"People Gather Here for Open Conversations, and Health Should Be in Our Open Conversations": Promoters of Black Men's Engagement in Diabetes Screenings at Local Barbershops.

Local barbershops, often racialized safe spaces, have long been used as sites of health interventions targeting Black American men. Here, we present findings from a barbershop intervention held in the Southeast where Black men were (1) approached using recruitment strategies informed by a community advisory board, (2) screened for type 2 diabetes, and interviewed to understand their levels of medical trust, motivation for testing in the barbershop, as well as the utility of barbershops in health promotion programming. The community advisory board consisted of five Black men from the city understudy. The intervention sample included 27 participants: 20 males and 7 females. Several men insisted on testing after their female spouses and two local women approached testers and were not denied access to screening. Themes that emerged for medical trust ranged from yes to no. Themes that emerged for motivation to screen included to know status (codes: for self, for loved ones), financial motivation (codes: free testing, incentives), risk (codes: family, race specific), referral (codes: other community member, barbershop), and convenience. Themes that emerged for the utility of barbershops in health interventions included access to people, trustworthy setting, location, and yes, they are useful with no explanation. Results show that barbershop interventions make a dynamic way to engage community members who otherwise may not trust medicine as a social structure. Results also show that future scholars and interventionists should consider gender dynamics, social class, and engaging community members as best practices in engaging Black men.

Meprin ß expression modulates the interleukin-6 mediated JAK2-STAT3 signaling pathway in ischemia/reperfusion-induced kidney injury.

Meprin metalloproteinases have been implicated in the pathophysiology of ischemia/reperfusion (IR)-induced kidney injury. Previous in vitro data showed that meprin ß proteolytically processes interleukin-6 (IL-6) resulting in its inactivation. Recently, meprin-ß was also shown to cleave the IL-6 receptor. The goal of this study was to determine how meprin ß expression impacts IL-6 and downstream modulators of the JAK2-STAT3-mediated signaling pathway in IR-induced kidney injury. IR was induced in 12-week-old male wild-type (WT) and meprin ß knockout (ßKO) mice and kidneys obtained at 24 h post-IR. Real-time PCR, western blot, and immunostaining/microscopy approaches were used to quantify mRNA and protein levels respectively, and immunofluorescence counterstaining with proximal tubule (PT) markers to determine protein localization. The mRNA levels for IL-6, CASP3 and BCL-2 increased significantly in both genotypes. Interestingly, western blot data showed increases in protein levels for IL-6, CASP3, and BCL-2 in the ßKO but not in WT kidneys. However, immunohistochemical data showed increases in IL-6, CASP3, and BCL-2 proteins in select kidney tubules in both genotypes, shown to be PTs by immunofluorescence counterstaining. IR-induced increases in p-STAT-3 and p-JAK-2 in ßKO at a global level but immunoflourescence counterstaining demonstrated p-JAK2 and p-STAT3 increases in select PT for both genotypes. BCL-2 increased only in the renal corpuscle of WT kidneys, suggesting a role for meprins expressed in leukocytes. Immunohistochemical analysis confirmed higher levels of leukocyte infiltration in WT kidneys when compared to ßKO kidneys. The present data demonstrate that meprin ß modulates IR-induced kidney injury in part via IL-6/JAK2/STAT3-mediated signaling.

Meprin-ß activity modulates the ß-catalytic subunit of protein kinase A in ischemia-reperfusion-induced acute kidney injury.

Meprin metalloproteases have been implicated in the progression of kidney injury. Previous work from our group has shown that meprins proteolytically process the catalytic subunit of protein kinase A (PKA-C), resulting in decreased PKA-C kinase activity. The goal of the present study was to determine the PKA-C isoforms impacted by meprin-ß and whether meprin-ß expression affects downstream mediators of the PKA signaling pathway in ischemia-reperfusion (IR)-induced kidney injury. IR was induced in 12-wk-old male wild-type (WT) and meprin-ß knockout (ßKO) mice. Madin-Darby canine kidney cells transfected with meprin-ß cDNA were also subjected to 2 h of hypoxia. Western blot analysis was used to evaluate levels of total PKA-C, PKA-Cα, PKA-Cß, phosphorylated (p-)PKA-C, and p-ERK1/2. Meprin-ß expression enhanced kidney injury as indicated by levels of neutrophil gelatinase-associated lipocalin and cystatin C. IR-associated decreases were observed in levels of p-PKA-C in kidney tissue from WT mice but not ßKO mice, suggesting that meprin-ß expression/activity is responsible for the in vivo reduction in kinase activity. Significant increases in levels of PKA-Cß were observed in kidney lysates for WT mice but not ßKO mice at 6 h post-IR. Proximal tubule PKA-Cß increases in WT but not ßKO kidneys were demonstrated by fluorescent microscopy. Furthermore, IR-induced injury was associated with significant increases in p-ERK levels for both genotypes. The present data demonstrate that meprin-ß enhances IR-induced kidney injury in part by modulating mediators of the PKA-Cß signaling pathway.

