A mobile app providing individually-tailored psychoeducation about sleep for older adults with chronic health conditions and low health literacy.

Objective: This paper reports on a study of a mobile app that provides tailored information about sleep to individuals aged 40 and older who have chronic health conditions and low health literacy. Methods: The sleep module was a part of a multitopic app focused on chronic disease self-management. Participants were randomly assigned to receive sleep psychoeducation at reading levels equivalent to 3rd, 6th or 8th grade. The primary outcome measure was the Pittsburgh Sleep Quality Index (PSQI), which was completed at baseline, after the intervention, and again three months later. Outcomes were assessed using repeated measures mixed effects models. Results: Most participants were Black, Indigenous, or Other Persons of Color (BIPOC; 87%); they had average reading level at the 7th grade. Health literacy, socioeconomic status, and number of health conditions were related to the PSQI. The PSQI score decreased over the course of the three study visits for all groups, consistent with a small to medium effect size (d = 0.40). No effect of treatment group was observed. Participants were positive about the usefulness and helpfulness of the app. Conclusion: Results suggest that a brief tailored information intervention may be beneficial for individuals aged 40 and older who have low health literacy and chronic health conditions. Further development of the intervention may enhance its clinical effectiveness.

Characteristics differentiating problem representation synthesis between novices and experts.

BACKGROUND: Formulating a thoughtful problem representation (PR) is fundamental to sound clinical reasoning and an essential component of medical education. Aside from basic structural recommendations, little consensus exists on what characterizes high-quality PRs. OBJECTIVES: To elucidate characteristics that distinguish PRs created by experts and novices. METHODS: Early internal medicine residents (novices) and inpatient teaching faculty (experts) from two academic medical centers were given two written clinical vignettes and were instructed to write a PR and three-item differential diagnosis for each. Deductive content analysis described the characteristics comprising PRs. An initial codebook of characteristics was refined iteratively. The primary outcome was differences in characteristic frequencies between groups. The secondary outcome was characteristics correlating with diagnostic accuracy. Mixed-effects regression with random effects modeling compared case-level outcomes by group. RESULTS: Overall, 167 PRs were analyzed from 30 novices and 54 experts. Experts included 0.8 fewer comorbidities (p < .01) and 0.6 more examination findings (p = .01) than novices on average. Experts were less likely to include irrelevant comorbidities (odds ratio [OR] = 0.4, 95% confidence interval [CI] = 0.2-0.8) or a diagnosis (OR = 0.3, 95% CI = 0.1-0.8) compared with novices. Experts encapsulated clinical data into higher-order terms (e.g., sepsis) than novices (p < .01) while including similar numbers of semantic qualifiers (SQs). Regardless of expertise level, PRs following a three-part structure (e.g., demographics, temporal course, and clinical syndrome) and including temporal SQs were associated with diagnostic accuracy (p < .01). CONCLUSIONS: Compared with novices, expert PRs include less irrelevant data and synthesize information into higher-order concepts. Future studies should determine whether targeted educational interventions for PRs improve diagnostic accuracy.

Developing a Web-Based Asynchronous Case Discussion Format on Social Media to Teach Clinical Reasoning: Mixed Methods Study.

