Haiping Mao

Distinct hsp70 Domains Mediate Apoptosis-inducing Factor Release and Nuclear Accumulation *

Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-ΔEEVD) or ATPase function (hsp70-ΔATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury. In contrast, mutants lacking either the chaperone function (hsp70-ΔEEVD) or the ATP hydrolytic domain (hsp70-ΔATPase) failed to prevent mitochondrial AIF release. Although hsp70-ΔEEVD did not inhibit bax activation or mitochondrial membrane injury after cell stress, this hsp70 mutant co-immunoprecipitated with leaked AIF in injured cells and decreased nuclear AIF accumulation. In contrast, hsp70-ΔATPase did not interact with AIF either in intact cells or in a cell-free system and furthermore, failed to prevent nuclear AIF accumulation. These results demonstrate that mitochondrial protection against bax-mediated injury requires both intact chaperone and ATPase functions, whereas the ATPase domain is critical for sequestering AIF in the cytosol.

Prevalence and risk factors associated with chronic kidney disease in an adult population from southern China.

BACKGROUND Population-based studies evaluating the prevalence of kidney damage in different communities have been limited in developing countries. We conducted a population-based screening study in the southern Chinese city of Guangzhou that aimed to identify the prevalence and associated risk factors of chronic kidney disease (CKD) in southern Chinese populations. METHODS We interviewed 6311 residents (>20 years) from six districts of Guangzhou from July 2006 to June 2007 and tested for haematuria, albuminuria and reduced renal function. Associations between age, gender, smoking, diabetes mellitus, hypertension, hyperuricaemia and kidney damage were examined. RESULTS There were 6311 subjects enrolled in this study. After adjustment for age and gender, the prevalence of albuminuria, haematuria and reduced estimated glomerular filtration rate (eGFR) was 6.6% [95% confidence interval (CI): 5.5-7.6%], 3.8% (95% CI: 3.4%, 4.3%) and 3.2% (95% CI: 2.4%, 3.3%), respectively. Approximately 12.1% (95% CI: 11.3%, 12.9%) of the sample population had at least one indicator of kidney damage. Age, diabetes mellitus, hypertension, central obesity, hyperlipidaemia and use of nephrotoxic medications were independently associated with albuminuria; hyperuricaemia, age, gender, hypertension and use of nephrotoxic medications were independently associated with reduced eGFR, and female gender was independently associated with haematuria. CONCLUSIONS In the general adult population from southern China, 12.1% has either proteinuria, haematuria and/or reduced eGFR, indicating the presence of kidney damage, with an awareness of only 9.6%. The high prevalence and low awareness of CKD in this population suggest an urgent need for CKD prevention programmes in China.

HSP72 attenuates renal tubular cell apoptosis and interstitial fibrosis in obstructive nephropathy

Although heat shock protein 72 kDa (HSP72) protects tubular epithelium from a variety of acute insults, its role in chronic renal injury and fibrosis is poorly characterized. In this study, we tested the hypothesis that HSP72 reduces apoptosis and epithelial-to-mesenchymal transition (EMT), important contributors to tubular cell injury in vitro and in vivo. In rats, orally administered geranylgeranylacetone (GGA), an agent that selectively induces HSP72, markedly reduced both apoptosis and cell proliferation in tubular epithelium and decreased both interstitial fibroblast accumulation and collagen I deposition after unilateral ureteric obstruction, a model of chronic renal tubulointerstitial fibrosis and dysfunction. In cultured renal NRK52E cells, exposure to TGF-beta1 induced EMT and apoptosis, major causes of renal fibrosis and tubular atrophy, respectively. Exposure to a pan-caspase inhibitor (ZVAD-FMK) prevented TGF-beta1-induced apoptosis but did not reduce EMT. In contrast, selective HSP72 expression in vitro inhibited EMT caused by TGF-beta1 as indicated by preserving the E-cadherin expression level and alpha-smooth muscle actin induction. Small interfering RNA directed against HSP72 blocked the cytoprotective effects of HSP72 overexpression on EMT in TGF-beta1-exposed cells. Taken together, our data indicate that HSP72 ameliorates renal tubulointerstitial fibrosis in obstructive nephropathy by inhibiting both renal tubular epithelial cell apoptosis and EMT.

Clinical Outcome of Hyperuricemia in IgA Nephropathy: A Retrospective Cohort Study and Randomized Controlled Trial

Background: Hyperuricemia is an independent risk factor for renal progression in IgA nephropathy (IgAN). However, no study has evaluated the effect of allopurinol on the clinical outcome in hyperuricemic IgAN. Methods: First,a retrospective cohort study of 353 IgAN patients was conducted to explore the relationship between uric acid (UA) and the progression of renal disease over a mean period of 5 years. Then, 40 hyperuricemic IgAN patients were randomized to receive allopurinol (100–300 mg/day) or usual therapy for 6 months. The study outcomes were renal disease progression and/or blood pressure. Results: Hyperuricemia independently predicted renal survival at 1, 3, and 5 years after adjustment for different baseline estimated glomerular filtration rates. In the randomized controlled trial, allopurinol did not significantly alter renal progression or proteinuria. The antihypertensive drug dosage was reduced in 7 of 9 cases with hypertension in the allopurinol group compared to 0 of 9 cases in the control group (p < 0.01). UA levels correlated with mean arterial pressure in normotensive patients (r = 0.388, p < 0.001). Conclusion: Hyperuricemia predicts the progression of IgAN independently of baseline estimated glomerular filtration rate. Allopurinol may improve the control of blood pressure. Further studies are required to explore the effects of lowering UA on renal protection in IgAN.