LC-MS-based metabolomics analysis to identify meprin-ß-associated changes in kidney tissue from mice with STZ-induced type 1 diabetes and diabetic kidney injury.

Meprin metalloproteases have been implicated in the pathophysiology of diabetic kidney disease (DKD). Single-nucleotide polymorphisms in the meprin-ß gene have been associated with DKD in Pima Indians, a Native American ethnic group with an extremely high prevalence of DKD. In African American men with diabetes, urinary meprin excretion positively correlated with the severity of kidney injury. In mice, meprin activity decreased at the onset of diabetic kidney injury. Several studies have identified meprin targets in the kidney. However, it is not known how proteolytic processing of the targets by meprins impacts the metabolite milieu in kidneys. In the present study, global metabolomics analysis identified differentiating metabolites in kidney tissues from wild-type and meprin-ß knockout mice with streptozotocin (STZ)-induced type 1 diabetes. Kidney tissues were harvested at 8 wk post-STZ and analyzed by hydrophilic interaction liquid chromatography ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Principal component analysis identified >200 peaks associated with diabetes. Meprin expression-associated metabolites with strong variable importance of projection scores were indoxyl sulfate, N-γ-l-glutamyl-l-aspartic acid, N-methyl-4-pyridone-3-carboxamide, inosine, and cis-5-decenedioic acid. N-methyl-4-pyridone-3-carboxamide has been previously implicated in kidney injury, and its isomers, 4-PY and 2-PY, are markers of peroxisome proliferation and inflammation that correlate with creatinine clearance and glucose tolerance. Meprin deficiency-associated differentiating metabolites with high variable importance of projection scores were cortisol, hydroxymethoxyphenylcarboxylic acid-O-sulfate, and isovaleryalanine. The data suggest that meprin-ß activity enhances diabetic kidney injury in part by altering the metabolite balance in kidneys, favoring high levels of uremic toxins such as indoxyl sulfate and N-methyl-pyridone-carboxamide.

Meprin ß metalloproteases associated with differential metabolite profiles in the plasma and urine of mice with type 1 diabetes and diabetic nephropathy.

BACKGROUND: Meprin metalloproteases are abundantly expressed in the brush border membranes of kidney proximal tubules and small intestines. Meprins are also expressed in podocytes and leukocytes (monocytes and macrophages). Meprins are implicated in the pathophysiology of diabetic nephropathy (DN) but underlying mechanisms are not fully understood. Single nucleotide polymophisms (SNPs) in the meprin ß gene were associated with DKD in human subjects. Furthermore, meprin α and ß double deficiency resulted in more severe kidney injury and higher mortality rates in mice with Streptozotocin (STZ)-induced type 1 diabetes. Identification of meprin substrates has provided insights on how meprins could modulate kidney injury. Meprin targets in the kidney include extracellular matrix (ECM) proteins, modulators of inflammation, and proteins involved in the protein kinase A (PKA) and PKC signaling pathways. The current study used a global metabolomics approach to determine how meprin ß expression impacts the metabolite milieu in diabetes and DKD. METHODS: Low dose STZ was used to induce type 1 diabetes in 8-week old wild-type (WT) and meprin ß knockout (ßKO) mice. Blood and urine samples were obtained at 4 and 8 weeks post-STZ injection. Assays for albumin, creatinine, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule - 1 (KIM-1), and cystatin C were used for biochemical assessment of kidney injury. Data for biomarkers of kidney injury utilized two-way ANOVA. Metabolomics data analysis utilized UPLC-QTOF MS and multivariate statistics. RESULTS: The number of metabolites with diabetes-associated changes in levels were significantly higher in the WT mice when compared to meprin ßKO counterparts. Annotated meprin ß expression-associated metabolites with strong variable importance in projection (VIP) scores play roles in lipid metabolism (LysoPC(16:1(9Z)), taurocholic acid), amino acid metabolism (indoxyl sulfate, hippuric acid), and neurotransmitter/stress hormone synthesis (cortisol, 3-methoxy-4-hydroxyphenylethylene glycolsulfate, homovanillic acid sulfate). Metabolites that associated with meprin ß deficiency include; 3,5-dihydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavone 3-glucuronide, pantothenic acid, and indoxyl glucuronide (all decreased in plasma). CONCLUSION: Taken together, the annotated metabolites suggest that meprin ß impacts complications of diabetes such as DKD by altering distinct metabolite profiles.