BACKGROUND: Case-based learning conferences are valuable to trainees, but growing clinical demands hinder consistent attendance. Social media increasingly acts as a venue for trainees to supplement their education asynchronously. We designed and implemented a web-based asynchronous clinical case discussion series on the Twitter social media platform to fill this educational gap. OBJECTIVE: The aim of this mixed methods study is to examine the nature of interactions among web-based case discussion participants and assess local attitudes regarding the educational intervention. METHODS: Starting in February 2018, we posted clinical vignettes to a dedicated Twitter account with the prompt "What else do you want to know?" to stimulate discussion. The authors replied in real time when case discussion participants requested additional details. Additional data about the case were posted at regular intervals to the discussion thread to advance the overall case discussion. Participants were asked to explain their reasoning and support their conclusions when appropriate. Web-based engagement was assessed using Twitter Analytics. Participants' posts were qualitatively analyzed for themes, with special attention to examples of using clinical reasoning skills. A codebook of types of participant posts and interactions was refined iteratively. Local engagement and attitudes at our institution were assessed by surveying internal medicine trainees (n=182) and faculty (n=165) after 6 months. RESULTS: Over a 6-month period, 11 live case discussions were engaged with by users 1773 times. A total of 86 Twitter profiles spanning 22 US states and 6 countries contributed to discussions among participants and the authors. Participants from all training levels were present, ranging from students to faculty. Interactions among participants and the case moderators were most commonly driven by clinical reasoning, including hypothesis-driven information gathering, discussing the differential diagnosis, and data interpretation or organization. Of 71 respondents to the local survey, 29 (41%) reported having a Twitter account. Of the 29 respondents with Twitter accounts, 17 (59%) reported participating in the case discussions. Respondents agreed that case participation increased both their clinical reasoning skills (15/17, 88%) and clinical knowledge (13/17, 76%). CONCLUSIONS: A social media-based serialized case discussion was a feasible asynchronous teaching method for engaging web-based learners of all levels in a clinical reasoning discussion. Further study should examine what factors drive trainee participation in web-based case discussions and under what circumstances asynchronous discussion might be preferred over in-person teaching activities.

A mobile app for chronic disease self-management for individuals with low health literacy: A multisite randomized controlled clinical trial.

Objective: The purpose of this study was to evaluate the effects of a mobile app designed to improve chronic disease self-management in older adult patients with low health literacy and who had at least one chronic health condition, and to assess the impact of delivering information at different levels of reading difficulty. Methods: A randomized controlled trial was completed at two sites. Individuals 40 years of age and older screened for low health literacy who had at least one chronic health condition were randomly assigned to a tailored information multimedia app with text at one of three grade levels. Four primary outcomes were assessed: patient activation, chronic disease self-efficacy, health-related quality of life, and medication adherence. Results: All groups showed overall increases in activation, self-efficacy, and health-related quality of life, but no change in medication adherence. No between-group differences were observed. Conclusions: The mobile app was effective in increasing participants' levels of several psychosocial variables, but reading difficulty level was not significantly related to outcomes.Registered at clinicaltrials.gov NCT02922439.

The use of podcasts as a tool to teach clinical reasoning: a pseudorandomized and controlled study.

OBJECTIVES: Podcasts have emerged as an efficient method for widespread delivery of educational clinical reasoning (CR) content. However, the impact of such podcasts on CR skills has not been established. We set out to determine whether exposure to expert reasoning in a podcast format leads to enhanced CR skills. METHODS: This is a pseudo-randomized study of third-year medical students (MS3) to either a control group (n=22) of pre-established online CR modules, or intervention group (n=26) with both the online modules and novel CR podcasts. The podcasts were developed from four "clinical unknown" cases presented to expert clinician educators. After completing these assignments in weeks 1-2, weekly history and physical (H&P) notes were collected and graded according to the validated IDEA rubric between weeks 3-7. A longitudinal regression model was used to compare the H&P IDEA scores over time. Usage and perception of the podcasts was also assessed via survey data. RESULTS: Ninety control and 128 intervention H&Ps were scored. There was no statistical difference in the change of average IDEA scores between intervention (0.92, p=0.35) and control groups (-0.33, p=0.83). Intervention participants positively received the podcasts and noted increased discussion of CR principles from both their ward (3.1 vs. 2.4, p=0.08) and teaching (3.2 vs. 2.5, p=0.05) attendings. CONCLUSIONS: This is the first objective, pseudo-randomized assessment of CR podcasts in undergraduate medical education. While we did not demonstrate significant improvement in IDEA scores, our data show that podcasts are a well-received tool that can prime learners to recognize CR principles.

Absent or diminished pedal pulses and estimated GFR decline in patients with diabetic kidney disease.

