The mechanisms by which angiotensin II (Ang II) promotes renal fibrosis remain incompletely understood. Ang II both stimulates TGFb signaling and activates the EGF receptor (EGFR), but the relative contribution of these pathways to renal fibrogenesis is unknown. Using a murine model with EGFRdeficient proximal tubules, we demonstrate that upstream activation of EGFR-dependent ERK signaling is critical for mediating sustained TGFb expression in renal fibrosis. Persistent activation of the Ang II receptor stimulated ROS-dependent phosphorylation of Src, leading to sustained EGFRdependent signaling for TGFb expression. Either genetic or pharmacologic inhibition of EGFR significantly decreased TGFb-mediated fibrogenesis. We conclude that TGFb-mediated tissue fibrosis relies on a persistent feed-forward mechanism of EGFR/ERK activation through an unexpected signaling pathway, highlighting EGFR as a potential therapeutic target for modulating tissue fibrogenesis.

Diabetic kidney disease, a leading cause of end-stage renal disease, has become a serious public health problem worldwide and lacks effective therapies. Autophagy is a highly conserved lysosomal degradation pathway that removes protein aggregates and damaged organelles to maintain cellular homeostasis. As important stress-responsive machinery, autophagy is involved in the pathogenesis of various diseases. Emerging evidence has suggested that dysregulated autophagy may contribute to both glomerular and tubulointerstitial pathologies in kidneys under diabetic conditions. This review summarizes the recent findings regarding the role of autophagy in the pathogenesis of diabetic kidney disease and highlights the regulation of autophagy by the nutrient-sensing pathways and intracellular stress signaling in this disease. The advances in our understanding of autophagy in diabetic kidney disease will facilitate the discovery of a new therapeutic target for the prevention and treatment of this life-threatening diabetes complication.

Patients with chronic kidney diseases, including diabetic nephropathy, are more susceptible to acute kidney injury (AKI) and have a worse prognosis following AKI. However, the underlying mechanism is unclear. Here we tested whether diabetic mice were more sensitive to AKI and show that renal ischemia-reperfusion induced significantly more severe AKI and higher mortality in the streptozotocin and the Akita diabetic mouse models. The severity of AKI in the mice correlated with their blood glucose levels. In vitro, high glucose-conditioned renal proximal tubular cells showed higher apoptosis and caspase activation following ATP-depletion and hypoxic injury, accompanied by a heightened mitochondrial accumulation of Bax and release of cytochrome c. In response to injury, both glucose-conditioned renal proximal tubular cells and diabetic kidney tissues showed markedly higher p53 induction. Suppression of p53 diminished the sensitivity of high glucose-conditioned cells to acute injury in vitro. Moreover, blockade of p53 by pifithrin-α, siRNA, or proximal tubule-targeted gene ablation reduced ischemic AKI in diabetic mice. Insulin reduced blood glucose in diabetic mice and largely attenuated their AKI sensitivity. Thus, our results suggest the involvement of hyperglycemia, p53 and mitochondrial pathway of apoptosis in the susceptibility of diabetic models to AKI.

In our present studies utilizing a well characterized proximal tubule cell line, LLCPKcl4, we determined that all four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) stimulated [ 3 H]thymidine incorporation, with 14,15-EET being the most potent. In contrast, no mitogenic effects were seen with arachidonic acid, other cP450 arachidonate metabolites (12R-hydroxyeicosatetraenoic acid (12R-HETE), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), or 20-HETE), or lipoxygenase metabolites (5S-HETE, leukotriene B 4 , or lipoxin A4). We found that their metabolically more stable sulfonimide (SI) analogs (11,12-EET-SI and 14,15-EET-SI) were also potent mitogens. In addition 14,15-EET-SI also increased cell proliferation as well as expression of both c-fos and egr-1 mRNA. The protein kinase C and A inhibitors, H-7 and H-8, or the cyclooxygenase inhibitor, indomethacin, had no effect upon 14,15-EET-induced Cytochrome P450 epoxygenase catalyzes the NADPHdependent epoxidation of arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) 1 in a regio-and stereo-selective manner. The EETs are produced predominantly by epoxygenases of the 2C family of cP450s, which have been localized in the kidney to the mammalian proximal tubule. The proximal tubule contains the highest concentration of cP450 within the mammalian kidney (1) and expresses minimal cyclooxygenase or lipoxygenase activity (2). The EETs or their hydration products have been implicated in modulation of vascular tone and renal glomerular hemodynamics (3), renal proximal tubule function, and regulation of mitogenesis (4, 5). Recently, EETs have been suggested to be an endothelialderived hyperpolarizing factor (6), although there is controversy about this issue (7,8). Direct administration of EETs inhibits amiloride-sensitive sodium transport (9) and 86 Rb uptake in LLC-PK1, a nontransformed, immortalized cell line from pig kidney with certain proximal tubule characteristics (10).EETs have also been proposed as second messengers for hormones and growth factors in the proximal tubule. We have shown that epidermal growth factor (EGF) stimulates EET production in rat proximal tubule suspensions and primary cultured rabbit proximal tubule cells, and EETs may mediate both EGF-stimulated calcium influx and mitogenesis in proximal tubules (11). Omata et al. also demonstrated EET production upon stimulation with angiotensin II (12). EETs may mediate the effect of high (Ͼ10 Ϫ7 M) angiotensin II to increase cytosolic calcium ([Ca 2ϩ ] i ) and decrease Na ϩ /H ϩ exchange activity in cultured rabbit proximal tubule cells and isolated rat proximal tubules (13)(14)(15). In the present studies, we examined mitogenic signaling mechanisms of EETs in renal epithelial cells. We determined the mitogenic effects of all four EET regioisomers and utilized metabolically more stable sulfonimide analogs to determine potential intracellular signaling mechanisms mediating mitogenic effects. We demonstrate here that EETs activate pp60 c-src and initiate a ty...