Inflammation and Kidney Injury in Diabetic African American Men.

African Americans are disproportionately burdened by diabetic kidney disease (DKD). However, little is known about the cellular and molecular mechanisms underlying the onset and progression of DKD in this population. The goal of the current study was to determine the association between specific inflammation markers and kidney injury in diabetic African American men. To this end, we recruited diabetic patients either with (n = 20) or without (n = 87) diagnosed kidney disease along with age-matched nondiabetic controls (n = 81). Urinary albumin-to-creatinine ratios (UACRs) and estimated glomerular filtration rates (eGFR) were used for biochemical assessment of kidney function. We then measured plasma and urinary levels of seven inflammatory markers, including adiponectin, C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), interleukin-6 (IL-6), and intercellular cell adhesion molecule-1 (ICAM-1). Plasma levels of TNF-α, TNFR1, and TNFR2 were significantly higher in diabetics with macroalbuminuria compared to nondiabetic controls and diabetics with normoalbuminuria or microalbuminuria. Likewise, urinary levels of ICAM-1 were higher in diabetics with macroalbuminuria compared to the other groups. Indeed, urinary ICAM-1, plasma TNF-α, and adiponectin had moderate positive correlations with UACR while plasma TNFR1 and TNFR2 levels were strongly correlated with kidney injury, indicated by multiple biomarkers of kidney injury. In contrast, though plasma CRP was elevated in diabetic subjects relative to nondiabetic controls, its levels did not correlate with kidney injury. Together, these data suggest that inflammation, particularly that mediated by the TNF-α/NF-κB signaling axis, may play a role in the pathogenesis of DKD in African American men.

Undiagnosed Kidney Injury in Uninsured and Underinsured Diabetic African American Men and Putative Role of Meprin Metalloproteases in Diabetic Nephropathy.

Diabetes is the leading cause of chronic kidney disease. African Americans are disproportionately burdened by diabetic kidney disease (DKD) and end stage renal disease (ESRD). Disparities in DKD have genetic and socioeconomic components, yet its prevalence in African Americans is not adequately studied. The current study used multiple biomarkers of DKD to evaluate undiagnosed DKD in uninsured and underinsured African American men in Greensboro, North Carolina. Participants consisted of three groups: nondiabetic controls, diabetic patients without known kidney disease, and diabetic patients with diagnosed DKD. Our data reveal undiagnosed kidney injury in a significant proportion of the diabetic patients, based on levels of both plasma and urinary biomarkers of kidney injury, namely, urinary albumin to creatinine ratio, kidney injury molecule-1, cystatin C, and neutrophil gelatinase-associated lipocalin. We also found that the urinary levels of meprin A, meprin B, and two kidney meprin targets (nidogen-1 and monocytes chemoattractant protein-1) increased with severity of kidney injury, suggesting a potential role for meprin metalloproteases in the pathophysiology of DKD in this subpopulation. The study also demonstrates a need for more aggressive tests to assess kidney injury in uninsured diabetic patients to facilitate early diagnosis and targeted interventions that could slow progression to ESRD.

Meprin Metalloprotease Deficiency Associated with Higher Mortality Rates and More Severe Diabetic Kidney Injury in Mice with STZ-Induced Type 1 Diabetes.

