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Gametophytic self-incompatibility (GSI) has been widely studied in flowering plants, but studies of the mechanisms underlying pollen tube growth arrest by self S-RNase in GSI species are limited. In the present study, two leucine-rich repeat extensin genes in pear (Pyrus bretschneideri), PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real-time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of the self-compatible cultivar 'Jinzhui' (harboring a spontaneous bud mutation) than in those of the self-incompatible cultivar 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 stimulated pollen tube growth and attenuated the inhibitory effects of self S-RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. These results indicate that abnormal expression of PbLRXA2.1 and PbLRXA2.2 is involved in the loss of self-incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by the ABRE-binding factor PbABF.D.2. Knockdown of PbABF.D.2 decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expression of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 was repressed by self S-RNase, suggesting that self S-RNase can arrest pollen tube growth by restricting the PbABF.D.2-PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide novel insight into pollen tube growth arrest by self S-RNase.

Senescent renal tubular cells may be linked to diabetic kidney disease (DKD)-related tubulopathy. We studied mice with or without diabetes in which hedgehog interacting protein (HHIP) was present or specifically knocked out in renal tubules (HhipLow-dose streptozotocin was employed to induce diabetes in both HhipCompared with HhipTubular HHIP deficiency prevented DKD-related tubulopathy, possibly via the inhibition of SGLT2 expression and cellular senescence.

Background: To investigate whether obesity with or without metabolic syndrome is prospectively associated with coronary artery calcium (CAC) progression and incident cardiovascular disease events. Methods: A total of 1730 participants from the CARDIA study (Coronary Artery Risk Development in Young Adults) were included (age, 40.1±3.6 years; 38.3% men), who completed computed tomography of CAC at baseline (year 15: 2000–2001) and follow-up (year 20 or 25). Metabolically healthy obesity (MHO) was defined as body mass index≥30 kg/m 2 without any metabolic syndrome components in our main analysis. Sensitivity analyses were conducted for several conditions characterizing 4 metabolic phenotypes. Results: During a mean follow-up of 9.1 years, 439 participants had CAC progression. MHO subjects had a significantly higher risk of CAC progression than their metabolically healthy normal weight counterparts (adjusted hazard ratios [95% CIs] from 1.761 [1.369–2.264] to 2.047 [1.380–3.036]) depending on the definition of MHO adopted. Obesity with unhealthy metabolic profile remained the highest significant risk of CAC progression and cardiovascular disease events whatever the definitions adopted for metabolically unhealthy status. Up to 60% of participants with MHO converted to metabolically unhealthy obesity from year 15 to year 20 or year 25. Further sensitivity analysis showed that MHO throughout carried a similar risk of incident cardiovascular disease events compared with metabolically healthy normal weight throughout. Conclusions: Different metabolic phenotypes of obesity beginning at a young age exhibit distinct risks of CAC progression and subsequent cardiovascular disease events in later midlife. MHO represents an intermediate phenotype between metabolically low- to high-risk obese individuals. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT00005130.

ObjectiveThe study aimed to assess the associations of physical activity (PA) trajectories across a 25-year span with coronary artery calcium (CAC) progression, and subsequent risk of cardiovascular disease (CVD) events.MethodsWe included 2497 participants from the Coronary Artery Disease Risk Development in Young Adults study who had computed tomography-assessment of CAC at baseline (year 15: 2000–2001) and follow-up (year 20 or 25) and at least three measures of PA from year 0 to year 25. Long-term PA trajectories were determined by latent class modelling using a validated questionnaire.ResultsAmong the included participants, 1120 (44.9%) were men, 1418 (56.8%) were white, and the mean (SD) age was 40.4 (3.6) years. We identified three distinct PA trajectories based on PA average levels and change patterns: low (below PA guidelines, n=1332; 53.3%); moderate (meeting and slightly over PA guidelines, n=919; 36.8%) and high (about three times PA guidelines or more, n=246; 9.9%). During a mean (SD) follow-up of 8.9 (2.1) years, 640 (25.6%) participants had CAC progression. Participants in the high PA trajectory group had a higher risk of CAC progression than those in the low PA trajectory group after adjustment for traditional cardiovascular risk factors (HR 1.51; 95% CI 1.18 to 1.94). However, high PA trajectory was not associated with an increased risk of incident CVD events (HR 1.01; 95% CI 0.44 to 2.31) and the incidence of CVD events in participants with CAC progression was similar across all three PA trajectory groups (p=0.736).ConclusionLong-term PA about three times the guidelines or more is independently associated with CAC progression; however, no additional risk of incident CVD events could be detected.

