CiteULike: jyuh's Wolf

Identification of renal injury in cardiac surgery: the role of kidney-specific proteins.

J Boldt — 2008-08-09T10:24:27-00:00

Journal of cardiothoracic and vascular anesthesia, Vol. 22, No. 1. (February 2008), pp. 122-132.

Which hospitals have significantly better or worse than expected mortality rates for acute myocardial infarction patients? Improved risk adjustment with present-at-admission diagnoses.

GJ Stukenborg — 2008-07-25T04:39:10-00:00

Circulation, Vol. 116, No. 25. (18 December 2007), pp. 2960-2968.

BACKGROUND: Public reports that compare hospital mortality rates for patients with acute myocardial infarction are commonly used strategies for improving the quality of care delivered to these patients. Fair comparisons of hospital mortality rates require thorough adjustments for differences among patients in baseline mortality risk. This study examines the effect on hospital mortality rate comparisons of improved risk adjustment methods using diagnoses reported as present-at-admission. METHODS AND RESULTS: Logistic regression models and related methods originally used by California to compare hospital mortality rates for patients with acute myocardial infarction are replicated. These results are contrasted with results obtained for the same hospitals by patient-level mortality risk adjustment models using present-at-admission diagnoses, using 3 statistical methods of identifying hospitals with higher or lower than expected mortality: indirect standardization, adjusted odds ratios, and hierarchical models. Models using present-at-admission diagnoses identified substantially fewer hospitals as outliers than did California model A for each of the 3 statistical methods considered. CONCLUSIONS: Large improvements in statistical performance can be achieved with the use of present-at-admission diagnoses to characterize baseline mortality risk. These improvements are important because models with better statistical performance identify different hospitals as having better or worse than expected mortality.

Present-at-admission diagnoses improved mortality risk adjustment among acute myocardial infarction patients.

GJ Stukenborg — 2008-07-25T04:37:05-00:00

Journal of clinical epidemiology, Vol. 60, No. 2. (February 2007), pp. 142-154.

OBJECTIVE: Hospital mortality outcomes for acute myocardial infarction (AMI) patients are a focus of quality improvement programs conducted by government agencies. AMI mortality risk-adjustment models using administrative data typically adjust for baseline differences in mortality risk with a limited set of common and definite comorbidities. In this study, we present an AMI mortality risk-adjustment model that adjusts for comorbid disease and for AMI severity using information from secondary diagnoses reported as present at admission for California hospital patients. STUDY DESIGN AND SETTING: AMI patients were selected from California hospital administrative data for 1996 through 1999 according to criteria used by the California Hospital Outcomes Project Report on Heart Attack Outcomes, a state-mandated public report that compares hospital mortality outcomes. We compared results for the new model to two mortality risk-adjustment models used to assess hospital AMI mortality outcomes by the state of California, and to two other models used in prior research. RESULTS: The model using present-at-admission diagnoses obtained substantially better discrimination between predicted survival and inpatient death than the other models we considered. CONCLUSION: AMI mortality risk-adjustment methods can be meaningfully improved using present-at-admission diagnoses to identify comorbid disease and conditions related closely to AMI.

The Framingham Heart Study 100K SNP genome-wide association study resource: overview of 17 phenotype working group reports.

LA Cupples — 2008-04-16T16:12:34-00:00

BMC medical genetics, Vol. 8 Suppl 1 (2007)

BACKGROUND: The Framingham Heart Study (FHS), founded in 1948 to examine the epidemiology of cardiovascular disease, is among the most comprehensively characterized multi-generational studies in the world. Many collected phenotypes have substantial genetic contributors; yet most genetic determinants remain to be identified. Using single nucleotide polymorphisms (SNPs) from a 100K genome-wide scan, we examine the associations of common polymorphisms with phenotypic variation in this community-based cohort and provide a full-disclosure, web-based resource of results for future replication studies.METHODS: Adult participants (n = 1345) of the largest 310 pedigrees in the FHS, many biologically related, were genotyped with the 100K Affymetrix GeneChip. These genotypes were used to assess their contribution to 987 phenotypes collected in FHS over 56 years of follow up, including: cardiovascular risk factors and biomarkers; subclinical and clinical cardiovascular disease; cancer and longevity traits; and traits in pulmonary, sleep, neurology, renal, and bone domains. We conducted genome-wide variance components linkage and population-based and family-based association tests.RESULTS: The participants were white of European descent and from the FHS Original and Offspring Cohorts (examination 1 Offspring mean age 32 +/- 9 years, 54% women). This overview summarizes the methods, selected findings and limitations of the results presented in the accompanying series of 17 manuscripts. The presented association results are based on 70,897 autosomal SNPs meeting the following criteria: minor allele frequency > or + 10%, genotype call rate > or = 80%, Hardy-Weinberg equilibrium p-value > or = 0.001, and satisfying Mendelian consistency. Linkage analyses are based on 11,200 SNPs and short-tandem repeats. Results of phenotype-genotype linkages and associations for all autosomal SNPs are posted on the NCBI dbGaP website at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007 webcite.CONCLUSION: We have created a full-disclosure resource of results, posted on the dbGaP website, from a genome-wide association study in the FHS. Because we used three analytical approaches to examine the association and linkage of 987 phenotypes with thousands of SNPs, our results must be considered hypothesis-generating and need to be replicated. Results from the FHS 100K project with NCBI web posting provides a resource for investigators to identify high priority findings for replication.

Renal injury due to renin-angiotensin-aldosterone system activation of the transforming growth factor-beta pathway.

G Wolf — 2008-07-01T09:37:22-00:00

Kidney international, Vol. 70, No. 11. (December 2006), pp. 1914-1919.

Glomerulosclerosis, interstitial fibrosis, and tubular atrophy occur with end-stage kidney failure, irrespective of the primary etiology. The transforming growth factor-beta (TGF-beta) is a key factor in these alterations either directly, by stimulating synthesis of extracellular matrix components and reducing collagenase production, or indirectly through other profibrogenic factors such as connective tissue growth factor (CTGF). TGF-beta is important for the proliferation of intrarenal fibroblasts and the epithelial-mesenchymal transition through which tubular cells become fibroblasts. Although several factors induce TGF-beta expression in the kidney, one very interesting aspect is the link between the renin-angiotensin-aldosterone (Aldo) system (RAAS) and TGF-beta. Angiotensin II (ANG II) stimulates TGF-beta expression in the kidney by various mechanisms and upregulates receptors for TGF-beta. ANG II can directly phosphorylate Smads without inducing TGF-beta. Recent data provide compelling evidence that other components of the RAAS including ANG III, renin, and Aldo also activate the TGF-beta system. As direct modulation of the TGF-beta system is not yet feasible in humans, angiotensin-converting enzyme (ACE) inhibitors and angiotensin type 1 (AT1)-receptor blockers are currently the most potential drugs to interfere with this ANG II-mediated TGF-beta expression. This review highlights some current aspects of the interaction between the RAAS and the TGF-beta axis.

Postprandial mineral metabolism and secondary hyperparathyroidism in early CKD.

T Isakova — 2008-06-30T01:53:32-00:00

Journal of the American Society of Nephrology : JASN, Vol. 19, No. 3. (March 2008), pp. 615-623.

Normophosphatemia and normocalcemia are maintained in chronic kidney disease (CKD) by increased levels of fibroblast growth factor-23 (FGF-23) and parathyroid hormone (PTH), but the stimuli for secretion of these hormones in early CKD are incompletely understood. Most human physiologic studies have focused on random or fasting measurements of phosphorus, calcium, FGF-23, and PTH, but in this study, the hypothesis was that measurements in the postprandial state may reveal intermittent stimuli that lead to increased FGF-23 and PTH levels. The 4-h postprandial response in 13 patients with CKD and fasting normophosphatemia and normocalcemia (mean GFR 41 +/- 8 ml/min per m(2)) was compared with 21 healthy volunteers. Compared with healthy subjects, fasting patients with CKD had significantly higher levels of FGF-23 and fractional excretion of phosphorus; lower fractional excretion of calcium; and no difference in serum calcium, phosphorus, and PTH levels. After standardized meals, urinary phosphorus excretion in both groups increased despite unchanged serum phosphorus and FGF-23 levels. Postprandial urinary calcium excretion also increased in both groups, and this was accompanied by significantly reduced serum calcium and increased PTH levels in patients with CKD only; therefore, FGF-23 does not seem to be an acute postprandial regulator of phosphaturia in CKD or in health, but inappropriate postprandial calciuria with episodic, relative hypocalcemia may represent a previously unreported mechanism of secondary hyperparathyroidism in CKD.