Background: Peripheral artery disease (PAD) is a complication of type 2 diabetes that leads to critical limb ischemia and amputation. We tested whether absent or diminished pedal pulses (ADPPs) predicts subsequent renal functional decline in patients with diabetic chronic kidney disease (CKD). We also examined the association between urinary biomarkers and ADPP as well as worsening CKD. Methods: Using a prospective longitudinal design, we studied 91 patients with type 2 diabetes and estimated glomerular filtration rate (eGFR) from 7 to 146 mL/min/1.73 m2. Baseline pedal pulses were assessed by standardized history and physical examination. The primary endpoint was decline in eGFR >30%. Potential confounders of the relationship between pedal pulses and eGFR were assessed by multivariable logistic regression. Results: Of 91 participants (median age 58 (range 30-83); median eGFR 72.4 ± 33.4 mL/min/1.73 m2), 43% had at least one ADPP. Baseline ADPP associated with increased risk of greater than 30% decline in eGFR (OR= 3.67, p = .004). This association remained significant (OR = 3.09, p = .029) after adjustment for traditional risk factors of renal function decline in diabetic kidney disease (DKD). In addition, urinary endothelin-1 (ET-1) was higher among patients with ADPP (p =.0006) and associated with eGFR decline greater than 30% (adjusted OR = 1.81, p = .035). Conclusions: ADPP is a strong predictor of decline in renal function in type 2 diabetes. Patients with type 2 diabetes and abnormal pedal pulses should be screened for DKD and monitored closely for progression of CKD.

A Mobile App for Chronic Disease Self-Management: Protocol for a Randomized Controlled Trial.

BACKGROUND: Health literacy is a critically important skill that helps people become active participants in their health care. Multiple studies in the United States and across the world have documented the association of health literacy with multiple health outcomes. In particular, the elderly and many members of minority groups have been shown to have low levels of health literacy; the same groups are disproportionately affected by chronic illnesses. These twin burdens affect the people most in need of the skills and knowledge required for coping with chronic illnesses. Chronic disease self-management (CDSM) is a logical target for a general health literacy intervention. In an approach that spans across specific diseases, CDSM targets problems and skills needed to cope with issues such as fatigue, pain, stress, depression, sleep disturbance, and treatment adherence. In a previous study, we showed that a computer-delivered tailored information intervention targeting health literacy could improve treatment and adherence and be cost effective, but it is not clear that this same strategy will be effective in persons with low health literacy and multiple chronic conditions. OBJECTIVE: The purpose of this study is to develop a computer-delivered mobile intervention that will provide individuals with chronic conditions the necessary information to cope with their conditions. METHODS: In this project, we will complete a qualitative study on the status and needs of individuals with more than one chronic condition. Results of this study will be used to develop a mobile tailored information app that will address self-management challenges in the areas of pain, sleep, fatigue, depression, anger, stress, memory problems, and treatment adherence. The impact of the intervention on patient quality of life, patient-provider relationships, health literacy, and patient activation will be assessed. We will also explore the extent to which health literacy mediates important outcomes, such as health-related quality of life and health service utilization. RESULTS: We are currently completing the preliminary qualitative and usability studies that will inform the content and design of the intervention. We anticipate that the intervention will be complete in 2017, and the clinical trial of its efficacy will also commence in 2017. CONCLUSIONS: Results will provide evidence on the usefulness of a mobile tailored information app for improving health literacy, patient activation, health-related quality of life, and self-reported health in patients with multiple chronic conditions. TRIAL REGISTRATION: Clinicaltrials.gov NCT02922439; https://clinicaltrials.gov/ct2/show/NCT02922439 (Archived by WebCite at http://www.webcitation.org/6pTiqDAyN).

Association of Urinary Biomarkers of Inflammation, Injury, and Fibrosis with Renal Function Decline: The ACCORD Trial.

