Fuyou Liu

Mitochondrial dynamics: regulatory mechanisms and emerging role in renal pathophysiology.

Mitochondria are a class of dynamic organelles that constantly undergo fission and fusion. Mitochondrial dynamics is governed by a complex molecular machinery and finely tuned by regulatory proteins. During cell injury or stress, the dynamics is shifted to fission, resulting in mitochondrial fragmentation, which contributes to mitochondrial damage and consequent cell injury and death. Emerging evidence has suggested a role of mitochondrial fragmentation in the pathogenesis of renal diseases including acute kidney injury and diabetic nephropathy. A better understanding of the regulation of mitochondrial dynamics and its pathogenic changes may unveil novel therapeutic strategies.

Low-dose paclitaxel ameliorates renal fibrosis in rat UUO model by inhibition of TGF- β /Smad activity

Transforming growth factor-β (TGF-β) has a pivotal function in the progression of renal fibrosis in a wide variety of renal diseases. Smad proteins have been identified to have an important function in regulating the expression of extracellular matrix (ECM) proteins through TGF-β signaling pathway. Aberrant TGF-β/Smad signaling can be modulated by stabilization of microtubules with paclitaxel. In this study, we investigated if paclitaxel can attenuate tubulointerstitial fibrosis in a rat model of unilateral ureteral obstruction (UUO). Rats in groups of six were subjected to UUO and received low-dose intraperitoneal injection of paclitaxel (0.3 mg/kg) twice a week. They were killed at day 7 and 14 after UUO or Sham operation. TGF-β signaling cascade and status of various ECM proteins were evaluated by RT–PCR, western blotting and immunohistochemical or immunofluorescence staining. The paclitaxel treatment markedly suppressed Smad2 and Smad3 phosphorylation. This was associated with attenuated expression of integrin-linked kinase, collagens I and III, fibronectin (FN) and α-smooth muscle actin, and a substantial decrease in renal fibrosis in animals that underwent UUO and received paclitaxel. These data indicate that the low-dose paclitaxel ameliorates renal tubulointerstitial fibrosis by modulating TGF-β signaling, and thus, the paclitaxel may have some therapeutic value in humans.

Low-dose paclitaxel ameliorates fibrosis in the remnant kidney model by down-regulating miR-192

Transforming growth factor (TGF)‐β has been shown to play a central role in the development of tubulointerstitial fibrosis, which can be corrected via treatment with paclitaxel. The biology of microRNA (miR) can be modulated by paclitaxel. We hypothesized that paclitaxel may attenuate renal fibrosis in a rat model of remnant kidney disease by inhibiting TGF‐β induced‐miRs. Rats in groups of 12 were subjected to 5/6 nephrectomy and received low‐dose intraperitoneal injection of paclitaxel. Renal functions were assessed at 8 weeks. The TGF‐β signalling cascade and ECM proteins were evaluated by real‐time polymerase chain reaction (TRT–PCR) and immunofluorescence microscopy. Animals with remnant kidneys developed hypertension, which was not relieved with paclitaxel treatment. However, paclitaxel treatment resulted in dampening the proteinuric response, reduction in serum BUN, creatinine levels and urine protein : creatinine ratio and normalization of creatinine clearance. These effects were accompanied by the inhibition of Smad2/3 activation, attenuation of renal fibrosis and normalization of integrin‐linked kinase (ILK), COL(I)A1, COL(IV)A2 and α‐SMA expression. Also, paclitaxel down‐regulated the expression of miR‐192, miR‐217 and miR ‐377, while miR‐15 was up‐regulated in the remnant kidney. In vitro, in tubular epithelial cells (NRK‐52E), paclitaxel also inhibited TGF‐β1‐induced Smad2/3 activation and normalized ILK, COL(I)A1, COL(IV)A2 and α‐SMA expression. Furthermore, ChIP analyses indicated that Taxol suppressed Smad3‐mediated miR‐192 transcriptional activity. Over‐expression of miR‐192 in NRK‐52E mimicked the changes seen in the remnant kidney, while inclusion of miR‐192 inhibitor in the culture medium blocked TGF‐β1‐induced COL(I)A1 and COL(IV)A2 expression, while ILK and α‐SMA were unaffected. These data suggest that low‐dose paclitaxel ameliorates renal fibrosis via modulating miR‐192 pathobiology and TGF‐β/Smad signalling. Copyright

Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts

Interstitial fibrosis is an inevitable outcome of all kinds of progressive chronic kidney disease (CKD). Emerging data indicate that rapamycin can ameliorate kidney fibrosis by reducing the interstitial infiltrates and accumulation of extra cellular matrix (ECM). However, the cellular mechanism that regulates those changes has not been well understood yet. In this study, we revealed the persistent activation of mammalian target of rapamycin (mTOR) signaling in the interstitial macrophages and myofibroblasts, but rarely in injured proximal epithelial cells, CD4+ T cells, neutrophils, or endothelial cells, during the development of kidney fibrosis. Administration of rapamycin to unilateral ureteral obstruction (UUO) mice significantly suppressed the immunoreactivity of mTOR signaling, which decreased the inflammatory responses and ECM accumulation in the obstructed kidneys. Isolated macrophages from rapamycin-treated obstructed kidneys presented less inflammatory activity than vehicle groups. In vitro study confirmed that rapamycin significantly inhibited the fibrogenic activation of cultured fibroblasts (NIH3T3 cells), which was induced by the stimulation of TGF-β1. Further experiment revealed that rapamycin did not directly inhibit the fibrogenesis of HK2 cells with aristolochic acid treatment. Our findings clarified that rapamycin can ameliorate kidney fibrosis by blocking the mTOR signaling in interstitial macrophages and myofibroblasts.

Mitochondrial dysregulation and protection in cisplatin nephrotoxicity

Abstract Nephrotoxicity is a major side effect of cisplatin in chemotherapy. Pathologically, cisplatin nephrotoxicity is characterized by cell injury and death in renal tubules. The research in the past decade has gained significant understanding of the cellular and molecular mechanisms of tubular cell death, revealing a central role of mitochondrial dysregulation. The pathological changes in mitochondria in cisplatin nephrotoxicity are mainly triggered by DNA damage response, pro-apoptotic protein attack, disruption of mitochondrial dynamics, and oxidative stress. As such, inhibitory strategies targeting these cytotoxic events may provide renal protection. Nonetheless, ideal approaches for renoprotection should not only protect kidneys but also enhance the anticancer efficacy of cisplatin in chemotherapy.

Tubular p53 Regulates Multiple Genes to Mediate AKI

A pathogenic role of p53 in AKI was suggested a decade ago but remains controversial. Indeed, recent work indicates that inhibition of p53 protects against ischemic AKI in rats but exacerbates AKI in mice. One intriguing possibility is that p53 has cell type-specific roles in AKI. To determine the role of tubular p53, we generated two conditional gene knockout mouse models, in which p53 is specifically ablated from proximal tubules or other tubular segments, including distal tubules, loops of Henle, and medullary collecting ducts. Proximal tubule p53 knockout (PT-p53-KO) mice were resistant to ischemic and cisplatin nephrotoxic AKI, which was indicated by the analysis of renal function, histology, apoptosis, and inflammation. However, other tubular p53 knockout (OT-p53-KO) mice were sensitive to AKI. Mechanistically, AKI associated with the upregulation of several known p53 target genes, including Bax, p53-upregulated modulator of apoptosis-α, p21, and Siva, and this association was attenuated in PT-p53-KO mice. In global expression analysis, ischemic AKI induced 371 genes in wild-type kidney cortical tissues, but the induction of 31 of these genes was abrogated in PT-p53-KO tissues. These 31 genes included regulators of cell death, metabolism, signal transduction, oxidative stress, and mitochondria. These results suggest that p53 in proximal tubular cells promotes AKI, whereas p53 in other tubular cells does not.

Rap1 ameliorates renal tubular injury in diabetic nephropathy

Rap1b ameliorates high glucose (HG)-induced mitochondrial dysfunction in tubular cells. However, its role and precise mechanism in diabetic nephropathy (DN) in vivo remain unclear. We hypothesize that Rap1 plays a protective role in tubular damage of DN by modulating primarily the mitochondria-derived oxidative stress. The role and precise mechanisms of Rap1b on mitochondrial dysfunction and of tubular cells in DN were examined in rats with streptozotocin (STZ)-induced diabetes that have Rap1b gene transfer using an ultrasound microbubble-mediated technique as well as in renal proximal epithelial tubular cell line (HK-2) exposed to HG ambiance. The results showed that Rap1b expression decreased significantly in tubules of renal biopsies from patients with DN. Overexpression of a constitutively active Rap1b G12V notably ameliorated renal tubular mitochondrial dysfunction, oxidative stress, and apoptosis in the kidneys of STZ-induced rats, which was accompanied with increased expression of transcription factor C/EBP-β and PGC-1α. Furthermore, Rap1b G12V also decreased phosphorylation of Drp-1, a key mitochondrial fission protein, while boosting the expression of genes related to mitochondrial biogenesis and antioxidants in HK-2 cells induced by HG. These effects were imitated by transfection with C/EBP-β or PGC-1α short interfering RNA. In addition, Rap1b could modulate C/EBP-β binding to the endogenous PGC-1α promoter and the interaction between PGC-1α and catalase or mitochondrial superoxide dismutase, indicating that Rap1b ameliorates tubular injury and slows the progression of DN by modulation of mitochondrial dysfunction via C/EBP-β–PGC-1α signaling.