Impact of Activated Vitamin D and Race on Survival among Hemodialysis Patients.

Myles Wolf — 2008-06-17T04:49:16-00:00

Journal of the American Society of Nephrology : JASN (9 April 2008)

Contrary to most examples of disparities in health outcomes, black patients have improved survival compared with white patients after initiating hemodialysis. Understanding potential explanations for this observation may have important clinical implications for minorities in general. This study tested the hypothesis that greater use of activated vitamin D therapy accounts for the survival advantage observed in black and Hispanic patients on hemodialysis. In a prospective cohort of non-Hispanic white (n = 5110), Hispanic white (n = 979), and black (n = 3214) incident hemodialysis patients, higher parathyroid hormone levels at baseline were the primary determinant of prescribing activated vitamin D therapy. Median parathyroid hormone was highest among black patients, who were most likely to receive activated vitamin D and at the highest dosage. One-year mortality was lower in black and Hispanic patients compared with white patients (16 and 16 versus 23%; P < 0.01), but there was significant interaction between race and ethnicity, activated vitamin D therapy, and survival. In multivariable analyses of patients treated with activated vitamin D, black patients had 16% lower mortality compared with white patients, but the difference was lost when adjusted for vitamin D dosage. In contrast, untreated black patients had 35% higher mortality compared with untreated white patients, an association that persisted in several sensitivity analyses. In conclusion, therapy with activated vitamin D may be one potential explanation for the racial differences in survival among hemodialysis patients. Further studies should determine whether treatment differences based on biologic differences contribute to disparities in other conditions.

Molecular and cellular approaches for the detection of proteinprotein interactions: latest techniques and current limitations

Lalonde — 2008-02-08T00:50:26-00:00

The Plant Journal, Vol. 53, No. 4. (February 2008), pp. 610-635.

Advanced glycation endproducts influence the mRNA expression of RAGE, RANKL and various osteoblastic genes in human osteoblasts.

S Franke — 2008-04-20T09:03:17-00:00

Archives of physiology and biochemistry, Vol. 113, No. 3. (June 2007), pp. 154-161.

Glycation reactions resulting in the generation and accumulation of advanced glycation endproducts (AGEs) are potential mechanisms by which bone protein may be altered in vivo. AGEs accumulate in the bone increasingly with age come into close contact with osteoblasts or osteoclasts. The direct effect of AGEs on bone cells has not been thoroughly investigated. This study aimed to examine whether glycated bovine serum albumin (AGE - BSA) as an AGE modulate the mRNA expression of various genes in primary human osteoblast cultures. The following parameters were included: RAGE (receptor for AGEs), alkaline phosphatase, osteocalcin, osterix and RANKL (receptor activator of nuclear factor-kappaB ligand). Primary human osteoblast cultures were obtained from bone specimens of six patients with osteoarthrosis. Human osteoblasts were treated in AGE - BSA or control-BSA (non-glycated BSA) containing medium (5 mg/ml each) over a time course of seven days. After RT-PCR the mRNA expression was measured by real-time PCR. Related to control - BSA exposure, the mRNA expression of RAGE, RANKL and osterix increased during AGE - BSA treament. For alkaline phosphatase and osteocalcin a tendency of down-regulation was found. In summary, the study presents evidence that advanced glycation end products accumulated in bone alter osteoblasts by activation the AGE - RAGE pathway (RAGE mRNA up-regulation), inducing enhanced osteoclastogenesis (RANKL mRNA up-regulation) and impaired matrix mineralization (down-regulation of alkaline phosphatase and osteocalcin mRNA). Thus, AGEs may play a functional role in the development of bone diseases (e.g. osteoporosis).

Cadmium and mercury cause an oxidative stress-induced endothelial dysfunction

Wolf — 2007-01-19T17:28:25-00:00

BioMetals, Vol. 20, No. 1. (February 2007), pp. 73-81.

Advanced glycation end-products induce cell cycle arrest and hypertrophy in podocytes

Christiane Ruster — 2008-03-18T03:54:35-00:00

Nephrol. Dial. Transplant. (14 March 2008), gfn085.

Background. Podocyte injury with loss of cells into the urine seems to be an early factor in diabetic nephropathy. Advanced glycation end-products (AGEs) are important mediators of structural and functional renal abnormalities in diabetic nephropathy. We and others have previously described that mice with a deletion in the gene for the cell cycle regulatory p27Kip1 are protected from some features of diabetic nephropathy. Methods. The present study investigates a potential influence of AGE-modified bovine serum albumin (AGE-BSA) on podocyte growth and p27Kip1 expression in culture. The p27Kip1 expression was measured by western blots and real-time PCR. Cell cycle analysis, cell hypertrophy, proliferation and various markers of apoptosis and necrosis were assessed. The p27Kip expression was inhibited by siRNA or was overexpressed in podocytes with an inducible expression system. Results. AGE-BSA was actively taken up into the cell as determined by immunohistochemistry, western blots and HPLC. Incubation with AGE-BSA induced in differentiated podocytes, but not in tubular cells, p27Kip1 mRNA and protein expression. This induction was associated with cell cycle arrest of podocytes, cell hypertrophy (as measured by increases in cell size and protein/cell number ratios) and an increase in necrotic, but not apoptotic cells. Inhibition of p27Kip1 expression with siRNA halted the AGE-BSA-mediated cell cycle arrest and hypertrophy, but did not interfere with AGE uptake into podocytes. In contrast, overexpression of p27Kip1 using an inducible expression system stimulated hypertrophy and cell cycle arrest of podocytes. Conclusion. Our data demonstrate that AGE-BSA-induced hypertrophy and damage of cultured podocytes occurs by a mechanism involving p27Kip1. This effect can contribute to the loss of podocytes in diabetic nephropathy. 10.1093/ndt/gfn085

Rapid development of severe end-organ damage in C57BL//6 mice by combining DOCA salt and angiotensin II

F Kirchhoff — 2008-02-19T09:22:56-00:00

Kidney Int, Vol. 73, No. 5. (21 November 2007), pp. 643-650.

Vitamin D levels and early mortality among incident hemodialysis patients.

M Wolf — 2008-02-18T03:20:40-00:00

Kidney Int, Vol. 72, No. 8. (October 2007), pp. 1004-1013.

Vitamin D deficiency is associated with cardiovascular disease, the most common cause of mortality in hemodialysis patients. To investigate the relation between blood levels of 25-hydroxyvitamin D (25D) and 1,25-dihydroxyvitamin D (1,25D) with hemodialysis outcomes, we measured baseline vitamin D levels in a cross-sectional analysis of 825 consecutive patients from within a prospective cohort of incident US hemodialysis patients. Of these patients, 78% were considered vitamin D deficient with 18% considered severely deficient. Calcium, phosphorus, and parathyroid hormone levels correlated poorly with 25D and 1,25D concentrations. To test the association between baseline vitamin D levels and 90-day mortality, we selected the next 175 consecutive participants who died within 90 days and compared them to the 750 patients who survived in a nested case-control analysis. While low vitamin D levels were associated with increased mortality, significant interaction was noted between vitamin D levels, subsequent active vitamin D therapy, and survival. Compared to patients with the highest 25D or 1,25D levels who received therapy, untreated deficient patients were at significantly increased risk for early mortality. Our study shows that among incident hemodialysis patients, vitamin D deficiency is common, correlates poorly with other components of mineral metabolism and is associated with increased early mortality.

Diabetes models by screen for hyperglycemia in phenotype-driven ENU mouse mutagenesis projects

Bernhard Aigner — 2008-02-11T09:59:21-00:00

Am J Physiol Endocrinol Metab, Vol. 294, No. 2. (1 February 2008), pp. E232-240.