BACKGROUND AND OBJECTIVES: Current measures for predicting renal functional decline in patients with type 2 diabetes with preserved renal function are unsatisfactory, and multiple markers assessing various biologic axes may improve prediction. We examined the association of four biomarker-to-creatinine ratio levels (monocyte chemotactic protein-1, IL-18, kidney injury molecule-1, and YKL-40) with renal outcome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We used a nested case-control design in the Action to Control Cardiovascular Disease Trial by matching 190 participants with ≥40% sustained eGFR decline over the 5-year follow-up period to 190 participants with ≤10% eGFR decline in a 1:1 fashion on key characteristics (age within 5 years, sex, race, baseline albumin-to-creatinine ratio within 20 µg/mg, and baseline eGFR within 10 ml/min per 1.73 m(2)), with ≤10% decline. We used a Mesoscale Multiplex Platform and measured biomarkers in baseline and 24-month specimens, and we examined biomarker associations with outcome using conditional logistic regression. RESULTS: Baseline and 24-month levels of monocyte chemotactic protein-1-to-creatinine ratio levels were higher for cases versus controls. The highest quartile of baseline monocyte chemotactic protein-1-to-creatinine ratio had fivefold greater odds, and each log increment had 2.27-fold higher odds for outcome (odds ratio, 5.27; 95% confidence interval, 2.19 to 12.71 and odds ratio, 2.27; 95% confidence interval, 1.44 to 3.58, respectively). IL-18-to-creatinine ratio, kidney injury molecule-1-to-creatinine ratio, and YKL-40-to-creatinine ratio were not consistently associated with outcome. C statistic for traditional predictors of eGFR decline was 0.70, which improved significantly to 0.74 with monocyte chemotactic protein-1-to-creatinine ratio. CONCLUSIONS: Urinary monocyte chemotactic protein-1-to-creatinine ratio concentrations were strongly associated with sustained renal decline in patients with type 2 diabetes with preserved renal function.

Serum ß-Trace Protein and ß2-Microglobulin as Predictors of ESRD, Mortality, and Cardiovascular Disease in Adults With CKD in the Chronic Renal Insufficiency Cohort (CRIC) Study.

BACKGROUND: Serum ß-trace protein (BTP) and ß2-microglobulin (B2M) are independently associated with end-stage renal disease (ESRD) and mortality in the general population and high-risk groups with diabetes or advanced chronic kidney disease (CKD). Less is known about their associations with outcomes and predictive ability in adults with moderate CKD. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 3,613 adults from the CRIC (Chronic Renal Insufficiency Cohort) Study (45% women; mean age, 57.9 years; 41.0% non-Hispanic black; 51.9% with diabetes). PREDICTORS: BTP and B2M levels with a reciprocal transformation to reflect their associations with filtration, creatinine-based estimated glomerular filtration rate (eGFRcr), measured GFR, and a 4-marker composite score combining BTP, B2M, creatinine, and cystatin C levels. Predictors were standardized as z scores for comparisons across filtration markers. OUTCOMES: ESRD, all-cause mortality, and new-onset cardiovascular disease. RESULTS: During a 6-year median follow-up, 755 (21%) participants developed ESRD, 653 died, and 292 developed new-onset cardiovascular disease. BTP, B2M, and the 4-marker composite score were independent predictors of ESRD and all-cause mortality, and B2M and the 4-marker composite score of cardiovascular events, after multivariable adjustment. These associations were stronger than those observed for eGFRcr (P vs eGFRcr≤0.02). The 4-marker composite score led to improvements in C statistic and 2.5-year risk reclassification beyond eGFRcr for all outcomes. LIMITATIONS: Filtration markers measured at one time point; measured GFR available in subset of cohort. CONCLUSIONS: BTP and B2M levels may contribute additional risk information beyond eGFRcr, and the use of multiple markers may improve risk prediction beyond this well-established marker of kidney function among persons with moderate CKD.

Relationship of urinary endothelin-1 with estimated glomerular filtration rate in autosomal dominant polycystic kidney disease: a pilot cross-sectional analysis.

