Zhanmei Zhou

Kindlin-2 Mediates Activation of TGF-β/Smad Signaling and Renal Fibrosis

Activation of TGF-β/Smad signaling plays a central role in the pathogenesis of tubulointerstitial fibrosis, but the mechanisms underlying the initial interaction of the TGF-β receptor with Smads, leading to their activation, remain unclear. Here, we found that Kindlin-2, an integrin-binding protein, physically mediated the interaction of the TGF-β type I receptor (TβRI) with Smad3 in human kidney tubular epithelial cells. Kindlin-2 bound to TβRI through its FERM domain and to Smad3 through its N terminus. Overexpression of Kindlin-2 increased TGF-β-induced Smad3 activation. Knockdown of Kindlin-2 significantly suppressed the engagement of TβRI with Smad3 and inhibited TGF-β-induced Smad3 activation, as well as the expression of its target genes. Neither transfection of a Kindlin-2 mutant incapable of binding to β1 integrin nor knockdown of β1 integrin influenced the effect of Kindlin-2 on TGF-β1-induced Smad3 activation, indicating that this effect is independent of integrin. Kindlin-2 expression was markedly increased, predominantly in renal tubular epithelial cells, both in the unilateral ureteral obstruction model of kidney fibrosis and in human tissue exhibiting tubulointerstitial fibrosis. Furthermore, in the unilateral ureteral obstruction model, knocking down Kindlin-2 significantly inhibited activation of TGF-β/Smad signaling, decreased the expression of matrix genes, and ameliorated fibrosis. In summary, Kindlin-2 physically interacts with both TβRI and Smad3, promoting the activation of TGF-β/Smad signaling and contributing to the pathogenesis of tubulointerstitial fibrosis. Blockade of Kindlin-2 might be a rational therapeutic strategy for the treatment of fibrotic kidney diseases.

A Salt-Induced Reno-Cerebral Reflex Activates Renin-Angiotensin Systems and Promotes CKD Progression

Salt intake promotes progression of CKD by uncertain mechanisms. We hypothesized that a salt-induced reno-cerebral reflex activates a renin-angiotensin axis to promote CKD. Sham-operated and 5/6-nephrectomized rats received a normal-salt (0.4%), low-salt (0.02%), or high-salt (4%) diet for 2 weeks. High salt in 5/6-nephrectomized rats increased renal NADPH oxidase, inflammation, BP, and albuminuria. Furthermore, high salt activated the intrarenal and cerebral, but not the systemic, renin-angiotensin axes and increased the activity of renal sympathetic nerves and neurons in the forebrain of these rats. Renal fibrosis was increased 2.2-fold by high versus low salt, but intracerebroventricular tempol, losartan, or clonidine reduced this fibrosis by 65%, 69%, or 59%, respectively, and renal denervation or deafferentation reduced this fibrosis by 43% or 38%, respectively (all P<0.05). Salt-induced fibrosis persisted after normalization of BP with hydralazine. These data suggest that the renal and cerebral renin-angiotensin axes are interlinked by a reno-cerebral reflex that is activated by salt and promotes oxidative stress, fibrosis, and progression of CKD independent of BP.

GQ5 Hinders Renal Fibrosis in Obstructive Nephropathy by Selectively Inhibiting TGF-β–Induced Smad3 Phosphorylation

TGF-β1, via Smad-dependent or Smad-independent signaling, has a central role in the pathogenesis of renal fibrosis. This pathway has been recognized as a potential target for antifibrotic therapy. Here, we identified GQ5, a small molecular phenolic compound isolated from the dried resin of Toxicodendron vernicifluum, as a potent and selective inhibitor of TGF-β1-induced Smad3 phosphorylation. In TGF-β1-stimulated renal tubular epithelial cells and interstitial fibroblast cells, GQ5 inhibited the interaction of Smad3 with TGF-β type I receptor (TβRI) by blocking binding of Smad3 to SARA, suppressed subsequent phosphorylation of Smad3, reduced nuclear translocation of Smad2, Smad3, and Smad4, and downregulated the transcription of major fibrotic genes such as α-smooth muscle actin (α-SMA), collagen I, and fibronectin. Notably, intraperitoneal administration of GQ5 in rats immediately after unilateral ureteral obstruction (UUO) selectively inhibited Smad3 phosphorylation in UUO kidneys, suppressed renal expression of α-SMA, collagen I, and fibronectin, and resulted in impressive renal protection after obstructive injury. Late administration of GQ5 also effectively attenuated fibrotic lesions in obstructive nephropathy. In conclusion, our results suggest that GQ5 hinders renal fibrosis in rats by selective inhibition of TGF-β1-induced Smad3 phosphorylation.

Reno-Cerebral Reflex Activates the Renin-Angiotensin System, Promoting Oxidative Stress and Renal Damage After Ischemia-Reperfusion Injury.

