This study provides the first evidence, to our knowledge, that Ang II induces O2. in cultured tubular cells. Ang II-mediated activation of membrane bound NAD(P)H oxidase, probably by an increase in p22phox transcripts, is likely responsible for this induction. Generation of O2. subsequently induces p27Kip1 expression and stimulates hypertrophy, suggesting a novel mechanism of how Ang II can modulate cell cycle regulation.

Hypertrophy of mesangial cells is an early hallmark of diabetic nephropathy. We have previously shown that murine mesangial cells (MMC), cultured in high-glucose medium, are arrested in the G1 phase of the cell cycle and undergo hypertrophy. This study was undertaken to test whether high glucose-containing medium influences the expression of p27Kip1, an inhibitor of G1 phase active cyclin-dependent kinases (CDK). Incubation of MMC, in the absence of other factors for 48-96 h, in medium containing high D-glucose (450 mg/dl), stimulated p27Kip1 protein expression but failed to influence mRNA abundance. These effects were independent of the osmolarity of the medium. High glucose-stimulated expression of p27Kip1 involved activation of protein kinase C and was partly dependent on induction of transforming growth factor-beta (TGF-beta). Immunoprecipitation experiments revealed that only small amounts of p27Kip1 protein from MMC grown in high-glucose medium preferentially associates with CDK2 but not with CDK4. The p27Kip1 antisense, but not missense, oligonucleotides inhibited high glucose-stimulated total protein synthesis and facilitated G1 phase exit. Our data showed for the first time that expression of p27Kip1 protein is pivotal in mesangial cell hypertrophy induced by high ambient glucose. These findings may be important in the deciphering of molecular processes causing diabetic glomerular hypertrophy.

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Our findings indicate that albumin up-regulates ligand-binding TGF-beta receptors on cultured proximal tubular cells. Albumin-induced activation of local Ang II production appears to be responsible for this effect. This may amplify the matrix-stimulatory actions of TGF-beta on tubular cells and could be a novel mechanism for how proteinuria exhibits pathophysiologic effects on tubular cells ultimately leading to tubulointerstitial fibrosis.

Early diabetic nephropathy is characterized by glomerular hypertrophy. Previous studies in vitro have demonstrated that mesangial cells exposed to high glucose are arrested in the G1-phase of the cell cycle and express increased levels of the cyclin-dependent kinase inhibitor p27Kip1. The present study was performed to investigate the renal expression of p27Kip1 in db/db mice, a model of diabetes mellitus type II. Glomerular p27Kip1 protein, but not mRNA expression, was strongly enhanced in diabetic db/db mice compared with non-diabetic db/+ littermates. Immunohistochemical studies revealed that this stimulated expression was mainly restricted to the nuclei of mesangial cells and podocytes, but glomerular endothelial cells occasionally also stained positively. Quantification of p27Kip1 positive glomerular cells showed a significant increase of these cells in db/db mice compared with non-diabetic db/+ animals. Although tubular cells revealed a positive staining for p27Kip1 protein, there was no difference between db/+ and db/db mice. Immunoprecipitation experiments revealed that p27Kip1 protein associates with Cdk2 and Cdk4, but not with Cdk6. To test for the influence of hyperglycemia on cell cycle arrest and p27Kip1 expression, mesangial cells were isolated from db/+ and db/db mice. There was a similar basal proliferation when these cells were grown in normal glucose-containing medium (100 mg/dl). However, raising the glucose concentration to 275 to 450 mg/dl induced cell cycle arrest in db/+ as well as db/db mesangial cells. Increasing the medium osmolarity with D-mannitol failed to induce p27Kip1 expression in mesangial cells. Transfection of cells with p27Kip1 antisense, but not missense, phosphorothioate oligonucleotides facilitated cell cycle progression equally well in db/+ and db/db mesangial cells. Furthermore, p27Kip1 expression was comparable in both cell lines in normal glucose, but increased in high glucose medium. Our studies demonstrate that p27Kip1 expression is enhanced in diabetic db/db animals. This induction appears to be due to hyperglycemia. Expression of p27Kip1 may be important in cell cycle arrest and hypertrophy of mesangial cells during early diabetic nephropathy.