Meprins are membrane-bound and secreted metalloproteinases consisting of α and/or ß subunits that are highly expressed in kidney epithelial cells and are differentially expressed in podocytes and leukocytes (macrophages and monocytes). Several studies have implicated meprins in the progression of diabetic nephropathy (DN) and fibrosis-associated kidney disease. However, the mechanisms by which meprins modulate DN are not understood. To delineate the role of meprins in DN, we subjected meprin αß knockout (αßKO) mice and their wild-type (WT) counterparts to streptozotocin-induced type 1 diabetes. The 18-week survival rates were significantly lower for diabetic meprin αßKO mice when compared to those for their WT counterparts. There were significant decreases in mRNA and protein levels for both meprin α and ß in diabetic WT kidneys. Furthermore, the blood urea nitrogen levels and urine albumin/creatinine ratios increased in diabetic meprin αßKO but not in diabetic WT mice, indicating that meprins may be protective against diabetic kidney injury. The brush border membrane levels of villin, a meprin target, significantly decreased in diabetic WT but not in diabetic meprin αßKO kidneys. In contrast, isoform-specific increases in cytosolic levels of the catalytic subunit of PKA, another meprin target, were demonstrated for both WT and meprin αßKO kidneys.

Hypoxia Associated Proteolytic Processing of OS-9 by the Metalloproteinase Meprin ß.

Meprin metalloproteases play a role in the pathology of ischemia/reperfusion- (IR-) induced renal injury. The endoplasmic reticulum-associated protein, osteosarcoma-9 (OS-9), has been shown to interact with the carboxyl-terminal tail of meprin ß. More importantly, OS-9 interacts with the hypoxia inducible factor-1α (HIF-1α) and the prolyl-hydroxylase, proteins which mediate the cell's response to hypoxia. To determine if OS-9 is a meprin substrate, kidney proteins from meprin αß knockout mice (αßKO) (which lack endogenous meprins) and purified human OS-9 were incubated with activated forms of meprin A and meprin B, and Western blot analysis was used to evaluate proteolytic processing of OS-9. Fragmentation of OS-9 was observed in reactions with meprin B, but not meprin A. To determine whether meprin B cleaves OS-9 in vivo, wild-type (WT) and meprin αßKO mice were subjected to IR-induced renal injury. Fragmentation of OS-9 was observed in kidney proteins from WT mice subjected to IR, but not in meprin αßKO counterparts. Transfection of kidney cells (MDCK and HEK293) with meprin ß cDNA prevented accumulation of OS-9 following exposure to the hypoxia mimic, CoCl2. These data suggest that meprin ß interaction with OS-9 plays a role in the hypoxia response associated with IR-induced renal injury.

Isoform-specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A: significance in acute and chronic kidney injury.

Meprin metalloproteases are abundantly expressed in the brush-border membranes of kidney proximal tubules. Meprins are implicated in ischemia-reperfusion (IR)-induced renal injury and diabetic nephropathy. The protein kinase A (PKA) signaling pathway modulates extracellular matrix metabolism in diabetic kidneys. The present study evaluated isoform-specific interactions between the catalytic subunit of PKA (PKA C) and meprins. To this end, cytosolic-enriched kidney proteins from meprin αß double knockout mice, and purified forms of recombinant mouse PKA Cα, Cß1, and Cß2, were incubated with activated forms of either homomeric meprin A or meprin B. The cleaved protein products were subjected to SDS-PAGE and analyzed by Coomassie staining and Western blot analysis. While meprin A only cleaved PKA Cß1, meprin B cleaved all three PKA C isoforms. Analysis of the proteolytic fragments by mass spectrometry revealed that meprin A and B cleave the PKA C isoforms at defined sites, resulting in unique cleavage products. Michaelis-Menten enzyme kinetics demonstrated that meprin B-mediated cleavage of PKA Cα occurs at a rate consistent with that of other physiologically relevant meprin substrates. Meprin cleavage decreased the kinase activity of PKA Cα, Cß1, and Cß2. PKA C levels were higher in diabetic kidneys, with evidence of in vivo fragmentation in wild-type diabetic kidneys. Confocal microscopy showed localization of meprin A in the glomeruli of diabetic kidneys. At 3 h post-IR, PKA C levels in proximal tubules decreased compared with distal tubules, which lack meprins. These data suggest that meprins may impact kidney injury, in part, via modulation of PKA signaling pathways.