More than 150 million people suffer from diabetes mellitus worldwide, and this number is expected to rise substantially within the next decades. Despite its high prevalence, the pathogenesis of diabetes mellitus is not completely understood. Therefore, appropriate experimental models are essential tools to gain more insight into the genetics and pathogenesis of the disease. Here, we describe the current efforts to establish novel diabetes models derived from unbiased, phenotype-driven, large-scale N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects started a decade ago using hyperglycemia as a high-throughput screen parameter. Mouse lines were established according to their hyperglycemia phenotype over several generations, thereby revealing a mutation as cause for the aberrant phenotype. Chromosomal assignment of the causative mutation and subsequent candidate gene analysis led to the detection of the mutations that resulted in novel alleles of genes already known to be involved in glucose homeostasis, like glucokinase, insulin 2, and insulin receptor. Additional ENU-induced hyperglycemia lines are under genetic analysis. Improvements in screen for diabetic animals are implemented to detect more subtle phenotypes. Moreover, diet challenge assays are being employed to uncover interactions between genetic and environmental factors in the pathogenesis of diabetes mellitus. The new mouse mutants recovered in phenotype-driven ENU mouse mutagenesis projects complement the available models generated by targeted mutagenesis of candidate genes, all together providing the large resource of models required for a systematic dissection of the pathogenesis of diabetes mellitus. 10.1152/ajpendo.00592.2007

Cell cycle regulation in diabetic nephropathy.

G Wolf — 2008-01-20T08:25:08-00:00

Kidney Int Suppl, Vol. 77 (September 2000)

Cell cycle regulation in diabetic nephropathy. Renal hypertrophy is one of the earliest abnormalities of diabetic nephropathy. Although selected cell populations. such as tubulointerstitial fibroblasts, may undergo sustained proliferation in the diabetic environment, most renal cells such as mesangial cells are arrested in the G1-phase of the cell cycle after actively leaving G0-phase and some self-limited early proliferation. High glucose, transforming growth factor-beta (TGF-beta), angiotensin II, and probably other factors induce inhibitors of cyclin-dependent kinases (CDK) including p21Cip1 and p27KiP1. These CDK-inhibitors bind to and inactivate G1-phase cyclin/CDK complexes. The consequence is a lack in kinase activity, underphosphorylation of the retinoblastoma gene protein, and a failure to initiate the G1-S-phase transit. The half-life of CDK-inhibitors may also be increased by serine phosphorylation mediated through activated MAP kinases. Treatment of diabetic rats with angiotensin-converting enzyme inhibitors attenuates glomerular hypertrophy and abolishes the glomerular expression of the CDK-inhibitors p16INK4 and p27KiP1, thus indicating that the cell cycle arrest can be therapeutically influenced. Cell cycle proteins may also be involved in these molecular events, leading to a limited degree of tubular apoptosis, which is a feature of diabetic nephropathy. Although not definitively proven, accumulating evidence suggests that early hypertrophy of renal cells may act as pacemaker for subsequent irreversible structural changes, such as glomerulosclerosis and tubulointerstitial fibrosis. Therefore, a better understanding of altered processes of cell cycle regulation is necessary to develop novel therapeutic strategies to prevent diabetic nephropathy. The recent observation that glomerular hypertrophy and proteinuria do not develop in diabetic p21CiP1 knockout mice indicates that this approach is feasible.

Advanced glycation end products and the kidney

Jurgen Bohlender — 2008-01-16T01:55:02-00:00

Am J Physiol Renal Physiol, Vol. 289, No. 4. (1 October 2005), pp. F645-659.

Advanced glycation end products (AGEs) are a heterogeneous group of protein and lipids to which sugar residues are covalently bound. AGE formation is increased in situations with hyperglycemia (e.g., diabetes mellitus) and is also stimulated by oxidative stress, for example in uremia. It appears that activation of the renin-angiotensin system may contribute to AGE formation through various mechanisms. Although AGEs could nonspecifically bind to basement membranes and modify their properties, they also induce specific cellular responses including the release of profibrogenic and proinflammatory cytokines by interacting with the receptor for AGE (RAGE). However, additional receptors could bind AGEs, adding to the complexity of this system. The kidney is both: culprit and target of AGEs. A decrease in renal function increases circulating AGE concentrations by reduced clearance as well as increased formation. On the other hand, AGEs are involved in the structural changes of progressive nephropathies such as glomerulosclerosis, interstitial fibrosis, and tubular atrophy. These effects are most prominent in diabetic nephropathy, but they also contribute to renal pathophysiology in other nondiabetic renal diseases. Interference with AGE formation has therapeutic potential for preventing the progression of chronic renal diseases, as shown from data of animal experiments and, more recently, the first clinical trials. 10.1152/ajprenal.00398.2004

Angiotensin II-stimulated expression of transforming growth factor beta in renal proximal tubular cells: attenuation after stable transfection with the c-mas oncogene.

G Wolf — 2008-01-14T04:31:16-00:00

Kidney Int, Vol. 48, No. 6. (December 1995), pp. 1818-1827.

Angiotensin II (Ang II) stimulates cellular hypertrophy of cultured murine proximal tubular cells (MCT cells). This Ang II-mediated hypertrophy depends on the endogenous induction and autocrine action of transforming growth factor-beta (TGF-beta). We have previously demonstrated that permanent transfection of MCT cells with the c-mas oncogene, whose protein product encodes a serpentine receptor-like moiety coupled to G proteins without an hitherto identified ligand, changes the hypertrophic actions of Ang II into a proliferative response (Am J Physiol 263: F931-F938, 1992). The present study demonstrated that Ang II failed to stimulate induction of TGF-beta 1 protein in c-mas transfected MCT cells under the control of SV 40 promoter/enhancer (pSV2mas) as measured by mink cell bioassay and specific ELISA for TGF-beta 1. Moreover, in contrast to either wild-type MCT cells or to cells permanently transfected with the SV 40 based expression plasmid only (pSV2 cells), Ang II stimulated gene transcription and mRNA expression of TGF-beta 1 were decreased in c-mas transfected cells. Our findings demonstrate that the Ang II-induced proliferation of c-mas transfected MCT cells most likely depends on failure of TGF-beta 1 induction in these cells. c-mas transfected cells are a useful tool to further investigate the complex relationships between activation of second messengers subsequent to binding of Ang II to AT1-receptors and gene regulation like transcription of TGF-beta 1.

The role of chemokines and chemokine receptors in diabetic nephropathy.

C Ruster — 2008-01-13T08:35:14-00:00

Front Biosci, Vol. 13 (2008), pp. 944-955.

Diabetic nephropathy is increasingly considered as an inflammatory disease characterized by leukocyte infiltration at every stage of renal involvement. Chemokines are important participators in the recruitment of specific subpopulations of inflammatory cells into renal compartments. MCP-1/CCL2 has been identified as having a key role in monocyte/macrophage recruitment in animal models of diabetic nephropathy, as well as in renal biopsies from patients with type 1 and 2 diabetes. Various factors of the diabetic milieu can induce renal expression of MCP-1/CCL2 and cell adhesion molecules, and thereby mediate the macrophage responses that ultimately cause renal injury. Possibly fractalkine/CX3CL1 functions as an arrest chemokine in monocyte/macrophage adhesion before migration into the kidney. T lymphocyte recruitment is influenced by up-regulation of RANTES/CCL5 throughout glomerular as well as tubulointerstitial structures as well as IP-10/CXCL10 mainly in the tubulointerstitium. Improved knowledge of gene polymorphisms of chemokines and their receptors could be useful to predict onset of diabetic nephropathy and define its progression. Blockade of the renin-angiotensin-aldosterone system is currently the only clinically used strategy to treat the inflammatory process in diabetic nephropathy. Newer strategies point to chemokine receptor antagonists and even to immunosuppressive therapy, but still remain in the experimental stage.

Angiotensin II is involved in the progression of renal disease: importance of non-hemodynamic mechanisms.

G Wolf — 2008-01-13T07:55:00-00:00

Nephrologie, Vol. 19, No. 7. (1998), pp. 451-456.