Insights into the Mechanisms Involved in the Expression and Regulation of Extracellular Matrix Proteins in Diabetic Nephropathy

Diabetic Nephropathy (DN) is believed to be a major microvascular complication of diabetes. The hallmark of DN includes deposition of Extracellular Matrix (ECM) proteins, such as, collagen, laminin and fibronectin in the mesangium and renal tubulo-interstitium of the glomerulus and basement membranes. Such an increased expression of ECM leads to glomerular and tubular basement membranes thickening and increase of mesangial matrix, ultimately resulting in glomerulosclerosis and tubulointerstitial fibrosis. The characteristic morphologic glomerular mesangial lesion has been described as Kimmelstiel-Wilson nodule, and the process at times is referred to as diabetic nodular glomerulosclerosis. Thus, the accumulation of ECM proteins plays a critical role in the development of DN. The relevant mechanism(s) involved in the increased ECM expression and their regulation in the kidney in diabetic state has been extensively investigated and documented in the literature. Nevertheless, there are certain other mechanisms that may yet be conclusively defined. Recent studies demonstrated that some of the new signaling pathways or molecules including, Notch, Wnt, mTOR, TLRs and small GTPase may play a pivotal role in the modulation of ECM regulation and expression in DN. Such modulation could be operational for instance Notch through Notch1/Jagged1 signaling, Wnt by Wnt/β- catenin pathway and mTOR via PI3-K/Akt/mTOR signaling pathways. All these pathways may be critical in the modulation of ECM expression and tubulo-interstitial fibrosis. In addition, TLRs, mainly the TLR2 and TLR4, by TLR2- dependent and TGF-β-dependent conduits, may modulate ECM expression and generate a fibrogenic response. Small GTPase like Rho, Ras and Rab family by targeting relevant genes may also influence the accumulation of ECM proteins and renal fibrosis in hyperglycemic states. This review summarizes the recent information about the role and mechanisms by which these molecules and signaling pathways regulate ECM synthesis and its expression in high glucose ambience in vitro and in vivo states. The understanding of such signaling pathways and the molecules that influence expression, secretion and amassing of ECM may aid in developing strategies for the amelioration of diabetic nephropathy.

NEPHROTOXICITY OF HIGH- AND LOW-OSMOLAR CONTRAST MEDIA: The protective role of amlodipine in a rat model

Purpose: To evaluate the nephrotoxicity of high- and low-osmolar contrast media (HOCM, LOCM) on kidneys in Sprague-Dawley rats. The protective role of amlodipine was studied. Material and Methods: Forty rats of both sexes were randomly divided into 5 groups (n = 8/group) and glycerine for inducing renal failure was given to all rats except controls. Results: In diatrizoate-injected rats, blood urea nitrogen (BUN) and serum creatinine (SCr) were increased; levels of phospholipase A2 (PLA2), lipid peroxide (LPO) and calcium were also increased in renal tissues. There was no significant difference between LOCM (iohexol) animals and glycerol controls either in the renal levels of PLA2, LPO and calcium or in the levels of BUN and SCr. The histologic changes were milder in the LOCM animals than in the HOCM animals. In the group pretreated with amlodipine, no increase in the levels of BUN or SCr was discovered and the renal content of PLA2, LPO and calcium were significantly lower than in the HOCM group; the renal injuries induced by diatrizoate were alleviated. Conclusion: The HOCM, diatrizoate, was more toxic to rat kidneys than the LOCM iohexol; PLA2, LPO and calcium load played a role in producing renal function impairment induced by diatrizoate meglumine; amlodipine protected the renal tissue from nephrotoxicity induced by diatrizoate.

The mitochondria-targeted antioxidant MitoQ ameliorated tubular injury mediated by mitophagy in diabetic kidney disease via Nrf2/PINK1.

Mitochondria play a crucial role in tubular injury in diabetic kidney disease (DKD). MitoQ is a mitochondria-targeted antioxidant that exerts protective effects in diabetic mice, but the mechanism underlying these effects is not clear. We demonstrated that mitochondrial abnormalities, such as defective mitophagy, mitochondrial reactive oxygen species (ROS) overexpression and mitochondrial fragmentation, occurred in the tubular cells of db/db mice, accompanied by reduced PINK and Parkin expression and increased apoptosis. These changes were partially reversed following an intraperitoneal injection of mitoQ. High glucose (HG) also induces deficient mitophagy, mitochondrial dysfunction and apoptosis in HK-2 cells, changes that were reversed by mitoQ. Moreover, mitoQ restored the expression, activity and translocation of HG-induced NF-E2-related factor 2 (Nrf2) and inhibited the expression of Kelch-like ECH-associated protein (Keap1), as well as the interaction between Nrf2 and Keap1. The reduced PINK and Parkin expression noted in HK-2 cells subjected to HG exposure was partially restored by mitoQ. This effect was abolished by Nrf2 siRNA and augmented by Keap1 siRNA. Transfection with Nrf2 siRNA or PINK siRNA in HK-2 cells exposed to HG conditions partially blocked the effects of mitoQ on mitophagy and tubular damage. These results suggest that mitoQ exerts beneficial effects on tubular injury in DKD via mitophagy and that mitochondrial quality control is mediated by Nrf2/PINK.