The role of Krüppel-like factor 4 in transforming growth factor-ß-induced inflammatory and fibrotic responses in human proximal tubule cells.

Krüppel-like factor 4 (KLF4) is known to mitigate inflammation in several cell types. Using human proximal tubule cells, the present study aimed to investigate the role of KLF4 in regulating transforming growth factor (TGF)-ß1 induced inflammatory and fibrotic responses. Human kidney proximal tubular cells were exposed to high glucose, or TGF-ß1 and KLF4 expressions were determined. Cells were then transfected with empty vector or KLF4 and exposed to 2-ng/mL TGF-ß1 for up to 72 h. Inflammatory proteins (macrophage migration inhibitory factor and monocyte chemoattractant protein-1) and pro-fibrotic proteins (fibronectin and collagen IV) were measured after 72 h by enzyme-linked immunosorbent assay and western blot, respectively. To determine the relevance to in vivo models of chronic kidney disease, KLF4 protein expression in streptozotocin-induced diabetic mice was determined. Krüppel-like factor 4 messenger RNA (mRNA) levels were significantly reduced in high glucose-treated human kidney proximal tubular cells. High glucose increased TGF-ß1 mRNA expression, which significantly increased migration inhibitory factor and monocyte chemoattractant protein-1 protein secretion. Transforming growth factor-ß1 significantly increased fibronectin and collagen IV protein expression. The overexpression of KLF4 significantly reduced TGF-ß-mediated increases in migration inhibitory factor and monocyte chemoattractant protein-1 but had no effect on TGF-ß-mediated fibronectin and collagen IV mRNA and protein expression. The levels of KLF4 mRNA were significantly reduced in the diabetic kidney, and diabetic animals had a significant reduction in renal tubular expression of KLF4 proteins. This data suggest that KLF4 reduces inflammation induced by TGF-ß1, suggesting a therapeutic role for KLF4 in diabetic nephropathy.

miR-124a is frequently down-regulated in glioblastoma and is involved in migration and invasion.

Glioblastoma (GBM) represents a formidable clinical challenge for both patients and treating physicians. Due to better local treatments and prolonged patient survival, remote recurrences are increasingly observed, underpinning the importance of targeting tumour migration and attachment. Aberrant expression of microRNA (miRNA) is commonly associated with cancer and loss of miR-124a has previously been implicated to function as a tumour suppressor. The assessment of miR-124a in clinical specimens has been limited and a potential role in migration and invasion has been unexplored until now. We measured the expression levels of mature miR-124a in a retrospective series of 119 cases of histologically confirmed GBM and found its expression was markedly lower in over 80% of the GBM clinical specimens compared to normal brain tissue. The level of reduction in the clinical cohort varied significantly and patients with lower than the average miR-124a expression levels displayed shorter survival times. Endogenous miR-124a expression and the protein expression of three of its targets; IQ motif containing GTPase activating protein 1 (IQGAP1), laminin γ1 (LAMC1) and integrin ß1 (ITGB1) were significantly reciprocally associated in the majority of the clinical cases. We confirmed this association in our in vitro model. Functionally, the ectopic expression of mature miR-124a in a GBM cell line resulted in significant inhibition of migration and invasion, demonstrating a role for miR-124a in promoting tumour invasiveness. Our results suggest that miR-124a may play a role in GBM migration, and that targeted delivery of miR-124a may be a novel inhibitor of GBM invasion.

Role of Kruppel-like factor 6 in transforming growth factor-beta1-induced epithelial-mesenchymal transition of proximal tubule cells.

Krüppel-like factor 6 (KLF6) is a DNA-binding protein containing a triple zinc-fingered motif and plays a key role in the regulation of cell proliferation, differentiation, and development. More recently it has been implicated in hepatic fibrosis via its binding to the transforming growth factor (TGF)-beta control element. In the kidney, epithelial-mesenchymal transition (EMT) is a major contributor to the pathogenesis of renal fibrosis, with TGF-beta1 being a key mediator of EMT. The present study aimed to determine the role of KLF6 and TGF-beta1 in EMT in proximal tubule cells. To determine the relevance in clinical disease, KLF6 was measured in kidneys of streptozotocin-induced diabetic Ren-2 rats and in cells exposed to high (30 mM) glucose. TGF-beta1 was confirmed to induce EMT by morphological change, loss of E-cadherin, and gain in vimentin expression. KLF6 mRNA expression was concomitantly measured. To determine the role of KLF6 in EMT, the above markers of EMT were determined in KLF6-silenced (small interfering RNA) and KLF6-overexpressing proximal tubule cells. KLF6 overexpression significantly promoted a phenotype consistent with EMT. High glucose induced KLF6 in proximal tubule cells (P < 0.05). This increase in KLF6 in response to high glucose was TGF-beta1 mediated. In an in vivo model of diabetic nephropathy KLF6 increased at week 8 (P < 0.05). KLF6 plays a permissive role in TGF-beta1-induced EMT in proximal tubule cells. Its upregulation in in vivo models of diabetic nephropathy suggests it as a potential therapeutic target.

High glucose induces macrophage inflammatory protein-3 alpha in renal proximal tubule cells via a transforming growth factor-beta 1 dependent mechanism.