BACKGROUND: The pathogenesis of progressive renal insufficiency in autosomal dominant polycystic kidney disease (ADPKD) is unclear. Evidence from experimental models of ADPKD suggests that elevated endothelin-1 (ET-1) drives cyst growth, renal fibrosis and loss of renal function, but whether ET-1 is elevated in humans with ADPKD is uncertain. METHODS: In a cross-sectional study of ADPKD we measured urinary ET-1, a surrogate for ET-1 in kidney cortex, in spot collections corrected for creatinine. The volume of each kidney was measured using MRI-based stereology. The relationship of urine ET-1 with MDRD eGFR and kidney volume was modeled by multiple linear regression with adjustment for clinical covariates. RESULTS: Patients with ADPKD were ages 18 to 53 with eGFRs (median, interquartile range) of 63.2 (43.5-80.2) ml/min/1.73 m(2) and albumin/creatinine ratios (ACR) of 115.0 (7.5-58.5) µg/mg. Urine ET-1 was inversely associated with eGFR (r = -0.480, P < 0.05) and positively (r = 0.407, P = 0.066) with ACR independent of age and female sex (P < 0.01). ET-1 appeared to be positively associated with total kidney volume (r = 0.426, P = 0.100), with a test for trend across urine ET-1 quartiles yielding z = 1.83, P = 0.068. ET-1 strongly correlated with NAGase (r = 0. 687, P = 0.001), a marker of tubular damage and a surrogate marker of renal disease progression in ADPKD. Of note, ET-1 levels in urine were not correlated with hypertension. CONCLUSIONS: In a translational study of patients with ADPKD, urinary ET-1 was inversely associated with eGFR and positively correlated with total kidney volume. Taken together with results from experimental models, these findings suggest that the role of ET-1 in ADPKD warrants further investigation.

Urine neutrophil gelatinase-associated lipocalin levels do not improve risk prediction of progressive chronic kidney disease.

Novel biomarkers may improve our ability to predict which patients with chronic kidney disease (CKD) are at higher risk for progressive loss of renal function. Here, we assessed the performance of urine neutrophil gelatinase-associated lipocalin (NGAL) for outcome prediction in a diverse cohort of 3386 patients with CKD in the Chronic Renal Insufficiency Cohort study. In this cohort, the baseline mean estimated glomerular filtration rate (eGFR) was 42.4 ml/min per 1.73 m(2), the median 24-h urine protein was 0.2 g/day, and the median urine NGAL concentration was 17.2 ng/ml. Over an average follow-up of 3.2 years, there were 689 cases in which the eGFR was decreased by half or incident end-stage renal disease developed. Even after accounting for eGFR, proteinuria, and other known CKD progression risk factors, urine NGAL remained a significant independent risk factor (Cox model hazard ratio 1.70 highest to lowest quartile). The association between baseline urine NGAL levels and risk of CKD progression was strongest in the first 2 years of biomarker measurement. Within this time frame, adding urine NGAL to a model that included eGFR, proteinuria, and other CKD progression risk factors led to net reclassification improvement of 24.7%, but the C-statistic remained nearly identical. Thus, while urine NGAL was an independent risk factor of progression among patients with established CKD of diverse etiology, it did not substantially improve prediction of outcome events.

The renal transcriptome of db/db mice identifies putative urinary biomarker proteins in patients with type 2 diabetes: a pilot study.

We sought to identify novel urinary biomarkers of kidney function in type 2 diabetes. We screened the renal transcriptome of db/db and db/m mice for differentially expressed mRNA transcripts that encode secreted proteins with human orthologs. Whether elevated urine levels of the orthologous proteins correlated with diminished glomerular filtration rate was tested in a cross-sectional study of n = 56 patients with type 2 diabetes. We identified 36 putative biomarker genes in db/db kidneys: 31 upregulated and 5 downregulated. Urinary protein levels of six selected candidates (endothelin-1, lipocalin-2, transforming growth factor-ß, growth and differentiation factor-15, interleukin-6, and macrophage chemoattractant protein-1) were elevated in type 2 diabetic patients with subnormal glomerular filtration rate (i.e., <90 ml·min(-1)·1.73 m(-2)), independent of microalbuminuria, age, sex, race, and use of angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists. In contrast, urinary levels of fibroblast growth factor were not increased. A composite variable of urine albumin and any of the six candidate markers was associated with subnormal estimated glomerular filtration rate more closely than albumin alone. In addition, urinary endothelin-1, growth and differentiation factor-15, and interleukin-6 were associated with a marker of proximal tubule damage, N-acetyl-ß-d-glucosaminidase activity. These results suggest that gene expression profiling in diabetic mouse kidney can complement existing proteomic-based approaches for renal biomarker discovery in humans.