Several recent studies have provided clear evidence that angiotensin-converting enzyme (ACE)-inhibitors slow the progression of renal disease. These effects are mainly independent from a comitant reduction in systemic blood pressure. Thus, angiotensin II (Ang II) exerts other effects on the kidney which are involved in the loss of renal function. Ang II induces proliferation of cultured mesangial and glomerular endothelial cells. Our group was the first to demonstrate that Ang II stimulates hypertrophy of cultured proximal tubular cells. Ang II stimulates bioactivation and expression of transforming growth factor-beta (TGF-beta) in tubular MCT cells. This Ang II-mediated expression of TGF-beta is due to an increase in transcriptional activity. A neutralizing anti-TGF-beta antibody attenuates the Ang II-induced increase in protein synthesis in MCT cells suggesting that the hypertrophy is mediated by synthesis and activation of endogenous TGF-beta. Proximal tubular cells undergoing Ang II-mediated hypertrophy are arrested in the G1-phase of the cell cycle and express typical G1-phase-associated genes. Induction of such G1-phase-associated early growth response genes have been also described in vivo after infusion of Ang II into the renal artery. This G1-phase arrest depends on the induction of the cyclin-dependent kinase (CdK) inhibitor p27Kip1. p27Kip1 expression is stimulated after incubation of LLC-PK1 cells with Ang II or TGF-beta and binds to cyclin D1-CdK4 complexes, inhibits their kinase activity, and hampers G1-phase exit. Ang II stimulates transcription of collagen type IV in MCT cells. In addition to the classical a1 (IV) chain, a3 (IV) collagen, which has normally a restricted localization in the kidney, is also induced. This stimulation is mediated by endogenous synthesis and autocrine action of TGF-beta because a neutralizing anti-TGF-beta antibody as well as TGF-beta antisense oligonucleotides attenuate Ang II-induced collagen type IV transcription and synthesis. In addition, Ang II exerts immunomodulatory effects on the kidney through the induction of chemokines such as MCP-1 and RANTES. In conclusion, Ang II has emerged as a multifunctional acting as a growth factor and a profibrogenic cytokine, and even having inflammatory properties.

Checklist for the qualitative evaluation of clinical studies with particular focus on external validity and model validity

Gudrun Bornhoft — 2006-12-11T19:31:37-00:00

BMC Medical Research Methodology, Vol. 6 (11 December 2006), 56.

Prevalence and Prognostic Impact of Subclinical Cardiovascular Disease in Individuals With the Metabolic Syndrome and Diabetes

Erik Ingelsson — 2008-01-06T03:56:27-00:00

Diabetes, Vol. 56, No. 6. (1 June 2007), pp. 1718-1726.

Data are limited regarding prevalence and prognostic significance of subclinical cardiovascular disease (CVD) in individuals with metabolic syndrome (MetS). We investigated prevalence of subclinical CVD in 1,945 Framingham Offspring Study participants (mean age 58 years; 59% women) using electrocardiography, echocardiography, carotid ultrasound, ankle-brachial blood pressure, and urinary albumin excretion. We prospectively evaluated the incidence of CVD associated with MetS and diabetes according to presence versus absence of subclinical disease. Cross-sectionally, 51% of 581 participants with MetS had subclinical disease in at least one test, a frequency higher than individuals without MetS (multivariable-adjusted odds ratio 2.06 [95% CI 1.672.55]; P < 0.0001). On follow-up (mean 7.2 years), 139 individuals developed overt CVD, including 59 with MetS (10.2%). Overall, MetS was associated with increased CVD risk (multivariable-adjusted hazards ratio [HR] 1.61 [95% CI 1.122.33]). Participants with MetS and subclinical disease experienced increased risk of overt CVD (2.67 [1.624.41] compared with those without MetS, diabetes, or subclinical disease), whereas the association of MetS with CVD risk was attenuated in absence of subclinical disease (HR 1.59 [95% CI 0.872.90]). A similar attenuation of CVD risk in absence of subclinical disease was observed also for diabetes. Subclinical disease was a significant predictor of overt CVD in participants without MetS or diabetes (1.93 [1.153.24]). In our community-based sample, individuals with MetS have a high prevalence of subclinical atherosclerosis that likely contributes to the increased risk of overt CVD associated with the condition. 10.2337/db07-0078

Enrichment and Detection of Molecules Secreted by Tumor Cells Using Magnetic Reversed-Phase Particles and LC-MALDI-TOF-MS.

Jochen F Peter — 2008-01-02T04:53:23-00:00

J Biomol Tech, Vol. 18, No. 5. (December 2007), pp. 287-297.

Tumor cells change their genetic expression pattern as they progress to states of increasing malignancy. Investigations at the DNA and RNA level alone cannot provide all the information resulting after the translation and processing of the corresponding proteins, which is one reason for a poor correlation between mRNA and the respective protein abundance. In diagnostics, differentially expressed peptides or proteins are important markers for the early detection of cancer. Unfortunately, tumor cells secrete peptides and proteins in only very low amounts, making mass spectrometric determination very difficult. In this publication, methods have been developed for the effective enrichment and cleanup of substances secreted by cultivated cancer cells. To obviate peptides from fetal calf serum used in cell culture, a serum surrogate was developed, which maintained growth of the cancer cells. After the binding of substances from cell-culture supernatants to custom-made magnetic reversed-phase particles, the substances were eluted and separated by capillary high-performance liquid chromatography. Fractions were spotted directly on a MALDI target, and MALDI-TOF mass spectrometric data acquisition was performed in automatic mode. This technology was used to detect substances secreted by two mammary carcinoma cell lines differing in their malignancy (MCF-7, MDA-MB(231)). Unequivocal differences in the peptide secretion patterns were observed. In conclusion, this system allows the sensitive investigation of peptides secreted by cancer cells in culture and provides a valuable tool for the investigation of cancer cells in different states of malignancy.

Effect of advanced glycation end products on oxidative stress in endothelial cells in culture: a warning on the use of cells studied in serum-free media.

YY Hui — 2007-12-29T08:42:15-00:00

Diabetologia, Vol. 44, No. 10. (October 2001), pp. 1310-1317.

AIMS/HYPOTHESIS: Alterations in vascular permeability and oxidative stress are characteristics of endothelial dysfunction in diabetic vascular disease. Since AGE-proteins have been hypothesized to mediate these effects, we studied the effects of AGE-bovine serum albumin on endothelial monolayer permeability and intracellular glutathione. METHODS: AGE-BSA was prepared by incubating BSA for 30 days at 37 degrees C with 0.5 mol/l glucose and 0.2 mol/l phosphate buffer, pH 7.4. Permeability to fluorescently labelled BSA was assessed in a bovine pulmonary artery endothelial cell monolayer preparation. Glutathione was measured by an enzymatic assay. RESULTS: AGE-BSA concentrations greater than 3 to 4 micromol/l produced maximal increases in permeability (6-8 times basal) within 3 to 4 h of incubation with the cells. This effect persisted for at least 48 h. However, BSA incubated in the absence of glucose produced similar effects. Dialysis of the AGE-BSA showed that low molecular weight components contained the permeability-increasing activity. Phosphate buffer used to prepare the AGE-BSA, at concentrations equivalent to those present in phosphate-buffered saline and in the AGE preparation (approximately 5 mmol/l), produced similar permeability increases at equivalent incubation times. Metal chelators (0.5 mmol/l) or inclusion of fetal bovine serum (10-20 %) blocked these permeability increases. These increases in permeability were associated with a decrease in endothelial glutathione, both inhibited by 10 mmol/l N-acetylcysteine, and a loss of cell-to-cell and cell-to-matrix adhesion molecules. CONCLUSION/INTERPRETATION: Trace amounts of redox-active metal ions in biological buffers could induce oxidative stress and alterations in cellular functions attributed to AGE-proteins in vitro. It is important to use metal-free phosphate and bicarbonate buffers in studies on cell biology in vitro, especially in serum-free media.

DNA fragmentation in chronic glomerulonephritis: an immunohistological analysis.