AIMS A kidney-brain interaction has been described in acute kidney injury, but the mechanisms are uncertain. Since we recently described a reno-cerebral reflex, we tested the hypothesis that renal ischemia-reperfusion injury (IRI) activates a sympathetic reflex that interlinks the renal and cerebral renin-angiotensin axis to promote oxidative stress and progression of the injury. RESULTS Bilateral ischemia-reperfusion activated the intrarenal and cerebral, but not the circulating, renin-angiotensin system (RAS), increased sympathetic activity in the kidney and the cerebral sympathetic regulatory regions, and induced brain inflammation and kidney injury. Selective renal afferent denervation with capsaicin or renal denervation significantly attenuated IRI-induced activation of central RAS and brain inflammation. Central blockade of RAS or oxidative stress by intracerebroventricular (ICV) losartan or tempol reduced the renal ischemic injury score by 65% or 58%, respectively, and selective renal afferent denervation or reduction of sympathetic tone by ICV clonidine decreased the score by 42% or 52%, respectively (all p < 0.05). Ischemia-reperfusion-induced renal damage and dysfunction persisted after controlling blood pressure with hydralazine. INNOVATION This study uncovered a novel reflex pathway between ischemic kidney and the brain that sustains renal oxidative stress and local RAS activation to promote ongoing renal damage. CONCLUSIONS These data suggest that the renal and cerebral renin-angiotensin axes are interlinked by a reno-cerebral sympathetic reflex that is activated by ischemia-reperfusion, which contributes to ischemia-reperfusion-induced brain inflammation and worsening of the acute renal injury. Antioxid. Redox Signal. 27, 415-432.

Numb contributes to renal fibrosis by promoting tubular epithelial cell cycle arrest at G2/M

Numb is a multifunctional protein involved in diverse cellular processes. However, the function of Numb in kidney remains unclear. Here, we reported that Numb is expressed in renal tubules and glomeruli in normal adult kidney. Numb expression was upregulated in fibrotic kidneys induced by unilateral ureteral obstruction (UUO) in mice as well as in human fibrotic kidney tissues. Numb overexpression in cultured proximal tubular cells increased the G2/M cell population and upregulated the expression of TGF-β1 and CTGF. Whereas, proximal tubule Numb knockout (PEPCK-Numb-KO) mice showed reduced G2/M arrest, decreased expression of TGF-β1 and CTGF, and attenuated fibrotic lesions due to either UUO or unilateral ischemia reperfusion nephropathy. Inhibiting p53 activity by pifithrin-β dramatically mitigated Numb-induced G2/M arrest, indicating that Numb potentiates G2/M arrest via stabilizing p53 protein. Together, these data suggest that Numb is a potential target for anti-fibrosis therapy.

WT1 ameliorates podocyte injury via repression of EZH2/β-catenin pathway in diabetic nephropathy

Abstract Epigenetic modulation of podocyte injury plays a pivotal role in diabetic nephropathy (DN). Wilms tumor 1 (WT1) has been found to have opposing roles with &bgr;‐catenin in podocyte biology. Herein, we asked whether the histone methyltransferase enzyme enhancer of zeste homolog 2 (EZH2) promotes WT1‐induced podocyte injury via &bgr;‐catenin activation and the underlying mechanisms. We found that WT1 antagonized EZH2 and ameliorated &bgr;‐catenin‐mediated podocyte injury as demonstrated by attenuated podocyte mesenchymal transition, maintenance of podocyte architectural integrity, decreased podocyte apoptosis and oxidative stress. Further, we provided mechanistical evidence that EZH2 was required in WT1‐mediated &bgr;‐catenin inactivation via repression of secreted frizzled‐related protein 1 (SFRP‐1), a Wnt antagonist. Moreover, EZH2‐mediated silencing of SFRP‐1 was due to increased histone 3 lysine 27 trimethylation (H3K27me3) on its promoter region. WT1 favored renal function and decreased podocyte injury in diabetic rats and DN patients. Notably, WT1 exhibited clinical and biological relevance as it was linked to dropped serum creatinine, decreased proteinuria and elevated estimated glomerular filtration rate (eGFR). We propose an epigenetic process via the WT1/EZH2/&bgr;‐catenin axis in attenuating podocyte injury in DN. Targeting WT1 and EZH2 could be potential therapeutic approaches for DN. HighlightsWT1 antagonized EZH2 and ameliorated &bgr;‐catenin‐mediated podocyte injury in DN.EZH2 was required in WT1‐mediated &bgr;‐catenin inactivation via repression of SFRP‐1.EZH2 and &bgr;‐catenin colocalization was identified in podocytes in vitro and in vivo.EZH2 is involved in the epigenetic modulation of WT1 and &bgr;‐catenin in podocytes in DN.