Loss of functioning nephrons stimulates the growth of residual kidney tissue to augment work capacity and maintain normal renal function. This growth largely occurs by hypertrophy rather than from hyperplasia of the remaining nephrons. The signaling mechanisms that increase RNA and protein synthesis during compensatory renal hypertrophy are unknown. This study found that the remaining kidney hypertrophied 42% by 16 d after unilateral nephrectomy (UNX) in DBA/2 mice. Immunoblotting analysis revealed increased phosphorylation of the 40S ribosomal protein S6 (rpS6) and the eukaryotic translation initiation factor (eIF) 4E-binding protein 1 (4E-BP1), the two downstream effectors of the mammalian target of rapamycin (mTOR). The highly specific mTOR inhibitor rapamycin blocked UNX-increased phosphorylation of both rpS6 and 4E-BP1. UNX increased the content of not only 40S and 60S ribosomal subunits but also 80S monosomes and polysomes in the remaining kidney. Administration of rapamycin decreased UNX-induced polysome formation and shifted the polysome profile in the direction of monosomes and ribosomal subunits. Pretreatment of the mice with rapamycin inhibited UNXinduced hypertrophy. These studies demonstrate that activation of the mTOR signaling pathway in the remaining kidney after UNX plays an essential role in modulating RNA and protein synthesis during development of compensatory renal hypertrophy.

A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regio-and enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regio-and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing These studies provide the first unequivocal evidence for a role for the AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated signaling and mitogenesis and provide compelling evidence for the PLA 2 -AA-EET pathway as an important intracellular-signaling pathway in cells expressing high levels of cytochrome P450 epoxygenase.Release of arachidonic acid secondary to phospholipase A 2 activation is a well recognized cellular response to a variety of growth factors, hormones, and cytokines (1, 2). Metabolites of arachidonic acid play important roles as intracellular second messengers, with well documented autocrine effects of cyclooxygenase and lipoxygenase metabolites (1, 3-5). There is now abundant suggestive evidence that arachidonate metabolites generated by cytochrome P450, the third pathway of arachidonic acid metabolism, may also serve as second messengers (6). These compounds have been implicated in the regulation of peptide secretion, distal nephron Na ϩ fluxes, cell Ca 2ϩ influx, and activation of Ca 2ϩ

The generation of reactive oxygen species (ROS), particularly superoxide, by damaged or dysfunctional mitochondria has been postulated to be an initiating event in the development of diabetes complications. The glomerulus is a primary site of diabetic injury, and podocyte injury is a classic hallmark of diabetic glomerular lesions. In streptozotocin-induced type 1 diabetes, podocyte-specific EGF receptor (EGFR) knockout mice (EGFR podKO ) and their wild-type (WT) littermates had similar levels of hyperglycemia and polyuria, but EGFR podKO mice had significantly less albuminuria and less podocyte loss compared with WT diabetic mice. Furthermore, EGFR podKO diabetic mice had less TGF-b1 expression, Smad2/3 phosphorylation, and glomerular fibronectin deposition. Immunoblotting of isolated glomerular lysates revealed that the upregulation of cleaved caspase 3 and downregulation of Bcl2 in WT diabetic mice were attenuated in EGFR podKO diabetic mice. Administration of the SOD mimetic mito-tempol or the NADPH oxidase inhibitor apocynin attenuated the upregulation of p-c-Src, p-EGFR, p-ERK1/2, p-Smad2/3, and TGF-b1 expression and prevented the alteration of cleaved caspase 3 and Bcl2 expression in glomeruli of WT diabetic mice. High-glucose treatment of cultured mouse podocytes induced similar alterations in the production of ROS; phosphorylation of c-Src, EGFR, and Smad2/3; and expression of TGF-b1, cleaved caspase 3, and Bcl2. These alterations were inhibited by treatment with mito-tempol or apocynin or by inhibiting EGFR expression or activity. Thus, results of our studies utilizing mice with podocyte-specific EGFR deletion demonstrate that EGFR activation has a major role in activating pathways that mediate podocyte injury and loss in diabetic nephropathy.

Bax and Bak, two pro-apoptotic Bcl-2 family proteins, have been implicated in acute kidney injury following renal ischemia/reperfusion; however, definitive evidence for a role of these genes in the disease process is lacking. Here we first examined two Bax-deficient mouse models and found that only conditional Bax-deletion specifically from proximal tubules could ameliorate ischemic acute kidney injury. Global (whole mouse) knockout of Bax enhanced neutrophil infiltration without significant effect on kidney injury. In contrast, global knockout of Bak protected mice from ischemic acute kidney injury with improved renal function. Interestingly, in these models, Bax or Bak knockout attenuated renal tubular cell apoptosis without significantly affecting necrotic tubular damage. Cytochrome c release in ischemic acute kidney injury was also suppressed in conditional Bax or global Bak-knockout mice. In addition, Bak deficiency prevented mitochondrial fragmentation in ischemic acute kidney injury. Thus, our gene-knockout studies support a critical role of Bax and Bak in tubular cell apoptosis in ischemic acute kidney. Furthermore, necrosis and apoptosis have distinguishable regulatory functions.