Villin and actin in the mouse kidney brush-border membrane bind to and are degraded by meprins, an interaction that contributes to injury in ischemia-reperfusion.

Meprins, metalloproteinases abundantly expressed in the brush-border membranes (BBMs) of rodent proximal kidney tubules, have been implicated in the pathology of renal injury induced by ischemia-reperfusion (IR). Disruption of the meprin ß gene and actinonin, a meprin inhibitor, both decrease kidney injury resulting from IR. To date, the in vivo kidney substrates for meprins are unknown. The studies herein implicate villin and actin as meprin substrates. Villin and actin bind to the cytoplasmic tail of meprin ß, and both meprin A and B are capable of degrading villin and actin present in kidney proteins as well as purified recombinant forms of these proteins. The products resulting from degradation of villin and actin were unique to each meprin isoform. The meprin B cleavage site in villin was Glu(744)-Val(745). Recombinant forms of rat meprin B and homomeric mouse meprin A had K(m) values for villin and actin of ∼1 µM (0.6-1.2 µM). The k(cat) values varied substantially (0.6-128 s(-1)), resulting in different efficiencies for cleavage, with meprin B having the highest k(cat)/K(m) values (128 M(-1)·s(-1) × 10(6)). Following IR, meprins and villin redistributed from the BBM to the cytosol. A 37-kDa actin fragment was detected in protein fractions from wild-type, but not in comparable preparations from meprin knockout mice. The levels of the 37-kDa actin fragment were significantly higher in kidneys subjected to IR. The data establish that meprins interact with and cleave villin and actin, and these cytoskeletal proteins are substrates for meprins.

The TATA binding protein associated factor 4b (TAF4b) mediates FSH stimulation of the IGFBP-3 promoter in cultured porcine ovarian granulosa cells.

We have established the gene for IGF binding protein-3 (IGFBP-3) as a target for FSH action. FSH effects on this gene require the PKA pathway as well as the PI-3 kinase and MAPK pathways. At the IGFBP-3 promoter, FSH effects depend on a site for TATA box binding protein (TBP) and formation of a high molecular weight transcription complex. To further elucidate FSH effects on the downstream events involving the TBP site, we cloned a pig TAF4b cDNA into a P-Flag expression vector. By co-transfecting granulosa cells with the IGFBP-3 promoter, we found that TAF4b mimics and enhances FSH induction of IGFBP-3 reporter activity. Using RT-PCR we showed that FSH stimulates expression of TAF4b. This would suggest that the role of TAF4b in follicular development is regulated by FSH. TAF4b may thus be the TFIID component that binds to the TBP site on the IGFBP-3 promoter and is essential for FSH induction of IGFBP-3.

Follicle-stimulating hormone induction of ovarian insulin-like growth factor-binding protein-3 transcription requires a TATA box-binding protein and the protein kinase A and phosphatidylinositol-3 kinase pathways.

The current study was done to elucidate the mechanism of the FSH stimulation of IGF-binding protein 3 (IGFBP-3) expression and map the FSH response element on the pig IGFBP-3 promoter. Forskolin induced IGFBP-3 reporter activity in transiently transfected granulosa cells. The protein kinase A (PKA) inhibitor [N-[2-(p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl] (and cotransfection with a PKA inhibitor expression vector), the phosphatidylinositol-3 kinase inhibitor [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], and the ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], all blocked FSH stimulation. Use of serial deletion constructs and site-directed mutagenesis show that a TATA box-binding protein site is required for FSH stimulation and that a specific protein 1 (Sp1) site is required for basal transcription. Gel shift assays of nuclear protein with a -61/-25 probe detected four protein-DNA complexes, with bands I and II having significantly higher intensities in FSH-treated cells than in controls. Mutation of the Sp1 site prevented formation of bands I and II whereas mutation of the TATA box-binding protein site prevented formation of band IV. Use of specific antibodies showed that Sp1 participates in formation of band I, Sp3 band II, and p300 in both I and II. Band III was nonspecifically competed out. We conclude that FSH stimulation of IGFBP-3 transcription is mediated by cAMP via the PKA pathway and requires the P1-3 kinase and likely the MAPK pathways.