U Ott — 2007-11-03T09:45:37-00:00

Nephron Clin Pract, Vol. 105, No. 1. (2007)

BACKGROUND: Experimental data suggest that apoptosis plays an important pathophysiological role in glomerulonephritis by restoring tissue structure after proliferation of intrinsic renal cells and infiltration of leukocytes. Relatively little is known of apoptosis in human glomerulonephritis, particularly in predicting renal function during follow-up. METHODS: In order to colocalize different markers for cell damage in renal tissue from patients with different forms of glomerulonephritis (GN), a series of semithin sections from 34 kidney biopsies were studied retrospectively. Normal kidney from a nephrectomy specimen with a small renal adenocarcinoma served as a control. DNA fragmentation, expression of tissue transglutaminase II, BAX and BCL-2 were visualized immunohistochemically. In some renal biopsies, immunohistochemical staining for activated caspase 3 was performed. Proinflammatory markers (C-reactive protein, leukocytes), serum creatinine, creatinine clearance, total proteinuria, albuminuria, alpha(1)-microglobulin and IgG excretion were determined at the time of biopsy. Serum creatinine and total proteinuria were assessed 6 and 12 months after renal biopsy. RESULTS: Nuclei with different degrees of DNA fragmentation were mainly found in epithelial cells of tubules, but also in glomerular cells, regardless of the form of GN studied. Transglutaminase II expression was found only in cells with a strong staining for DNA fragmentation. DNA fragmentation localized to glomerular cells was more pronounced in proliferative than in non-proliferative forms of GN, being most abundant in patients with rapid progressive GN. Staining for activated caspase 3 in selected biopsies confirmed the presence of apoptosis. BAX and BCL-2 staining was detected within the same cells, but exhibited a different intracellular distribution. In proliferative GN, the extent of DNA damage in tubular epithelial cells significantly corresponds with the concentration of serum creatinine (p < 0.04) and with urinary excretion of alpha(1)-microglobulin (p < 0.01) at the time of biopsy. A significant correlation (p < 0.01) was seen between glomerular DNA fragmentation and follow-up total proteinuria 12 months after biopsy for proliferative forms of GN. The damaged glomerular area (e.g. mesangial sclerosis) significantly correlated with DNA fragmentation in proliferative, but not in nonproliferative GN at the time of biopsy. Furthermore, glomerular damaged showed a significant correlation with tubular DNA damage in proliferative GN. CONCLUSION: In glomerular cells, apoptosis may be important for the clearance of proliferating cells whereas in tubules, cell damage showed dependence on the degree of tubular injury mediated by inflammation and/or proteinuria. Although the degree of apoptosis in tubular cells correlates with serum creatinine in proliferative GN at the time of biopsy, it is of limited use to predict future renal function.

Erk 1,2 phosphorylates p27(Kip1): Functional evidence for a role in high glucose-induced hypertrophy of mesangial cells.

G Wolf — 2007-10-21T13:57:30-00:00

Diabetologia, Vol. 46, No. 8. (August 2003), pp. 1090-1099.

AIMS/HYPOTHESIS. Mesangial cell hypertrophy is one of the earliest morphological abnormalities of diabetic nephropathy. We have previously shown that high glucose induces p27(Kip1) by a post-transcriptional mechanism and that mesangial cell hypertrophy depends on G(1)-phase arrest mediated by this CDK-inhibitor. However, it remains poorly understood how high glucose stimulates p27(Kip1) expression in mesangial cells. METHODS. Mesangial cells were isolated from p27(Kip1) +/+ and -/- mice and characterized by light microscopy and immunohistochemistry. It was tested by Western blotting and autoradiography whether high glucose medium activates Erk 1,2 and whether this activation phosphorylates p27(Kip1). The three consensus phosphorylation sites of p27(Kip1) were mutated and these constructs were expressed in p27(Kip1) -/- mesangial cells. Hypertrophy was assessed by different methods. RESULTS. High glucose stimulates phosphorylation of MAP kinases Erk 1,2 in p27(Kip1 )+/+ and -/- mesangial cells. Activation of Erk 1,2 leads to phosphorylation of p27(Kip1 )in vitro and in vivo. Mutations of serine(10) or threonine(187) still supported high glucose-induced hypertrophy. In contrast, a mutation of serine(178) converted the hypertrophic response into a proliferative phenotype. Mutation of serine(178) leads to the attenuated expression of p27(Kip1) protein in the presence of high glucose. CONCLUSIONS/INTERPRETATION. Our study shows that high glucose stimulates Erk 1,2 that phosphorylate p27(Kip1) at serine(178) increasing its expression. This is an important molecular mechanism of high glucose-induced hypertrophy of mesangial cells.

Collagen type VIII expression in human diabetic nephropathy

Gerth — 2007-10-10T02:59:00-00:00

European Journal of Clinical Investigation, Vol. 37, No. 10. (October 2007), pp. 767-773.

Magnesium and neoplasia: from carcinogenesis to tumor growth and progression or treatment.

FI Wolf — 2007-10-15T04:05:53-00:00

Arch Biochem Biophys, Vol. 458, No. 1. (1 February 2007), pp. 24-32.

Magnesium is involved in a wide range of biochemical reactions that are crucial to cell proliferation, differentiation, angiogenesis, and apoptosis. Changes in magnesium availability have been shown to influence biological responses of immuno-inflammatory cells. Equally plausible seems to be an involvement of magnesium in the multistep and interconnected processes that lead to tumor formation and development; however, the "how" and "when" of such an involvement remain to be defined. Here, we reviewed in vitro and in vivo data that indicated a role for magnesium in many biological and clinical aspects of cancer (from neoplastic transformation to tumor growth and progression or pharmacologic treatment). In adopting this approach we went through a full circle from molecular aspects to observational or epidemiological studies that could reconcile in a unifying picture the otherwise fragmentary or puzzling data currently available on the role of magnesium in cancer.

Cellular and molecular mechanisms of proteinuria in diabetic nephropathy.

G Wolf — 2007-10-04T04:09:53-00:00

Nephron Physiol, Vol. 106, No. 2. (2007)

One of the earliest clinically detectable abnormalities in diabetic nephropathy is microalbuminuria that eventually progresses to proteinuria. The degree of proteinuria correlates with the progression of glomerulosclerosis and tubulointerstitial fibrosis. In the glomerulus, a typical podocytopathy develops that participates in the initiation of glomerulosclerosis and the accelerated plasma protein leakage across the glomerular basement membrane (GBM) into Bowman's space. Downstream into the tubular compartment, the proteinuria induces proinflammatory and profibrogenetic injury in tubular cells which can facilitate the development of interstitial fibrosis and tubular atrophy. It has long been held that hemodynamic changes and the loss of negatively charged proteoglycans in the GBM are important mediators of proteinuria. More recently, biopsy studies in humans with diabetic kidney disease have provided strong evidence that podocytes are injured very early in the course of nephropathy. This podocytopathy--which is characterized by decreased podocyte number and/or density, GBM thickening and altered matrix composition, and foot process effacement--correlates closely with the development and progression of albuminuria. Components of the diabetic milieu (high glucose, accumulation of glycated proteins, high intrarenal angiotensin II (ANG II), and hypertension-induced mechanical stress) result in activation of cytokine systems, the most important of which are transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor-A (VEGF-A). ANG II-stimulated podocyte-derived VEGF, through a novel autocrine signaling loop, appears to be a major cause of nephrin downregulation and the development of proteinuria. Nephrin is an important protein of the slit diaphragm with anti-apoptotic signaling properties. TGF-beta1 causes podocyte apoptosis and an increase in extracellular matrix deposition. As a consequence, the denuded GBM adheres to Bowman's capsule initiating the development of glomerulosclerosis. Good control of hyperglycemia and hypertension and maximal inhibition of ANG II are essential steps in preventing the development and progression of diabetic nephropathy.

Cyclosporine A induces senescence in renal tubular epithelial cells.

P Jennings — 2007-09-26T09:31:55-00:00

Am J Physiol Renal Physiol, Vol. 293, No. 3. (September 2007)

The nephrotoxic potential of the widely used immunosuppressive agent cyclosporine A (CsA) is well recognized. However, the mechanism of renal tubular toxicity is not yet fully elucidated. Chronic CsA nephropathy and renal organ aging share some clinical features, such as renal fibrosis and tubular atrophy, raising the possibility that CsA may exert some of its deleterious effects via induction of a stress-induced senescent phenotype. We investigated this hypothesis in HK-2 cells and primary proximal tubular cells in vitro. CsA induced the production of H(2)O(2), caused cell cycle arrest in the G0/G1 phase, and inhibited DNA synthesis. Furthermore, CsA exposure lead to a reduction of telomere length, increased p53 serine 15 phosphorylation, and caused an upregulation of the cell cycle inhibitor p21(Kip1) (CDKN1A) mRNA levels. CsA caused an increase in p16(INK4a) (CDKN2A) expression after a 13-day exposure in primary proximal tubular cells but not in HK-2 cells. Coincubation of cells with CsA and catalase was able to prevent telomere shortening and partially restored DNA synthesis. In summary, CsA induces cellular senescence in human renal tubular epithelial cells, which can be attenuated by scavenging reactive oxygen species.

A network analysis of changes in molecular interactions in cellular signaling.