AIMS/HYPOTHESIS: We previously reported that renal tubule-specific deletion of Heterogeneous nuclear ribonucleoprotein F (Hnrnpf) results in upregulation of renal angiotensinogen (Agt) and downregulation of sodium-glucose co-transporter 2 (Sglt2) in Hnrnpf(RT) knockout (KO) mice. Non-diabetic Hnrnpf(RT) KO mice develop hypertension, renal interstitial fibrosis and glycosuria with no renoprotective effect from downregulated Sglt2 expression. Here, we investigated the effect of renal tubular Hnrnpf deletion on hyperfiltration and kidney injury in Akita mice, a model of type 1 diabetes. METHODS: Akita Hnrnpf(RT) KO mice were generated through crossbreeding tubule-specific (Pax8)-Cre mice with Akita floxed-Hnrnpf mice on a C57BL/6 background. Male non-diabetic control (Ctrl), Akita, and Akita Hnrnpf(RT) KO mice were studied up to the age of 24 weeks (n=8/group). RESULTS: Akita mice exhibited elevated systolic blood pressure as compared with Ctrl mice, which was significantly higher in Akita Hnrnpf(RT) KO mice than Akita mice. Compared with Akita mice, Akita Hnrnpf(RT) KO mice had lower blood glucose levels with increased urinary glucose excretion. Akita mice developed kidney hypertrophy, glomerular hyperfiltration (increased glomerular filtration rate), glomerulomegaly, mesangial expansion, podocyte foot process effacement, thickened glomerular basement membranes, renal interstitial fibrosis and increased albuminuria. These abnormalities were attenuated in Akita Hnrnpf(RT) KO mice. Treatment of Akita Hnrnpf(RT) KO mice with a selective A1 adenosine receptor inhibitor resulted in an increase in glomerular filtration rate. Renal Agt expression was elevated in Akita mice and further increased in Akita Hnrnpf(RT) KO mice. In contrast, Sglt2 expression was increased in Akita and decreased in Akita Hnrnpf(RT) KO mice. CONCLUSIONS/INTERPRETATION: The renoprotective effect of Sglt2 downregulation overcomes the renal injurious effect of Agt when these opposing factors coexist under diabetic conditions, at least partly via the activation of tubuloglomerular feedback.

Background: Remnant cholesterol (RC) contributes to residual risk of atherosclerotic cardiovascular disease, but population-based evidence on the prospective relationship between RC and coronary artery calcium (CAC) progression is rare. Methods: We pooled data obtained from 6544 atherosclerotic cardiovascular disease–free individuals from the CARDIA study (Coronary Artery Risk Development in Young Adults; n=2635) and MESA (Multi-Ethnic Study of Atherosclerosis; n=3909), with a mean±SD age of 47.2±19.8 years; 3019 (46.1%) were men who completed computed tomography of CAC at baseline. RC was measured as total cholesterol minus HDL (high-density lipoprotein) cholesterol minus calculated LDL (low-density lipoprotein) cholesterol (LDL-C) estimated by using the Martin/Hopkins equation. Adjusted Cox models were used to assess the relationships between RC levels and CAC progression. We also performed discordance analyses examining the risk of CAC progression in RC versus LDL-C discordant/concordant groups using median cut points and clinically relevant LDL-C targets. Results: During a median follow-up of 8.6 years, 2778 (42.5%) participants had CAC progression. After multivariable adjustment for demographics and cardiovascular risk factors, a 1-mg/dL increase in RC levels was associated with a 1.3% higher risk of CAC progression (hazard ratio, 1.013 [95% CI, 1.008–1.017]). Results were similar when we categorized individuals by RC quartiles. Furthermore, the discordant high RC/low LDL-C group had a significantly higher risk of CAC progression than the concordant low RC/LDL-C group regarding their medians (hazard ratio, 1.195 [95% CI, 1.063–1.343]) or when setting the clinical LDL-C cut points at 100 and 130 but not 70 mg/dL. The association remained robust across a series of sensitivity analyses. Conclusions: Elevated RC levels were associated with an increased risk of CAC progression independent of traditional cardiovascular risk factors, even in individuals with optimal LDL-C levels. Registration: URL: https://www.clinicaltrials.gov ; Unique identifiers: NCT00005130 (CARDIA) and NCT00005487 (MESA).