Background: Angiotensin II (ANG II) inhibits proliferation and induces differentiation in PC 12 cells via AT₂ receptor activation. Using differential display analysis, we previously isolated SM-20/PHD3 as a key factor, which is downregulated by ANG II treatment. Subsequently, it turned out that SM-20/PHD3 is a rat homologue of PHD3, a key prolyl hydroxylase involved in the initial steps fostering the degradation of hypoxia-inducible factor (HIF). The present study was undertaken to investigate whether the ANG-II-mediated suppression of SM-20/PHD3/PHD3 may be associated with an increase in HIF-1α. Methods: HIF-1α protein expression was assessed by Western blots. mRNA levels for HIF-1α were measured by real-time PCR and for SM-20/PHD3 by Northern blots. Binding of HIF-1α to consensus oligonucleotides in vitro was determined with gel shift analysis. SM-20/PHD3 was transiently overexpressed in PC 12 cells using an inducible expression system. Results: ANG II stimulated HIF-1α protein expression. This effect was already detected after 30 min and peaked at 6 h, but was not detectable anymore after 24– 48 h of stimulation. PD 123177, but not losartan, antagonized this effect, indicating transduction through AT₂ receptors. Real-time PCR failed to show a significant increase in HIF-1α transcripts after ANG II challenge at any time point. Gel shift analysis revealed that ANG-II-induced nuclear HIF-1α protein binds to consensus sites. A reduction in SM-20/PHD3 mRNA expression paralleled the increase in HIF-1α. Overexpression of SM-20/PHD3 transiently resulted in a decrease in HIF-1α protein concentrations under basal conditions as well as after stimulation with ANG II. Conclusion: ANG II stimulates HIF-1α expression by a posttranscriptional mechanism via AT₂ receptors. This increase is likely caused by a downregulation of SM-20/PHD3. The ANG-II-mediated increase in HIF-1α expression could be potentially involved in physiological as well as pathophysiological processes such as differentiation, growth inhibition, and remodeling.

Our data collectively suggest that the Ang-II-mediated increase in type IV collagen in MCT cells is mediated by endogenous synthesis and autocrine action of TGF-beta1. These findings may be important in changes of the tubulointerstitial architecture during the progression of renal disease.

Transforming growth factor beta (TGF-beta) plays an important role in the development of tubulointerstitial fibrosis in chronic renal disease. We were interested whether interference with oxygen radicals may modulate TGF-beta expression. Unexpectedly, we discovered that diphenylene iodine (DIP), an inhibitor of NADP(H) oxidase, induces a robust increase in TGF-beta transcript expression in cultured mouse proximal tubular cells (MCT cells). A similar increase was seen with EUK-8, a synthetic salen-manganese complex with high oxyradical scavenger activities. This induction of TGF-beta1 mRNA was paralleled by increasing protein expression. Transient transfection of MCT cells with a reporter construct in which murine TGF-beta1 enhancer/promoter elements were cloned in front of the luciferase gene, revealed that DIP, EUK-8, and Tiron all stimulated transcription of the TGF-beta1 gene whereas exogenous H2O2 suppressed transcription. Antisense oligonucleotides against p22phox, but not sense oligonucleotides, also increased transcriptional activity of TGF-beta1. Mutagenesis of Sp1 binding sites in the mouse TGF-beta1 enhancer/promoter abolished the stimulatory effect of the antioxidants. Gel shift experiments revealed that DIP as well as EUK-8 activated binding of nuclear proteins to Sp1 consensus sequence. Our data provide evidence that TGF-beta1 transcription is negatively regulated in MCT cells under basal conditions by NADP(H) oxidase-mediated oxygen radicals. Thus, antioxidant therapy may increase local synthesis of TGF-beta1 in the tubulointerstitium.