Oda Stoevesandt — 2007-03-04T05:46:50-00:00

Mol Cell Proteomics (26 December 2006)

Multiprotein complexes play an essential role in the propagation and integration of cellular signals. However, systems level analyses of signaling-dependent changes in the pattern of molecular interactions are still missing. Signaling in T lymphocytes is one prominent example, in which multiprotein complexes orchestrate signal transduction. We have implemented peptide microarrays comprising a set of interaction motifs of signaling proteins for network-based analyses of signaling-dependent changes in molecular interactions. Lysates of resting or stimulated cells are incubated on these arrays and the binding of signaling proteins is detected by immunofluorescence. Signaling-dependent complex formation leads to changes of signals on the microarrays in two ways: (1) Masking of a binding site of a signaling protein for a peptide on the array results in a signal decrease. (2) Interaction of a protein with a second protein, that in turn binds to a peptide on the array results in a signal increase for the first protein. Dissipation of complexes leads to the reverse changes. Competition with peptides corresponding to interaction motifs provides detailed information on the architecture of complexes; lack of individual signaling proteins reveals the functional interdependence of interactions in the network. We show that complex formation through phosphorylation of the scaffolding protein LAT acts as a signal amplifier. PLC1 deficiency increases the resting state levels of LAT-dependent complexes and augments the recruitment of the phosphatase SHPTP2 into complexes. For the analysis of signaling networks, the parallel detection of changes in interactions enables the identification of functional interdependencies with minimum a priori knowledge.

Leptin stimulates type I collagen production in db/db mesangial cells: glucose uptake and TGF-beta type II receptor expression.

DC Han — 2007-09-17T09:29:35-00:00

Kidney Int, Vol. 59, No. 4. (April 2001), pp. 1315-1323.

BACKGROUND: Serum leptin levels correlate with fat cell mass and are elevated in patients with massive obesity and type 2 diabetes mellitus, which are strong risk factors for the development of glomerulosclerosis. We have previously shown in cultured glomerular endothelial cells that leptin stimulates cellular proliferation and expression of the prosclerotic cytokine transforming growth factor-beta1 (TGF-beta1). Although the effect of leptin on the hypothalamus to regulate energy homeostasis is well known, the effect of leptin on the kidney, and specifically on the glomerular mesangial cell, is unclear. METHODS: The obese, diabetic db/db mouse, which lacks the functional full-length Ob-Rb leptin receptor, is a suitable model to assess the effects of hyperleptinemia on peripheral tissues that express other receptor isoforms. The effects of leptin on glucose uptake, the TGF-beta system, and type I collagen production were evaluated in db/db mouse mesangial cells in culture. A phosphatidylinositol-3 kinase (PI-3K) inhibitor was used to assess the role of PI-3K in mediating the effects of leptin. RESULTS: A short form of the leptin receptor (Ob-Ra), but not Ob-Rb, was present by reverse transcription-polymerase chain reaction in the kidney and mesangial cells of both nondiabetic db/m and diabetic db/db mice. In db/db mesangial cells, leptin increased 2-deoxy-D-glucose (2DOG) uptake dose dependently and stimulated gene expression of TGF-beta type II receptor (TbetaRII) and alpha1(I) collagen, but not TGF-beta1. Protein production of type I collagen (enzyme-linked immunosorbent assay) was also increased by leptin. Both leptin-stimulated 2DOG uptake and type I collagen production were suppressed by a PI-3K inhibitor, LY294002. Mesangial cells pretreated with leptin exhibited increased responsiveness to exogenous TGF-beta1, as evidenced by a greater production of type I collagen protein in leptin-pretreated cells exposed to low-dose TGF-beta1 (0.5 ng/mL). The addition of both TGF-beta1 (2 ng/mL) and leptin (100 ng/mL) increased type I collagen production more than addition of either TGF-beta1 or leptin alone. CONCLUSIONS: Leptin increases glucose uptake and type I collagen in db/db mesangial cells through a PI-3K-dependent pathway. We postulate that increased leptin levels may transmit a signal through the short-form leptin receptor to up-regulate TbetaRII and activate the intraglomerular TGF-beta system, which may contribute to the glomerulosclerosis of obesity or type 2 diabetes.

The hypertrophic effect of transforming growth factor-beta is reduced in the absence of cyclin-dependent kinase-inhibitors p21 and p27.

T Monkawa — 2007-09-17T09:31:00-00:00

J Am Soc Nephrol, Vol. 13, No. 5. (May 2002), pp. 1172-1178.

Transforming growth factor-beta (TGF-beta) has both antiproliferative and hypertrophic effects on mesangial cells (MC). However, it is not known if these processes are independent or if they share common signaling pathways. Proliferation and hypertrophy are regulated by specific cell-cycle regulatory proteins, where the cyclin-dependent kinase (CDK) inhibitors inhibit target cyclin-CDK complexes. This study examined whether the growth regulatory effects of TGF-beta were determined by the CDK inhibitors p21 and p27. Accordingly, cultured MC from wild type (+/+) and single and double null (-/-) p21 and p27 mice were grown in 5% serum in the presence or absence of TGF-beta1 (2 ng/ml). Proliferation ([(3)H]-thymidine incorporation, cell number, cell cycle) and hypertrophy ([(3)H]-leucine incorporation, total protein content, forward light scatter) were measured after 24 h, 48 h, and 96 h. TGF-beta inhibited proliferation in +/+ and p21/p27 double -/- MC to a similar extent. TGF-beta induced hypertrophy in +/+ MC (18.0% increase at 48 h), and to lesser extent in p21 -/- (12.8%) and p27 -/- MC (11.5%) measured by forward light scatter analysis. In p21/p27 double -/-, the hypertrophic effects of TGF-beta were significantly reduced (3.9% at 48 h). Similar results were obtained by measuring hypertrophy by total protein and [(3)H]-leucine incorporation. In conclusion, the CDK inhibitors p21 and p27 are not required for the antiproliferative effects of TGF-beta. However, the hypertrophic growth effects of TGF-beta are reduced in the absence of both p21 and p27. These data suggest that the regulation of the antiproliferative and hypertrophic effects of TGF-beta may be distinct processes.

The advanced glycation end product N(epsilon)-carboxymethyllysine is not a predictor of cardiovascular events and renal outcomes in patients with type 2 diabetic kidney disease and hypertension.

M Busch — 2007-09-13T10:03:44-00:00

Am J Kidney Dis, Vol. 48, No. 4. (October 2006), pp. 571-579.

BACKGROUND: Advanced glycation end products (AGEs) are implicated in the pathogenesis of vascular damage, especially in patients with diabetes and renal insufficiency. The oxidatively formed AGE N(epsilon)-carboxymethyllysine (CML) is thought to be a marker of oxidative stress. METHODS: Four hundred fifty patients with type 2 diabetes and nephropathy from the Irbesartan in Diabetic Nephropathy Trial cohort (mean age, 58 +/- 8.2 years; 137 women, 313 men) with a mean glomerular filtration rate of 48.2 mL/min (0.80 mL/s; Modification of Diet in Renal Disease formula) were followed up for 2.6 years. Serum CML was measured by using an enzyme-linked immunosorbent assay. Relationships between CML levels, traditional risk factors, and cardiovascular and renal events were tested in Cox proportional hazards models. RESULTS: Mean serum CML level was 599.9 +/- 276.0 ng/mL, and mean hemoglobin A1c level was 7.5% +/- 1.6%. One hundred forty-three first cardiovascular events occurred during follow-up; 74 patients died, 44 of cardiovascular causes. Final multivariate analysis showed age (relative risk [RR], 1.87; confidence interval [CI], 1.13 to 3.11; P = 0.016 for the highest compared with lowest quartile), history of prior cardiovascular events (RR, 1.96; CI, 1.35 to 2.85; P < 0.0005), and 24-hour urinary albumin-creatinine ratio (RR, 1.29; CI, 1.11 to 1.50 per doubling; P < 0.0005) to be independent risk factors for a first cardiovascular event, but not CML level. CML level also did not correlate significantly with renal outcome. CONCLUSION: Serum CML level could not be identified as an independent risk factor for cardiovascular or renal outcomes in the examined population. This suggests that traditional risk factors might have a more important role for these end points or that other AGE compounds, as well as tissue AGE levels, might be of greater relevance compared with serum levels, which remains open to further study.

Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis.