Endothelin-1 increases collagen accumulation in renal mesangial cells by stimulating a chemokine and cytokine autocrine signaling loop.

Endothelin-1 (ET-1), a potent vasoconstrictor, has been implicated in the pathogenesis of collagen accumulation, extracellular matrix remodeling, and renal and cardiac fibrosis in diabetes. However, the mechanism by which ET-1 promotes collagen accumulation remains unclear. Here, we analyzed the gene expression profile of ET-1-stimulated mesangial cells to identify determinants of collagen accumulation. In human mesangial cells (a microvascular pericyte that secretes excess collagen in diabetic glomerulosclerosis), ET-1 increased mRNA and protein for MCP-1 (macrophage chemoattractant protein-1) and IL-6. ET-1-induced MCP-1 and IL-6 mRNAs and proteins were blocked by an ET(A) (but not ET(B)) receptor antagonist. ET-1/ET(A) receptor signaling evoked a 7.4-fold increase in collagen accumulation. Exogenous addition of either recombinant MCP-1 or IL-6 increased collagen accumulation by 3.5-fold. Co-stimulation with both MCP-1 and IL-6 did not elevate collagen accumulation further. Neither an MCP-1-neutralizing antibody nor an MCP-1 receptor antagonist inhibited ET-1-induced collagen accumulation. Similarly, neutralizing antibodies against IL-6 or the gp130 subunit of the IL-6 receptor did not attenuate ET-1-induced collagen accumulation. However, co-incubation with MCP-1- and IL-6-neutralizing antibodies inhibited ET-1-induced collagen accumulation by 52%, suggesting a robust autocrine loop wherein MCP-1 and IL-6 are redundant. Taken together, these results demonstrate that an autocrine signaling loop involving MCP-1 and IL-6 contributes to ET-1-induced collagen accumulation.

AMP-activated protein kinase inhibits transforming growth factor-beta-induced Smad3-dependent transcription and myofibroblast transdifferentiation.

In wound healing, myofibroblast transdifferentiation (MFT) is a metaplastic change in phenotype producing profibrotic effector cells that secrete and remodel the extracellular matrix. Unlike pathways that induce MFT, the molecular mechanisms that negatively regulate MFT are poorly understood. Here, we report that AMP-activated protein kinase (AMPK) blocks MFT in response to transforming growth factor-beta (TGFbeta). Pharmacological activation of AMPK inhibited TGFbeta-induced secretion of extracellular matrix proteins collagen types I and IV and fibronectin. AMPK activation also prevented induction of the myofibroblast phenotype markers alpha-smooth muscle actin and the ED-A fibronectin splice variant. AMPK activators did not prevent MFT in cells transduced with an adenovirus expressing dominant negative, kinase-dead AMPKalpha2. Moreover, AMPK activators did not inhibit MFT induction in AMPK(alpha1,2)(-/-) fibroblasts, demonstrating a requirement for AMPK(alpha) expression. Adenoviral transduction of constitutively active AMPK(alpha2) was sufficient to prevent TGFbeta-induced collagen I, alpha-smooth muscle actin, and ED-A fibronectin. AMPK did not reduce TGFbeta-stimulated Smad3 COOH-terminal phosphorylation and nuclear translocation, which are necessary for MFT. However, AMPK activation inhibited TGFbeta-induced transcription driven by Smad3-binding cis-elements. Consistent with a role for AMPK in transcriptional regulation, nuclear translocation of AMPKalpha2 correlated with the appearance of active AMPKalpha in the nucleus. Collectively, these results demonstrate that AMPK inhibits TGFbeta-induced transcription downstream of Smad3 COOH-terminal phosphorylation and nuclear translocation. Furthermore, activation of AMPK is sufficient to negatively regulate MFT in vitro.

