +/- 0.7% in control cells vs. 21.6 +/- 3.8% in SNP [500 microM]-treated cells; n = 4-9; p < 0.01). SNP-mediated tubule cell apoptosis was not dependent on the activation of caspases or p53 but was possibly related to the generation of reactive oxygen species by SNP. Thus, in TKPTS cells induction of iNOS and generation of NO by LPS does not lead to tubular epithelial cell death.

The role of nitric oxide in reflux nephropathy

Reflux nephropathy accounts for about 25% of cases of end-stage renal failure, but the mechanism by which reflux produces renal scars is still not clear. The authors examined nitric oxide (NO), which regulates arteriolar tone, mesangial cell proliferation, and extracellular matrix production by staining of NOS, an important enzyme in the synthese of NO. They differentiated the isoforms inducing NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS), the first a proinflammatory agent secreted by macrophages and mediating apoptosis, the others thought to have beneficial effects in relaxing vascular tone and ameliorating tubulointerstitial fibrosis. Kidney specimen from children with severe reflux nephropathy (RN) obtained at nephrectomy were compared with normal kidney tissue from adults undergoing surgery for small kidney tumors. Staining for NOS and iNOS in the proximal tubules of control kidneys was weak compared with RN specimen. nNOS and eNOS were lacking in control tissue, but were strongly visible in the tubulointerstitial spaces and in the glomeruli in RN kidneys. The authors correlated the markedly increased iNOS activity with the impressive histologic increase in proximal tubule apoptosis and atrophy, interpreting this as an effect of a macrophage-stimulated proinflammatory cytokine overproduction. eNOS may buffer iNOS effects on fibrosis by an increase of regional blood flow and relaxation of mesangial vessels. nNOS, probably stimulated by angiotensin II receptors, may reduce interstitial fibrosis by leading to an increased cyclic GMP level. Therefore, blockage of iNOS and induction of eNOS and nNOS may be helpful in preventing parenchymal injuries in RN.—Peter Schmittenbecher

Acute Renal Failure and Proximal Tubule Lesions after Trichosanthin Injection in Rats

The structural basis of the recently recognized renal impairment after infusion of trichosanthin (TCS), a type I ribosome inactivating protein, is uncertain, but functionally it appears to be related to a lesion in the renal tubules. In this study, renal dysfunction in experimental rats was induced by a single dose of TCS. Creatinine clearance and tubular proteinuria were used to assess renal function. Light microscopy and ultrastructure of the kidneys were examined and apoptosis in proximal tubules was evaluated by thein situTdT-mediated nick end labeling technique. TCS-treated rats demonstrated a significant dose-dependent decrease in creatinine clearance together with a mild degree of low-molecular-weight proteinuria. The proximal convoluted tubule was the site of lesions showing individual tubular cell death, which was more abundant in rats receiving high doses of TCS. Apoptotic cell death, together with heterophagosomes and large residual bodies, was observed. DNA fragmentation was confirmed by thein situtechnique. There was also a dose-dependent density of apoptotic cells. Other portions of the nephron were spared, and it was not accompanied by any inflammatory infiltrate. In conclusion, these findings are consistent with TCS-induced proximal tubular toxicity resulting in reduction of glomerular filtration rate and tubular proteinuria. The extent of injury is dosage dependent. Both necrotic cell death and apoptosis participated in the loss of cells from the proximal tubules. Such toxicity may be mediated through intracellular events induced by trichosanthin.