SJ Shankland — 2007-09-05T08:24:38-00:00

Am J Physiol Renal Physiol, Vol. 278, No. 4. (April 2000)

The response to glomerular and tubulointerstitial cell injury in most forms of renal disease includes changes in cell number (proliferation and apoptosis) and cell size (hypertrophy). These events typically precede and may be responsible for the accumulation of extracellular matrix proteins that leads to a decrease in renal function. There is increasing evidence showing that positive (cyclins and cyclin-dependent kinases) and negative (cyclin-dependent kinase inhibitors) cell cycle regulatory proteins have a critical role in regulating these fundamental cellular responses to immune and nonimmune forms of injury. Data now show that altering specific cell cycle proteins affects renal cell proliferation and improves renal function. Equally exciting is the expanding body of literature showing novel biological roles for cell cycle proteins in the regulation of cell hypertrophy and apoptosis. With increasing understanding of the role for cell cycle regulatory proteins in renal disease comes the hope for potential therapeutic interventions.

Angiotensin II and cell cycle regulation.

G Wolf — 2007-09-05T08:24:51-00:00

Hypertension, Vol. 43, No. 4. (April 2004), pp. 693-698.

Angiotensin II has emerged as an important growth factor for vascular, cardiac, and renal cells. Depending on the specific cell type and presence of other growth factors, angiotensin II induces proliferation (replication of DNA with subsequent successful division of cells), hypertrophy (increase in cell size, cell protein, and mRNA content without DNA replication), apoptosis (programmed cell death), or differentiation. Such angiotensin II-mediated modulation of growth process may underlie various pathophysiological processes such as atherosclerosis, vascular and cardiac remodeling, and progression of chronic renal disease. Clearly, angiotensin II-induced proliferation requires complete cell progression through the various steps of the cell cycle. In contrast, cells undergoing angiotensin II-mediated hypertrophy are arrested in the G1-phase. Upregulation of cell cycle-dependent kinase inhibitors (eg, p27Kip1) plays an important role in this process. Although accumulating evidence suggests that apoptosis is cell cycle-dependent, only few data are currently available concerning the interaction of angiotensin II with the cell cycle machinery in apoptosis. We review the various angiotensin II-mediated growth processes and their relationship to events governing cell cycle regulation.

High glucose-induced hypertrophy of mesangial cells requires p27(Kip1), an inhibitor of cyclin-dependent kinases.

G Wolf — 2007-09-05T08:18:27-00:00

Am J Pathol, Vol. 158, No. 3. (March 2001), pp. 1091-1100.

Hypertrophy of mesangial cells is one of the earliest morphological alterations in the kidney after the onset of diabetes mellitus. We have previously shown that cultured mesangial cells exposed to high ambient glucose arrest in the G1 phase of the cell cycle and that this is associated with an increased expression of inhibitors of the cyclin-dependent kinase (CDK)-inhibitors p21(Cip) and p27(Kip1). To further investigate a potential role of p27Kip1 in the development of glucose-induced hypertrophy, mesangial cells from p27Kip1 wild-type (+/+) and knockout (-/-) mice were established. High glucose medium (450 mg/dl) increased p21(Cip1) protein in p27Kip1+/+ and -/- mesangial cells, and increased p27Kip1 protein levels in p27Kip1+/+ cells. In contrast to high glucose increasing de novo protein synthesis in p27Kip1+/+ cells, high glucose did not increase protein synthesis in p27Kip1-/- cells. High glucose also reduced DNA synthesis and caused cell cycle arrest in p27Kip1+/+ cells. In contrast, despite an increase in transforming growth factor (TGF)-beta mRNA and protein expression, DNA synthesis and cell cycle progression were increased by high glucose in p27Kip1-/- cells. Exogenous TGF-beta comparably induced fibronectin mRNA in p27Kip1+/+ and -/- cells suggesting intact TGF-beta receptor transduction. In addition, high glucose failed to increase the total protein/cell number ratio in p27Kip1-/- cells. However, in the presence of high glucose, reconstituting p27Kip1 expression by transient or stable transfection in p27Kip1-/- cells, using an inducible expression system, increased the de novo protein synthesis and restored G1-phase arrest. These results show that p27Kip1 is required for glucose-induced mesangial cell hypertrophy and cell cycle arrest.

Glomerular expression of p27Kip1 in diabetic db/db mouse: role of hyperglycemia.

G Wolf — 2007-09-05T08:18:34-00:00

Kidney Int, Vol. 53, No. 4. (April 1998), pp. 869-879.

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.

Angiotensin II-stimulated hypertrophy of LLC-PK1 cells depends on the induction of the cyclin-dependent kinase inhibitor p27Kip1.

G Wolf — 2007-09-05T08:19:17-00:00

Kidney Int, Vol. 50, No. 6. (December 1996), pp. 2112-2119.

Angiotensin II (Ang II) induces hypertrophy of cultured proximal tubular epithelial cells including the LLC-PK1 cell line. We have previously shown that this hypertrophy appears in the G1-phase of the cell cycle. Since progression through the cell cycle is controlled by a series of cyclin and cyclin-dependent kinase (CdK) complexes that may be inactivated by CdK inhibitors, we studied the expression of the CdK-inhibitor p27Kip1 in LLC-PK1 cells challenged with Ang II. Compared to cells grown in serum-free medium, Ang II treatment enhanced p27Kip1 protein, but not mRNA expression. This p27Kip1 induction was mediated through AT1-receptors. Exogenous TGF-beta also stimulated p27Kip1 protein expression. Immunoprecipitation experiments revealed that p27Kip1 preferentially associated with CdK4 in Ang II-treated LLC-PK1 cells and that the activity of this kinase was inhibited after Ang II-treatment, an effect that may be generated by increased p27Kip1 binding to cyclin D1-CdK4 complexes. In contrast, p27Kip1 was not associated with cyclin E-CdK2 complexes in Ang II-stimulated cells. Treatment of LLC-PK1 cells with p27Kip1 antisense, but not missense, oligonucleotides abolished the Ang II-mediated cell hypertrophy as measured by de novo protein synthesis and total protein content, and facilitated entry into the S-phase of the cell cycle. Our findings suggest that Ang II stimulates p27Kip1 expression in renal cells. Furthermore, this induction of the CdK-inhibitor appears pivotal in the hypertrophy induced by Ang II and elucidates the molecular mechanisms associated with this growth response in proximal tubular cells.

Atrial natriuretic peptide attenuates ANG II-induced hypertrophy of renal tubular cells.

T Hannken — 2007-09-05T08:22:37-00:00

Am J Physiol Renal Physiol, Vol. 281, No. 1. (July 2001)

ANG II arrests LLC-PK1 cells in the G1 phase of the cell cycle and induces hypertrophy, an effect mediated by induction of p27Kip1. We studied whether atrial natriuretic peptide (ANP) may modulate ANG II-induced hypertrophy and p27Kip1 expression in tubular LLC-PK1 cells. ANP, through its fragments 3---28 and 4---27, prevented ANG II-induced cell cycle arrest. ANP inhibited >80% of ANG II-induced p27Kip1 protein expression (Western blots). ANP stimulated expression of MKP-1, a phosphatase involved in dephosphorylation of p44/42 mitogen-activated protein (MAP) kinase, up to 12 h. ANP prevented the ANG II-mediated phosphorylation peak of MAP kinase after 12 h of stimulation. 8-Bromo-cGMP mimicked all the effects of ANP. Transfection with MKP-1 antisense, but not sense, oligonucleotides abolished the modifying role of ANP on ANG II-mediated cell cycle arrest. The effect of ANP on ANG II-mediated hypertrophy of LLC-PK1 cells is regulated on the level of MAP kinase phosphorylation, a key step in the induction of p27Kip1. Although ANP and ANG II both stimulate generation of reactive oxygen species, ANP additionally induces expression of MKP-1, leading to interference with ANG II-mediated MAP kinase phosphorylation.

Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals.

T Hannken — 2007-09-05T08:19:21-00:00

Kidney Int, Vol. 54, No. 6. (December 1998), pp. 1923-1933.