Oleate induces a myofibroblast-like phenotype in mesangial cells.

OBJECTIVE: High circulating free fatty acids, commonly associated with obesity and insulin resistance, impair structure and function of the microvasculature. However, the mechanisms by which fatty acids cause microvascular remodeling are unclear. Using the mesangial cell model of microvascular pericytes, we demonstrate that the monounsaturated free fatty acid oleate induces a myofibroblast phenotype, an important cell fate transition in fibrotic remodeling of the extracellular matrix. MATERIALS AND RESULTS: Oleate induced a time- and dose-dependent increase in secretion of collagen I and fibronectin. Oleate also induced the myofibroblast phenotype markers alpha smooth muscle actin and ED-A fibronectin, and the magnitude of marker protein expression was similar to that for transforming growth factor (TGF)-beta. Oleate raised TGF-beta secretion 2.2-fold, and processing of latent to bioactive TGF-beta was also elevated. Oleate rapidly stimulated extracellular signal-regulated kinase1/2, and a pharmacological MEK inhibitor blocked TGF-beta secretion and conversion to the myofibroblast phenotype. A neutralizing TGF-beta antibody and a TGF-beta receptor kinase inhibitor blocked oleate-induced collagen I, alpha smooth muscle actin, and ED-A fibronectin, suggesting that oleate-stimulated TGF-beta was necessary for inducing myofibroblasts. CONCLUSIONS: Collectively, these results demonstrate that oleate can induce a myofibroblast phenotype in mesangial cells, which suggests a mechanism whereby elevated free fatty acids might promote microvascular remodeling in vivo.

TGF-beta-regulated collagen type I accumulation: role of Src-based signals.

Transforming growth factor-beta (TGF-beta) stimulates myofibroblast transdifferentiation, leading to type I collagen accumulation and fibrosis. We investigated the function of Src in TGF-beta-induced collagen I accumulation. In human mesangial cells, PTyr416 Src (activated Src) was 3.3-fold higher in TGF-beta-treated cells than in controls. Src activation by TGF-beta was blocked by rottlerin and by a dominant negative mutant of protein kinase Cdelta (PKCdelta), showing that TGF-beta activates Src by a PKCdelta-based mechanism. Pharmacological inhibitors and a dominant negative Src mutant prevented the increase in collagen type I secretion in cells exposed to TGF-beta. Similarly, on-target Src small interference RNA (siRNA) prevented type I collagen secretion in response to TGF-beta, but off-target siRNA complexes had no effect. It is well established in mesangial cells that upregulation of type I collagen by TGF-beta requires extracellular signal-regulated kinase 1/2 (ERK1/2), and we found that activation of ERK1/2 by TGF-beta requires Src. In conclusion, these results suggest that stimulation of collagen type I secretion by TGF-beta requires a PKCdelta-Src-ERK1/2 signaling motif.

Oxidative signaling in renal epithelium: Critical role of cytosolic phospholipase A2 and p38(SAPK).