β-Catenin Promotes Survival of Renal Epithelial Cells by Inhibiting Bax

Ischemia activates Bax, a proapoptotic BCL2 protein, as well as the prosurvival beta-catenin/Wnt signaling pathway. To test the hypothesis that beta-catenin/Wnt signaling regulates Bax-mediated apoptosis after induction of metabolic stress, which occurs during renal ischemia, we infected immortalized and primary proximal tubular epithelial cells with adenovirus to express either constitutively active or dominant negative beta-catenin constructs. Constitutively active beta-catenin significantly decreased apoptosis and improved cell survival after metabolic stress. Furthermore, active beta-catenin decreased Bax activation, oligomerization, and translocation to mitochondria, and reduced both organelle membrane injury and apoptosis. Dominant negative beta-catenin had the opposite effects. Because Akt regulates Bax, we examined the effects of the beta-catenin mutants on Akt expression and activation. Constitutively active beta-catenin increased Akt-1 expression and activation before and after stress, and treatment with a phosphatidylinositol-3 kinase inhibitor antagonized the protective effects of beta-catenin on Akt activation, Bax inhibition, and cell survival. In addition, beta-catenin significantly increased the rate of phosphorylation at Bax serine(184), an Akt-specific target. Taken together, these results suggest that beta-catenin/Wnt signaling promotes survival of renal epithelial cells after metabolic stress, in part by inhibiting Bax in a phosphatidylinositol-3 kinase/Akt-dependent manner.

GSK3β Promotes Apoptosis after Renal Ischemic Injury

The mechanism by which the serine-threonine kinase glycogen synthase kinase-3beta (GSK3beta) affects survival of renal epithelial cells after acute stress is unknown. Using in vitro and in vivo models, we tested the hypothesis that GSK3beta promotes Bax-mediated apoptosis, contributing to tubular injury and organ dysfunction after acute renal ischemia. Exposure of renal epithelial cells to metabolic stress activated GSK3beta, Bax, and caspase 3 and induced apoptosis. Expression of a constitutively active GSK3beta mutant activated Bax and decreased cell survival after metabolic stress. In contrast, pharmacologic inhibition (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione [TDZD-8]) or RNA interference-mediated knockdown of GSK3beta promoted cell survival. Furthermore, RNA interference-mediated knockdown of Bax abrogated the cell death induced by constitutively active GSK3beta. In a cell-free assay, TDZD-8 inhibited the phosphorylation of a peptide containing the Bax serine(163) site targeted by stress-activated GSK3beta. In rats, TDZD-8 inhibited ischemia-induced activation of GSK3beta, Bax, and caspase 3; ameliorated tubular and epithelial cell damage; and significantly protected renal function. Taken together, GSK3beta-mediated Bax activation induces apoptosis and tubular damage that contribute to acute ischemic kidney injury.

HSP72 Inhibits Smad3 Activation and Nuclear Translocation in Renal Epithelial-to-Mesenchymal Transition

Although heat shock protein 72 (HSP72) ameliorates renal tubulointerstitial fibrosis by inhibiting epithelial-to-mesenchymal transition (EMT), the underlying mechanism is unknown. Because Smad proteins transduce TGF-beta signaling from the cytosol to the nucleus and HSP72 assists in protein folding and facilitates nuclear translocation, we investigated whether HSP72 inhibits TGF-beta-induced EMT by modulating Smad expression, activation, and nuclear translocation. To evaluate the roles of distinct HSP72 structural domains in these processes, we constructed vectors that expressed wild-type HSP72 or mutants lacking either the peptide-binding domain (HSP72-DeltaPBD), which is responsible for substrate binding and refolding, or the nuclear localization signal (HSP72-DeltaNLS). Overexpression of wild-type HSP72 or HSP72-DeltaNLS inhibited TGF-beta1-induced EMT, but HSP72-DeltaPBD did not, suggesting a critical role for the PBD in this inhibition. HSP72 overexpression inhibited TGF-beta1-induced phosphorylation and nuclear translocation of Smad3 and p-Smad3, but not Smad2; these inhibitory effects required the PBD but not the NLS. Coimmunoprecipitation assays suggested a physical interaction between Smad3 and the PBD. siRNA knockdown of endogenous HSP72 enhanced both TGF-beta1-induced Smad3 phosphorylation and EMT and confirmed the interaction of HSP72 with both Smad3 and p-Smad3. In vivo, induction of HSP72 by geranylgeranylacetone suppressed Smad3 phosphorylation in renal tubular cells after unilateral ureteral obstruction. In conclusion, HSP72 inhibits EMT in renal epithelial cells primarily by exerting domain-specific effects on Smad3 activation and nuclear translocation.

hsp72 inhibits focal adhesion kinase degradation in ATP-depleted renal epithelial cells.

