JNK1 ablation improves pancreatic β-cell mass and function in db/db diabetic mice without affecting insulin sensitivity and adipose tissue inflammation

The cJun N-terminal Kinases (JNK) emerged as a major link between obesity and insulin resistance, but their role in the loss of pancreatic β-cell mass and function driving the progression from insulin resistance to type-2 diabetes and in the complications of diabetes was not investigated to the same extent. Furthermore, it was shown that pan-JNK inhibition exacerbates kidney damage in the db/db model of obesity-driven diabetes. Here we investigate the role of JNK1 in the db/db model of obesity-driven type-2 diabetes. Mice with systemic ablation of JNK1 (JNK1−/−) were backcrossed for more than 10 generations in db/+ C57BL/KS mice to generate db/db-JNK1−/− mice and db/db control mice. To define the role of JNK1 in the loss of β-cell mass and function occurring during obesity-driven diabetes we performed comprehensive metabolic phenotyping, evaluated steatosis and metabolic inflammation, performed morphometric and cellular composition analysis of pancreatic islets, and evaluated kidney function in db/db-JNK1−/− mice and db/db controls. db/db-JNK1−/− mice and db/db control mice developed insulin resistance, fatty liver, and metabolic inflammation to a similar extent. However, db/db-JNK1−/− mice displayed better glucose tolerance and improved insulin levels during glucose tolerance test, higher pancreatic insulin content, and larger pancreatic islets with more β-cells than db/db mice. Finally, albuminuria, kidney histopathology, kidney inflammation and oxidative stress in db/db-JNK1−/− mice and in db/db mice were similar. Our data indicate that selective JNK1 ablation improves glucose tolerance in db/db mice by reducing the loss of functional β-cells occurring in the db/db mouse model of obesity-driven diabetes, without significantly affecting metabolic inflammation, steatosis, and insulin sensitivity. Furthermore, we have found that, differently from what previously reported for pan-JNK inhibitors, selective JNK1 ablation does not exacerbate kidney dysfunction in db/db mice. We conclude that selective JNK1 inactivation may have a superior therapeutic index than pan-JNK inhibition in obesity-driven diabetes

Protein Kinase C Delta (PKCδ) Affects Proliferation of Insulin-Secreting Cells by Promoting Nuclear Extrusion of the Cell Cycle Inhibitor p21Cip1/WAF1

BACKGROUND:High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. METHODOLOGY AND PRINCIPAL FINDINGS:Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21(Cip1/WAF1). This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21(Cip1/WAF1) at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21(Cip1/WAF1) was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21(Cip1/WAF1) with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. CONCLUSIONS AND SIGNIFICANCE:These observations disclose PKCδ as negative regulator of p21(Cip1/WAF1), which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role

Renal tubular HIF-2α expression requires VHL inactivation and causes fibrosis and cysts.

The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated α-subunits HIF-1α or HIF-2α. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1α is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2α is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2α expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2α expression. Continuous transgenic expression of HIF-2α by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2α in renal epithelia. Unphysiological expression of HIF-2α in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2α expression. However, HIF-2α overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms

A Molecular Signature of Proteinuria in Glomerulonephritis

Proteinuria is the most important predictor of outcome in glomerulonephritis and experimental data suggest that the tubular cell response to proteinuria is an important determinant of progressive fibrosis in the kidney. However, it is unclear whether proteinuria is a marker of disease severity or has a direct effect on tubular cells in the kidneys of patients with glomerulonephritis. Accordingly we studied an in vitro model of proteinuria, and identified 231 “albumin-regulated genes” differentially expressed by primary human kidney tubular epithelial cells exposed to albumin. We translated these findings to human disease by studying mRNA levels of these genes in the tubulo-interstitial compartment of kidney biopsies from patients with IgA nephropathy using microarrays. Biopsies from patients with IgAN (n = 25) could be distinguished from those of control subjects (n = 6) based solely upon the expression of these 231 “albumin-regulated genes.” The expression of an 11-transcript subset related to the degree of proteinuria, and this 11-mRNA subset was also sufficient to distinguish biopsies of subjects with IgAN from control biopsies. We tested if these findings could be extrapolated to other proteinuric diseases beyond IgAN and found that all forms of primary glomerulonephritis (n = 33) can be distinguished from controls (n = 21) based solely on the expression levels of these 11 genes derived from our in vitro proteinuria model. Pathway analysis suggests common regulatory elements shared by these 11 transcripts. In conclusion, we have identified an albumin-regulated 11-gene signature shared between all forms of primary glomerulonephritis. Our findings support the hypothesis that albuminuria may directly promote injury in the tubulo-interstitial compartment of the kidney in patients with glomerulonephritis