Previous studies from this laboratory have demonstrated a critical role of cytosolic phospholipase A2 (cPLA2) and arachidonic acid in angiotensin II (Ang II) AT2 receptor-mediated signal transduction in renal epithelium. In primary proximal tubular epithelial cells exposed to hydrogen peroxide (H2O2), both the selective cPLA2 inhibitors and the cPLA2 antisense oligonucleotides significantly attenuated H2O2-induced arachidonic acid liberation and activation of p38(SAPK), ERK1/2, and Akt1. This H2O2-induced kinase activation was significantly attenuated by a Src kinase inhibitor PP2, or by transient transfection of carboxyl-terminal Src kinase (CSK) that maintained Src in the dormant form. Under basal conditions, Src coimmunoprecipitated with epidermal growth factor receptor (EGFR), while H2O2 increased EGFR phosphorylation in the complex. We observed that inhibition of EGFR kinase activity with AG1478 significantly attenuated H2O2-induced p38(SAPK) and ERK1/2 activation, but did not inhibit Akt1 activation. Furthermore, it seems that p38(SAPK) is upstream of ERK1/2 and Akt1, since a p38(SAPK) inhibitor SB203580 significantly blocked H2O2-induced activation of ERK1/2 and Akt1. Interestingly, overexpression of the dominant-negative p38(SAPK) isoform alpha inhibited ERK1/2 but not Akt1 activation. Our observations demonstrate that in these nontransformed cells, activation of cPLA2 is a converging point for oxidative stress and Ang II, which share common downstream signaling mechanisms including Src and EGFR. In addition, p38(SAPK) provides a positive input to both growth and antiapoptotic signaling pathways induced by acute oxidative stress.

Association between endothelin-1 and collagen deposition in db/db diabetic mouse kidneys.

Endothelin-1 has been implicated in diabetic kidney injury, but there are few firm data establishing the temporal and spatial expression of kidney endothelin-1 in diabetes. We performed an immunohistochemical and histopathological analysis to determine endothelin-1 peptide expression in the kidneys of diabetic db/db mice and non-diabetic db/m controls. Diabetic mice were studied at 8 weeks, before histological damage is evident, and again at 16 weeks, when significant glomerular injury has occurred. Urinary endothelin-1 was 6.2- and 3.6-fold higher in 8- and 16-week diabetic mice compared to age-matched controls (P<0.01 db/db vs. db/m). Compared to non-diabetic kidneys, immunoreactive endothelin-1 was first elevated 2.5-fold (P=0.02) in the tubulointerstitial compartment at 8-week and remained high (3.8-fold, P<0.01) at 16 weeks. In contrast, glomerular endothelin-1 was elevated 3.2-fold (P=0.03) only in 16-week diabetic mice. Glomerular and tubulointerstitial endothelin-1 were unchanged in 8- and 16-week non-diabetic mice. Elevated endothelin-1 in diabetic mice associated temporally and spatially with collagen deposition, especially in the tubulointerstitial compartment. The localization of kidney endothelin-1 is consistent with a role for this peptide in renal fibrogenesis. These results also highlight the potential role of ET-1 in the pathogenesis of early tubulointerstitial changes in diabetes.

Cell cycle signaling by endothelin-1 requires Src nonreceptor protein tyrosine kinase.

Cross-talk between G protein-coupled receptors and protein tyrosine kinases is well established, but the phenotypic consequences of these signaling interactions are not completely understood. To investigate the role of Src family kinases in mitogenic signaling by G protein-coupled receptors, we used genetic and pharmacological inhibition of Src to study cell growth in response to endothelin-1. We found that dominant-negative Src and COOH-terminal Src kinase blocked mesangial cell growth in response to endothelin-1, whereas growth induced by v-Ras was unaffected. Endothelin-1-induced cell growth was blocked by the pharmacological Src antagonist 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) but not by the inactive analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine. RNA interference knockdown of Src with on-target but not with off-target small interfering RNAs also inhibited growth in cells treated with endothelin-1. Dominant-negative Src prevented growth in cells activated by platelet-derived growth factor alone or in combination with endothelin-1, which suggests that Src integrates mitogenic signals from diverse classes of cell surface receptors. To further explore the role of Src in mitogenic signaling by G protein-coupled receptors, we sought to determine whether endothelin-1 induced cyclin D1 by a Src-based mechanism. We found that endothelin-1 increased cyclin D1 protein, which was blocked by preincubation with the Src antagonist PP2 and with the protein kinase C antagonist bisindolylmaleimide I. These results provide evidence for a Src- and protein kinase C-based pathway of mitogenic signaling by endothelin-1 receptors that involves cyclin D1.