The angiotensin II receptor type 2 (AT2R) may be a potential therapeutic target for the treatment of hypertension and chronic kidney disease (CKD). The expression and function of AT2R in the vasculature and kidney appear sexually dimorphic. We hypothesised that Agtr2 knockout dams (AT2RKO) with gestational diabetes would program their offspring for subsequent hypertension and CKD in a sex-dependent manner. Age- and sex-matched offspring of non-diabetic and diabetic dams of wild-type (WT) and AT2RKO mice were followed from 4 to 20 weeks of age and were monitored for development of hypertension and nephropathy; a mouse podocyte cell line (mPOD) was also studied. Body weight was progressively lower in female compared with male offspring throughout the lifespan. Female but not male offspring from diabetic AT2RKO dams developed insulin resistance. Compared with the offspring of non-diabetic dams, the progeny of diabetic dams had developed more hypertension and nephropathy (apparent glomerulosclerosis with podocyte loss) at 20 weeks of age; this programming was more pronounced in the offspring of AT2RKO diabetic dams, particularly female AT2RKO progeny. Female AT2RKO offspring had lower basal ACE2 glomerular expression, resulting in podocyte loss. The aberrant ACE2/ACE ratio was far more diminished in glomeruli of female progeny of diabetic AT2RKO dams than in male progeny. Knock-down of Agtr2 in mPODs confirmed the in vivo data. AT2R deficiency accelerated kidney programming in female progeny of diabetic dams, possibly due to loss of protective effects of ACE2 expression in the kidney.

Clinical trials indicate that sodium/glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) improve kidney function, yet, the molecular regulation of SGLT2 expression is incompletely understood. Here, we investigated the role of the intrarenal renin–angiotensin system (RAS) on SGLT2 expression. In adult non-diabetic participants in the Nephrotic Syndrome Study Network (NEPTUNE, n=163), multivariable linear regression analysis showed SGLT2 mRNA was significantly associated with angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE) mRNA levels (P

Background: Recent studies show that sodium-glucose cotransporter 2 inhibitors (SGLT2i), originally approved for glycemic control in patients with type 2 diabetes, also exert renoprotective effects independently from effects on dysglycemia. Moreover, recent work indicates that SGLT2i treatment may be effective in patients with nondiabetic chronic kidney disease, including primary and secondary glomerular diseases. Summary: SGLT2i lower blood glucose by blocking glucose resorption in the early renal proximal tubule through the glucose transporter, SGLT2, leading to enhanced urinary glucose excretion. Recent studies indicate that SGLT2i may have pleiotropic effects on cells other than proximal tubular cells. SGLT2i reduce the glomerular workload by decreasing the intraglomerular pressure, thus ameliorating hyperfiltration, if present, and may also decrease systemic blood pressure. SGLT2i may also act directly on endothelial cells, possibly via modulating the effects of adhesion molecules and reducing inflammatory cytokines and reactive oxygen species. SGLT2i may have direct anti-inflammatory and antifibrotic effects on renal tubules. Some reports suggest direct protective effects on podocytes and mesangial cells as well. Here, we provide a review of the potential mechanisms of renoprotection, therapeutic utility, and potential side effects of SGLT2i in patients with nondiabetic glomerular diseases, based on data from studies carried out in cells, experimental animals, and humans. Key Messages: SGLT2i may be a promising addition to the glomerular disease treatment armamentarium. However, it is unclear at what point of the natural history of specific glomerular diseases (whether this is immune or nonimmune mediated) SGLT2i can be beneficial. Additionally, further studies are needed to assess the long-term efficacy and safety of SGLT2i in patients with nondiabetic glomerular diseases.

We investigated the impact of nuclear factor erythroid 2-related factor 2 (Nrf2) overexpression in renal proximal tubular cells (RPTCs) on blood glucose, kidney injury and sodium-glucose co-transporter 2 (Sglt2) expression in diabetic Akita Nrf2-/-/Nrf2RPTC transgenic (Tg) mice. Immortalized human RPTCs (HK2) stably transfected with plasmid containing the SGLT2 promoter, human kidneys from patients with diabetes were also studied. Nrf2 overexpression was associated with increased blood glucose, glomerular filtration rate, urinary albumin-creatinine ratio, tubulointerstitial fibrosis and Sglt2 expression in Akita Nrf2-/-/Nrf2RPTC Tg mice compared to their Akita Nrf2-/- littermates. In vitro, oltipraz or transfection of NRF2 cDNA stimulated SGLT2 expression and SGLT2 promoter activity in HK2, and these effects were inhibited by trigonelline or NRF2 small interfering RNA. The deletion of the NRF2-responsive element (NRF2-RE) in the SGLT2 promoter abolished the stimulatory effect of oltipraz on SGLT2 promoter activity. NRF2 binding to the NRF2-RE of the SGLT2 promoter was confirmed by gel mobility shift assay and chromatin immunoprecipitation assays. Kidneys from patients with diabetes exhibited higher levels of NRF2 and SGLT2 in the RPTCs than kidneys from patients without diabetes. These results suggest a link by which NRF2 mediates hyperglycemia-stimulation of SGLT2 expression and exacerbates blood glucose and kidney injury in diabetes.