BACKGROUND: Hyperglycaemia is a causative factor in the pathogenesis of diabetic nephropathy, known to induce chemokines in the kidney. Macrophage inflammatory protein-3 alpha (MIP-3 alpha) is a CC chemokine that has been reported to attract memory T lymphocytes. Our previous microarray study showed significant increased level of MIP-3 alpha in high glucose-induced transcriptional profile in renal proximal tubule cells. Transforming growth factor-beta1 (TGF-beta1) is a key regulator in inflammation and fibrosis in diabetes mellitus setting. METHODS: This study aimed to determine the role of TGF beta 1 in high glucose-induced MIP-3 alpha expression. An in vitro model of human proximal tubular cells (HK-2 cells) and an in vivo model of the transgenic (mRen-2)27 diabetic rat, well characterized as a model of human diabetic nephropathy, were used. Small interfering RNA technology was used to silence TGF-beta1 gene in HK-2 cells and subsequent experiments were performed to measure mRNA and protein levels of MIP-3 alpha using real time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to measure the protein level of MIP-3 alpha and CD3 a marker of T lymphocytes in the in vivo model. RESULTS: MIP-3 alpha mRNA and protein expression was increased in HK-2 cells by high glucose and TGF-beta1. MIP-3 alpha was up-regulated in the dilated tubules of diabetic rats compared with non-diabetic control animals and CD3 was found to be present around the dilated tubules expressing MIP-3 alpha. This up-regulation was attenuated in the presence of an angiotensin-converting enzyme (ACE) inhibitor. MIP-3 alpha expression significantly decreased in cells in which the TGF-beta1 gene was silenced using small interfering RNA. Furthermore, exposure to high glucose did not induce MIP-3 alpha expression in TGF-beta1 gene silenced cells compared with wild-type cells. CONCLUSIONS: In summary, we have uniquely demonstrated that high glucose increases MIP-3 alpha through a TGF beta 1 dependent pathway, suggesting the centrality of TGF-beta1 in both the inflammatory and previously demonstrated fibrotic responses in diabetic nephropathy.

Transforming growth factor-beta1 differentially mediates fibronectin and inflammatory cytokine expression in kidney tubular cells.

Transforming growth factor-beta(1) (TGF-beta(1)) is not only an important fibrogenic but also immunomodulatory cytokine in the human kidney. We have recently demonstrated that TGF-beta(1) induces interleukin-8 (IL-8), macrophage chemoattractant protein-1 (MCP-1), and fibronectin production in renal proximal tubular (HK-2) cells. However, the unique dependence of IL-8, MCP-1, and fibronectin on TGF-beta(1) expression is unknown. The TGF-beta(1) gene was effectively silenced in HK-2 cells using small-interference (si) RNA. Basal secretion of IL-8 and MCP-1 decreased (both P < 0.05) but, paradoxically, fibronectin increased (P < 0.05) in TGF-beta(1)-silenced cells compared with cells transfected with nonspecific siRNA. Significant increases were observed in mRNA for the TGF-beta(2) (P < 0.05), TGF-beta(3) (P < 0.05) isoforms and pSmad2 (P < 0.05), which were reflected in protein expression. Concurrent exposure to pan-specific TGF-beta antibody reversed the observed increase in fibronectin expression, suggesting that TGF-beta(2) and TGF-beta(3) isoforms mediate the increased fibronectin expression in TGF-beta(1)-silenced cells. An increase in the DNA binding activity of activator protein-1 (AP-1; P < 0.05) was also observed in TGF-beta(1)-silenced cells. In contrast, nuclear factor-kappaB (NF-kappaB) DNA binding activity was significantly decreased (P < 0.0005). These studies demonstrate that TGF-beta(1) is a key regulator of IL-8 and MCP-1, whereas fibronectin expression is regulated by a complex interaction between the TGF-beta isoforms in the HK-2 proximal tubular cell line. Decreased expression of TGF-beta(1) reduces chemokine production in association with reduced NF-kappaB DNA binding activity, suggesting that immunomodulatory pathways in the kidney are specifically dependent on TGF-beta(1). Conversely, decreased expression of TGF-beta(1) results in increased TGF-beta(2), TGF-beta(3), AP-1, and pSmad2 that potentially mediates the observed increase in fibronectin.

Rapamycin worsens renal function and intratubular cast formation in protein overload nephropathy.

BACKGROUND: Rapamycin (sirolimus) is associated with functional nephrotoxicity in some patients with nephrotic glomerular diseases but the pathophysiologic mechanisms are not known. This study investigated the effects of rapamycin on renal function and structure in protein overload nephropathy. METHODS: Rats with protein overload nephropathy [induced by bovine serum albumin (BSA), 2.1 g by daily intraperitoneal injection, day 0 to day 3] received daily intraperitoneal injections of either vehicle [dimethyl sulfoxide (DMSO)], rapamycin (0.2 mg/kg, an inhibitor of mammalian target of rapamycin), or roscovitine (3.5 mg/kg, a small molecule cyclin-dependent kinase inhibitor) (N= 9 each) from day -3 to day 3. RESULTS: In protein overload nephropathy, rapamycin caused severe acute renal failure and mild hypercholesterolemia (both P < 0.05). Rapamycin dramatically increased intratubular cast formation, and proximal tubular epithelial cells were swollen and engorged with increased cytoplasmic protein droplets. The number of 5-bromo-2'-deoxyuridine (BrdU)-positive tubular epithelial cells increased by more than 20-fold on day 3 in protein overload nephropathy, and this was attenuated by 65% with rapamycin (P < 0.05), whereas roscovitine was ineffective. Rapamycin increased the protein expression of p27(kip1) in tubular epithelial cells, but did not alter D-type cyclin expression or apoptosis. CONCLUSION: Rapamycin caused a specific pattern of acute renal injury characterized by increased intratubular cast formation in protein overload nephropathy. This could be due to disruption of a potentially important compensatory mechanism in nephrotic glomerular diseases involving tubular epithelial cell protein endocytosis and proliferation.