Comparison of distinctive models for calculating an interlobar emphysema heterogeneity index in patients prior to endoscopic lung volume reduction

Dorothea Theilig,1 Felix Doellinger,1 Alexander Poellinger,1 Vera Schreiter,1 Konrad Neumann,2 Ralf-Harto Hubner31Department of Radiology, Charité Campus Virchow Klinikum, Charité, Universitätsmedizin Berlin, Berlin, Germany; 2Institute of Biometrics and Clinical Epidemiology, Charité Campus Benjamin Franklin, Charité, Universitätsmedizin Berlin, Berlin, Germany; 3Department of Pneumology, Charité Campus Virchow Klinikum, Charité, Universitätsmedizin Berlin, Berlin, GermanyBackground: The degree of interlobar emphysema heterogeneity is thought to play an important role in the outcome of endoscopic lung volume reduction (ELVR) therapy of patients with advanced COPD. There are multiple ways one could possibly define interlobar emphysema heterogeneity, and there is no standardized definition.Purpose: The aim of this study was to derive a formula for calculating an interlobar emphysema heterogeneity index (HI) when evaluating a patient for ELVR. Furthermore, an attempt was made to identify a threshold for relevant interlobar emphysema heterogeneity with regard to ELVR.Patients and methods: We retrospectively analyzed 50 patients who had undergone technically successful ELVR with placement of one-way valves at our institution and had received lung function tests and computed tomography scans before and after treatment. Predictive accuracy of the different methods for HI calculation was assessed with receiver-operating characteristic curve analysis, assuming a minimum difference in forced expiratory volume in 1 second of 100 mL to indicate a clinically important change.Results: The HI defined as emphysema score of the targeted lobe (TL) minus emphysema score of the ipsilateral nontargeted lobe disregarding the middle lobe yielded the best predicative accuracy (AUC =0.73, P=0.008). The HI defined as emphysema score of the TL minus emphysema score of the lung without the TL showed a similarly good predictive accuracy (AUC =0.72, P=0.009). Subgroup analysis suggests that the impact of interlobar emphysema heterogeneity is of greater importance in patients with upper lobe predominant emphysema than in patients with lower lobe predominant emphysema.Conclusion: This study reveals the most appropriate ways of calculating an interlobar emphysema heterogeneity with regard to ELVR. Keywords: CT-quantitative, COPD, emphysema heterogeneity, endoscopic lung volume reductio

Pulmonary lymphangioleiomyomatosis: analysis of disease manifestation by region-based quantification of lung parenchyma

PURPOSE Lymphangioleiomyomatosis (LAM) is characterized by proliferation of smooth muscle tissue that causes bronchial obstruction and secondary cystic destruction of lung parenchyma. The aim of this study was to evaluate the typical distribution of cystic defects in LAM with quantitative volumetric chest computed tomography (CT). MATERIALS AND METHODS CT examinations of 20 patients with confirmed LAM were evaluated with region-based quantification of lung parenchyma. Additionally, 10 consecutive patients were identified who had recently undergone CT imaging of the lung at our institution, in which no pathologies of the lung were found, to serve as a control group. Each lung was divided into three regions (upper, middle and lower thirds) with identical number of slices. In addition, we defined a "peel" and "core" of the lung comprising the 2 cm subpleural space and the remaining inner lung area. Computerized detection of lung volume and relative emphysema was performed with the PULMO 3D software (v3.42, Fraunhofer MEVIS, Bremen, Germany). This software package enables the quantification of emphysematous lung parenchyma by calculating the pixel index, which is defined as the ratio of lung voxels with a density <-950HU to the total number of voxels in the lung. RESULTS Cystic changes accounted for 0.1-39.1% of the total lung volume in patients with LAM. Disease manifestation in the central lung was significantly higher than in peripheral areas (peel median: 15.1%, core median: 20.5%; p=0.001). Lower thirds of lung parenchyma showed significantly less cystic changes than upper and middle lung areas combined (lower third: median 13.4, upper and middle thirds: median 19.0, p=0.001). CONCLUSION The distribution of cystic lesions in LAM is significantly more pronounced in the central lung compared to peripheral areas. There is a significant predominance of cystic changes in apical and intermediate lung zones compared to the lung bases