BACKGROUND: Angiotensin II (Ang II) induces hypertrophy of cultured proximal tubular cells. We have previously demonstrated that this Ang II-mediated hypertrophy occurs in the G1-phase of the cell cycle and depends on the induction of p27Kip1, an inhibitor of G1-phase cyclin/cyclin-dependent kinase complexes. The present study was undertaken to investigate whether Ang II may stimulate superoxide anions (O2.) formation in cultured LLC-PK1 and cultured mouse proximal tubule (MCT) cells, and to gain further insight into a potential relationship between O2. and cell cycle regulation. METHODS: Reactive oxygen species were measured with the lucigenin method in intact cells. The effects of various inhibitors were tested on Ang II-induced O2. production. Cells were transiently transfected with phosphorothioate-modified rat p22phox antisense oligonucleotides to investigate the potential role of NAD(P)H oxidase. Expression of p22phox mRNA after Ang II-treatment was detected with Northern blots. Incorporation of [3H]leucine into de novo synthesized proteins was used as a parameter of cell hypertrophy. Expression of p27Kip1 was evaluated in cell lysates by Western blotting. RESULTS: Ang II stimulated the accumulation of O2. in tubular cells; however, an addition of two different antioxidants completely abolished measurable O2. This effect was transduced by angiotensin receptor type-1 (AT1) and was inhibited by a flavoprotein inhibitor (DIP) or p22phox antisense oligonucleotides, indicating the involvement of membrane NAD(P)H oxidase. Ang II-stimulated de novo protein synthesis was attenuated by DIP, antioxidants, and p22phox antisense oligonucleotides. The Ang II-induced expression of p27Kip1 protein and cellular hypertrophy were reduced by similar treatments. Generation of O2. by xanthine supplementation also stimulated p27Kip1 expression and induced hypertrophy in LLC-PK1 cells. CONCLUSIONS: 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.

Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1): role in angiotensin II-mediated hypertrophy of proximal tubular cells.

T Hannken — 2007-09-05T08:19:54-00:00

J Am Soc Nephrol, Vol. 11, No. 8. (August 2000), pp. 1387-1397.

Angiotensin II (AngII) induces G(1) phase arrest and hypertrophy of cultured renal proximal tubular cells. In previous studies, it was shown that these effects depend on oxygen radical-mediated induction of p27(Kip1), an inhibitor of cyclin-dependent kinases. The present study was undertaken to investigate whether mitogen-activated protein (MAP) kinases serve as signaling intermediates between AngII-induced oxidative stress and induction of p27(Kip1). AngII (10(-7) M) induces a biphasic phosphorylation pattern of p44/42 MAP kinase with an early phosphorylation after 2 min and a later, second phosphorylation peak after prolong incubation (12 h) in cultured proximal tubular cells from two different species (MCT and LLC-PK(1) cells). Total protein expression of MAP kinase was not changed by AngII. These phosphorylation patterns of p44/42 MAP kinase caused activation of the enzyme, as detected by phosphorylated MAP substrate Elk-1 after immuno-precipitation of MAP kinase. Exogenous H(2)O(2) also stimulates a biphasic phosphorylation of p44/42 MAP kinase. The flavoprotein inhibitor diphenylene iodinium, as well as the antioxidant N-acetylcysteine, prevented AngII-induced p44/42 MAP kinase phosphorylation, indicating involvement of reactive oxygen species generated by membrane-bound NAD(P)H oxidase. The MAP kinase kinase inhibitor PD98059 completely inhibits AngII-induced p27(Kip1) expression and (3)[H]leucine incorporation into proteins as a previously established marker of cell hypertrophy. PD98059 did not attenuate AngII-stimulated intracellular synthesis of oxygen radicals. Transient transfection with p44/42 MAP kinase antisense, but not sense, phosphorothioate-modified oligonucleotides also prevented AngII-induced MAP kinase phosphorylation, p27(Kip1) expression, and cell hypertrophy. Furthermore, induction of p27(Kip1) by H(2)O(2) was also abolished in the presence of PD98059. Although AngII induces phosphorylation of the stress-activated p38 MAP kinase, inhibition of this enzyme with SB203580 failed to attenuate induced p27(Kip1) expression and hypertrophy. These data provide evidence that AngII- mediated oxygen stress leads to the phosphorylation of p44/42 MAP kinase in proximal tubular cells. Activation of this enzyme is essential for p27(Kip1) expression, G(1) phase arrest, and hypertrophy of proximal tubular cells. These findings may lead to new concepts concerning interference of the development of proximal tubular hypertrophy, which may eventually turn into a maladaptive process in vivo leading ultimately to tubular atrophy and tubulointerstitial fibrosis.

Angiotensin II induces p27(Kip1) expression in renal tubules in vivo: role of reactive oxygen species.

G Wolf — 2007-09-05T08:19:28-00:00

J Mol Med, Vol. 79, No. 7. (July 2001), pp. 382-389.

Previous studies have demonstrated that angiotensin II (ANG II) mediates cell cycle arrest of cultured renal tubular cells by induction of p27(Kip1), an inhibitor of cyclin-dependent kinases. However, it is not known whether ANG II exerts similar effects in vivo. Infusion of ANG II into naive rats for 7 days increased formation of reactive oxygen species in tubular cells of the kidney. Furthermore, ANG II infusion stimulated protein expression of p27(Kip1) as detected by western blotting of tubular lysates and immunohistochemistry. Infusion of ANG II reduced tubular proliferation as detected by proliferating-cell nuclear antigen (PCNA) immunohistochemistry. The increase in p27(Kip1) expression was not due to an increase in mRNA. Immunoprecipitation experiments revealed that the increased p27(Kip1) protein associates with cyclin-dependent kinase 2. Coadministration of the radical scavenger dimethylthiourea abolished this ANG II mediated p27(Kip1) expression without reducing systemic blood pressure. Furthermore, dimethylthiourea infusion attenuates the ANG II mediated G(1)-phase arrest of tubular cells. However, infusion of norepinephrine did not induce reactive oxygen species or p27(Kip1) expression, despite a significant increase in blood pressure. Thus ANG II induces p27(Kip1) expression in renal tubular cells in vivo. This effect is mediated by reactive oxygen species. Since tubular hypertrophy depends on G(1)-phase arrest and may promote subsequent development of interstitial fibrosis, administering oxygen radical scavenger may be a therapeutic tool to counteract ANG II dependent remodeling of renal tubular cells.

Angiotensin II-induced hypertrophy of proximal tubular cells requires p27Kip1.

G Wolf — 2007-09-05T08:18:35-00:00

Kidney Int, Vol. 64, No. 1. (July 2003), pp. 71-81.

BACKGROUND: Angiotensin II (Ang II), as a single factor, induces hypertrophy of cultured proximal tubular cells of various species. Cells undergoing hypertrophy are arrested in the G1 phase of the cell cycle. Ang II also stimulated the expression of p27Kip1, an inhibitor of cyclin-dependent kinases (CDK). Although previous studies inhibiting p27Kip1 expression with antisense oligonucleotides suggested that this CDK inhibitor is important for Ang II-induced hypertrophy of proximal tubular cells, nonspecific effects of antisense technology, and the inability to transfect 100% of cells raised concerns about the true role of p27Kip1 in tubular hypertrophy. METHODS: Proximal tubular cells were isolated and cultured from wild-type (p27Kip1+/+) and knockout (p27Kip1-/-) mice. p27Kip1 genomic and protein expression was evaluated. Proximal tubular cell origin was confirmed by expression of various markers [3M-1 antigen, gamma-glutamyltransferase, angiotensin-converting enzyme (ACE)]. Cell proliferation (cell number, 3[H]thymidine incorporation) and hypertrophy (de novo protein synthesis as measured by 3[H]leucine incorporation, hypertrophy index, cell size) were evaluated. CDK2 and CDK4 activities were determined by an in vitro kinase assay. In addition, cell cycle analysis was performed by flow cytometry. p27Kip1 expression was reconstituted in two different clones of p27Kip1-/- proximal tubular cells using an inducible vector system based on ecdysone response elements. RESULTS: In accordance with previous studies, 10-7 mol/L Ang II induces hypertrophy and cell cycle arrest of p27Kip1+/+ proximal tubular cells. In contrast, Ang II facilitated cell cycle progression of two p27Kip1-/- proximal tubular cell lines without inducing hypertrophy. Ang II activates CDK4/cyclin D kinase activity in p27Kip1+/+ and -/- tubular cells, but stimulates CDK2/cyclin E activity only in wild-type cells. However, in the presence of Ang II, reconstituting p27Kip1 expression in p27Kip1-/- tubular cells using an inducible expression system, restored G1 phase arrest and the hypertrophic phenotype. Ang II did not induce apoptosis of either p27Kip1+/+ or -/- tubular cells. CONCLUSION: Our findings are the first clear evidence that p27Kip1 is required for Ang II-induced hypertrophy of proximal tubular cells. However, although p27Kip1 expression is an absolute requirement for this hypertrophy, reconstitution experiments revealed that other factors induced by Ang II contribute to this hypertrophy.