Prior heat stress (HS) or the selective overexpression of hsp72 prevents apoptosis caused by exposure to metabolic inhibitors by protecting the mitochondrial membrane and partially reducing caspase-3 activation. Focal adhesion kinase (FAK), a tyrosine kinase, exhibits anti-apoptotic properties and is a potential target for degradation by caspase-3. This study tested the hypothesis that hsp72 interacts with FAK, preventing caspase-3-mediated degradation during ATP depletion. ATP depletion (5 mmNaCN and 5 mm 2-deoxy-d-glucose in the absence of medium glucose) caused FAK degradation within 15 min. FAK degradation was completely prevented by a caspase-3-specific inhibitor. HS induced the accumulation of hsp72, increased the interaction between hsp72 and FAK, and significantly inhibited FAK degradation during ATP depletion. Selective overexpression of wild-type hsp72 (but not hsp72ΔEEVD) reproduced the protective effects of HS on FAK cleavage. Purified hsp72 prevented the degradation of FAK by caspase-3 in vitro in a dose-dependent manner without affecting caspase-3 activity. Interaction between hsp72 and FAK is critical because both exogenous ATP and deletion of the substrate-binding site decreased protection of FAK by hsp72. These data indicate that FAK is an early target of injury in cells exposed to metabolic inhibitors and demonstrate that hsp72 reduces caspase-3-mediated proteolysis of FAK, an anti-apoptotic protein.

A Crosstalk between the Smad and JNK Signaling in the TGF-β-Induced Epithelial-Mesenchymal Transition in Rat Peritoneal Mesothelial Cells

Transforming growth factor β (TGF-β) induces the process of epithelial-mesenchymal transition (EMT) through the Smad and JNK signaling. However, it is unclear how these pathways interact in the TGF-β1-induced EMT in rat peritoneal mesothelial cells (RPMCs). Here, we show that inhibition of JNK activation by introducing the dominant-negative JNK1 gene attenuates the TGF-β1-down-regulated E-cadherin expression, and TGF-β1-up-regulated α-SMA, Collagen I, and PAI-1 expression, leading to the inhibition of EMT in primarily cultured RPMCs. Furthermore, TGF-β1 induces a bimodal JNK activation with peaks at 10 minutes and 12 hours post treatment in RPMCs. In addition, the inhibition of Smad3 activation by introducing a Smad3 mutant mitigates the TGF-β1-induced second wave, but not the first wave, of JNK1 activation in RPMCs. Moreover, the inhibition of JNK1 activation prevents the TGF-β1-induced Smad3 activation and nuclear translocation, and inhibition of the TGF-β1-induced second wave of JNK activation greatly reduced TGF-β1-induced EMT in RPMCs. These data indicate a crosstalk between the JNK1 and Samd3 pathways during the TGF-β1-induced EMT and fibrotic process in RPMCs. Therefore, our findings may provide new insights into understanding the regulation of the TGF-β1-related JNK and Smad signaling in the development of fibrosis.

Heat Shock Protein 72 Enhances Autophagy as a Protective Mechanism in Lipopolysaccharide-Induced Peritonitis in Rats

Peritoneal dialysis-related peritonitis causes the denudation of mesothelial cells and, ultimately, membrane integrity alterations and peritoneal dysfunction. Because heat shock protein 72 (HSP72) confers protection against apoptosis and because autophagy mediates survival in response to cellular stresses, we examined whether autophagy contributes to HSP72-mediated cytoprotection in lipopolysaccharide (LPS)-induced peritonitis. Exposure of cultured peritoneal mesothelial cells to LPS resulted first in autophagy and later, apoptosis. Inhibition of autophagy by 3-methyladenine or Beclin-1 small-interfering RNA sensitized cells to apoptosis and abolished the antiapoptotic effect of HSP72, suggesting that autophagy activation acts as a prosurvival mechanism. Overexpression of HSP72 augmented autophagy through c-Jun N-terminal kinase (JNK) phosphorylation and Beclin-1 up-regulation. Suppression of JNK activity reversed HSP72-mediated Beclin-1 up-regulation and autophagy, indicating that HSP72-mediated autophagy is JNK dependent. In a rat model of LPS-associated peritonitis, autophagy occurred before apoptosis in peritoneum. Up-regulation of HSP72 by geranylgeranylacetone increased autophagy, inhibited apoptosis, and attenuated peritoneal injury, and these effects were blunted by down-regulation of HSP72 with quercetin. Additionally, blocking autophagy by chloroquine promoted apoptosis and aggravated LPS-associated peritoneal dysfunction. Thus, HSP72 protects peritoneum from LPS-induced mesothelial cells injury, at least in part by enhancing JNK activation-dependent autophagy and inhibiting apoptosis. These findings imply that HSP72 induction might be a potential therapy for peritonitis.