Abrogation of Protein Uptake through Megalin-Deficient Proximal Tubules Does Not Safeguard against Tubulointerstitial Injury

Sustained proteinuria and tubulointerstitial damage have been closely linked with progressive renal failure. Upon excess protein endocytosis, tubular epithelial cells are thought to produce mediators that promote inflammation, tubular degeneration, and fibrosis. This concept was tested in a transgenic mouse model with megalin deficiency. Application of an anti-glomerular basement membrane serum to transgenic megalin-deficient mice [Cre(+)/GN] and megalin-positive littermates [Cre(-)/GN] produced the typical glomerulonephritis (GN) with heavy proteinuria in both groups. Tubulointerstitial damages correlated closely with glomerular damages in pooled Cre(+)/GN and Cre(-)/GN mice. Owing to a mosaic pattern of megalin expression in the mutant mice, Cre(+)/GN kidneys permitted side-by-side analysis of megalin-deficient and megalin-positive tubules in the same kidney. Protein endocytosis was found only in megalin-positive cells. TGF-beta, intercellular adhesion molecule, vascular cellular adhesion molecule, endothelin-1, and cell proliferation were high in megalin-positive cells, whereas apoptosis, heat-shock protein 25, and osteopontin were enhanced in megalin-deficient cells. No fibrotic changes were associated with either phenotype. Tubular degeneration with interstitial inflammation was found only in nephrons with extensive crescentic lesions at the glomerulotubular junction. In sum, enhanced protein endocytosis indeed led to an upregulation of profibrotic mediators in a megalin-dependent way; however, there was no evidence that endocytosis played a pathogenetic role in the development of the tubulointerstitial disease

Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis

Human glomerulonephritis (GN) is characterized by sustained proteinuria, sodium retention, hypertension, and edema formation. Increasing quantities of filtered protein enter the renal tubule, where they may alter epithelial transport functions. Exaggerated endocytosis and consequent protein overload may affect proximal tubules, but intrinsic malfunction of distal epithelia has also been reported. A straightforward assignment to a particular tubule segment causing salt retention in GN is still controversial. We hypothesized that 1) trafficking and surface expression of major transporters and channels involved in volume regulation were altered in GN, and 2) proximal tubular endocytosis may influence locally as well as downstream expressed tubular transporters and channels. Effects of anti-glomerular basement membrane GN were studied in controls and megalin-deficient mice with blunted proximal endocytosis. Mice displayed salt retention and elevated systolic blood pressure when proteinuria had reached 10-15 mg/24 h. Surface expression of proximal Na(+)-coupled transporters and water channels was in part [Na(+)-P(i) cotransporter IIa (NaPi-IIa) and aquaporin-1 (AQP1)] increased by megalin deficiency alone, but unchanged (Na(+)/H(+) exchanger 3) or reduced (NaPi-IIa and AQP1) in GN irrespective of the endocytosis defect. In distal epithelia, significant increases in proteolytic cleavage products of alpha-epithelial Na(+) channel (ENaC) and gamma-ENaC were observed, suggesting enhanced tubular sodium reabsorption. The effects of glomerular proteinuria dominated over those of blunted proximal endocytosis in contributing to ENaC cleavage. Our data indicate that ENaC-mediated sodium entry may be the rate-limiting step in proteinuric sodium retention. Enhanced proteolytic cleavage of ENaC points to a novel mechanism of channel activation which may involve the action of filtered plasma proteases