Wnt9a Promotes Renal Fibrosis by Accelerating Cellular Senescence in Tubular Epithelial Cells

Cellular senescence is associated with renal disease progression, and accelerated tubular cell senescence promotes the pathogenesis of renal fibrosis. However, the underlying mechanism is unknown. We assessed the potential role of Wnt9a in tubular cell senescence and renal fibrosis. Compared with tubular cells of normal subjects, tubular cells of humans with a variety of nephropathies and those of several mouse models of CKD expressed high levels of Wnt9a that colocalized with the senescence-related protein p16INK4A Wnt9a expression level correlated with the extent of renal fibrosis, decline of eGFR, and expression of p16INK4A Furthermore, ectopic expression of Wnt9a after ischemia-reperfusion injury (IRI) induced activation of β-catenin and exacerbated renal fibrosis. Overexpression of Wnt9a exacerbated tubular senescence, evidenced by increased detection of p16INK4A expression and senescence-associated β-galactosidase activity. Conversely, shRNA-mediated knockdown of Wnt9a repressed IRI-induced renal fibrosis in vivo and impeded the growth of senescent tubular epithelial cells in culture. Notably, Wnt9a-induced renal fibrosis was inhibited by shRNA-mediated silencing of p16INK4A in the IRI mouse model. In a human proximal tubular epithelial cell line and primary renal tubular cells, Wnt9a remarkably upregulated levels of senescence-related p16INK4A, p19ARF, p53, and p21 and decreased the phosphorylation of retinoblastoma protein. Wnt9a also induced senescent tubular cells to produce TGF-β1, which promoted proliferation and activation in normal rat kidney fibroblasts. Thus, Wnt9a drives tubular senescence and fibroblast activation. Furthermore, the Wnt9a-TGF-β pathway appears to create a reciprocal activation loop between senescent tubular cells and activated fibroblasts that promotes and accelerates the pathogenesis of renal fibrosis.

QiShenYiQi Attenuates Renal Interstitial Fibrosis by Blocking the Activation of β-Catenin

Chronic kidney disease (CKD) is becoming a worldwide problem. However, current treatment options are limited. In the current study we showed that QiShenYiQi (QSYQ), a water-ethanol extract from several Chinese medicines, is a potent inhibitor of renal interstitial fibrosis. QSYQ inhibited transforming growth factor-β1 (TGF-β1)-responsive α-smooth muscle actin (α-SMA), collagen I, and fibronectin up-regulation in obstructive nephropathy and cultured cells. Administration of QSYQ also inhibited the established renal interstitial fibrosis in obstructive nephropathy. Interestingly, QSYQ selectively inhibited TGF-β1-induced β-catenin up-regulation and downstream gene transcription. Taken together, our study suggests that QSYQ selectively inhibits TGF-β1-induced β-catenin up-regulation and might have significant therapeutic potential for the treatment of renal fibrosis.

Long noncoding RNA lnc-TSI inhibits renal fibrogenesis by negatively regulating the TGF-β/Smad3 pathway

A human long noncoding RNA targeting the TGF-β/Smad pathway reduced fibrogenesis in mouse models of renal fibrosis. A target for renal fibrosis Transforming growth factor–β (TGF-β) is a known regulator of fibrosis but has remained difficult to target. Wang et al. identified a kidney-specific long noncoding RNA, lnc-TSI, that inhibited Smad3 activation and downstream profibrogenic gene expression in human tubular epithelial cells (TECs). Ectopic lnc-TSI expression in mouse TECs confirmed inhibition of TGF-β signaling, and delivery of lnc-TSI in unilateral ureteral obstruction and ischemic reperfusion injury–induced fibrosis models resulted in reduced renal fibrogenesis. Expression of lnc-TSI negatively correlated with fibrosis and renal failure in IgA nephropathy patients, raising the possibility that the lncRNA could be a potential therapeutic target. Transforming growth factor–β (TGF-β) is a well-established central mediator of renal fibrosis, a common outcome of almost all progressive chronic kidney diseases. Here, we identified a poorly conserved and kidney-enriched long noncoding RNA in TGF-β1–stimulated human tubular epithelial cells and fibrotic kidneys, which we termed TGF-β/Smad3-interacting long noncoding RNA (lnc-TSI). Lnc-TSI was transcriptionally regulated by Smad3 and specifically inhibited TGF-β–induced Smad3 phosphorylation and downstream profibrotic gene expression. Lnc-TSI acted by binding with the MH2 domain of Smad3, blocking the interaction of Smad3 with TGF-β receptor I independent of Smad7. Delivery of human lnc-TSI into unilateral ureteral obstruction (UUO) mice, a well-established model of renal fibrosis, inhibited phosphorylation of Smad3 in the kidney and attenuated renal fibrosis. In a cohort of 58 patients with biopsy-confirmed IgA nephropathy (IgAN), lnc-TSI renal expression negatively correlated with the renal fibrosis index (r = −0.56, P < 0.001) after adjusting for cofounders. In a longitudinal study, 32 IgAN patients with low expression of renal lnc-TSI at initial biopsy had more pronounced increases in their renal fibrosis index and experienced stronger declines in renal function at repeat biopsy at a mean of 48 months of follow-up. These data suggest that lnc-TSI reduced renal fibrogenesis through negative regulation of the TGF-β/Smad pathway.