Zinc is considered protective against atherosclerosis; however, the association between dietary zinc intake and cardiovascular disease remains debated. We investigated whether dietary zinc intake was associated with coronary artery calcium (CAC) progression in the Multi-Ethnic Study of Atherosclerosis (MESA). This analysis included 5186 participants aged 61.9 ± 10.2 years (48.8% men; 41.3% white, 25.0% black, 21.6% Hispanic, and 12.1% Chinese American) from the MESA. Dietary zinc intake was assessed by a self-administered, 120-item food frequency questionnaire at baseline (2000–2002). Baseline and follow-up CAC were measured by computed tomography. CAC progression was defined as CAC > 0 at follow-up for participants with CAC = 0 at baseline; and an annualized change of 10 or percent change of ≥ 10% for those with 0 0.05). In this multiethnic population free of clinically apparent cardiovascular disease, higher dietary zinc intake from non-red meat sources was independently associated with a lower risk of CAC progression. The MESA trial was registered at clinicaltrials.gov as NCT00005487.

Podocyte damage and loss are the early event in the development of focal segmental glomerulosclerosis (FSGS). Podocytes express angiotensin II type-2-receptor (AT2R), which may play a key role in maintaining kidney integrity and function. Here, we examined the effects of AT2R deletion and AT2R agonist compound 21 (C21) on the evolution of FSGS. FSGS was induced by adriamycin (ADR) injection in both male wild-type (WT) and AT2R knockout (KO) mice. C21 was administered to WT-FSGS mice either one day before or 7 days after ADR (Pre-C21 or Post-C21), using two doses of C21 at either 0.3 (low dose, LD) or 1.0 (high dose, HD) mg/kg/day. ADR-induced FSGS was more severe in AT2RKO mice compared with WT-FSGS mice, and included profound podocyte loss, glomerular fibrosis, and albuminuria. Glomerular cathepsin L expression increased more in AT2RKO-FSGS than in WT-FSGS mice. C21 treatment ameliorated podocyte injury, most significantly in the Pre C21-HD group, and inhibited glomerular cathepsin L expression. In vitro, Agtr2 knock-down in mouse podocyte cell line given ADR confirmed the in vivo data. Mechanistically, C21 inhibited cathepsin L expression, which protected synaptopodin from destruction and stabilized actin cytoskeleton. C21 also prevented podocyte apoptosis. In conclusion, AT2R activation by C21 ameliorated ADR-induced podocyte injury in mice by the inhibition of glomerular cathepsin L leading to the maintenance of podocyte integrity and prevention of podocyte apoptosis.

Calcific aortic valve disease (CAVD) is a highly prevalent condition that comprises a disease continuum, ranging from microscopic changes to profound fibro-calcific leaflet remodeling, culminating in aortic stenosis, heart failure, and ultimately premature death. Ferroptosis has been hypothesized to contribute to the pathogenesis of CAVD. We aimed to study the association between ferroptosis genes and CAVD and reveal the potential roles of ferroptosis in CAVD. CAVD-related differentially expressed genes (DEGs) were identified via bioinformatic analysis of Datasets GSE51472 and GSE12644 obtained from Gene Expression Omnibus. A ferroptosis dataset containing 259 genes was obtained from the Ferroptosis Database. We then intersected with CAVD-related DEGs to identify the ferroptosis DEGs. Subsequently, protein–protein interaction networks and functional enrichment analyses were performed for ferroptosis DEGs. Then, we used miRWalk3.0 to predict the target pivotal microRNAs. An in vitro model of CAVD was constructed using human aortic valve interstitial cells. The qRT-PCR and western blotting methods were used to validate the ferroptosis DEGs identified by the microarray data. A total of 21 ferroptosis DEGs in CAVD containing 12 upregulated and nine downregulated genes were identified. The results of the Gene Set Enrichment Analysis (GSEA) and analysis of the KEGG pathway by WebGestalt indicated that the ferroptosis DEGs were enriched in six signaling pathways among which NAFLD (including IL-6, BID, and PRKAA2 genes) and HIF-1 (including IL-6, HIF-1, and HMOX1 genes) signaling pathways were also verified by DAVID and/or Metascape. Finally, the in vitro results showed that the mRNA and protein expression levels of IL-6, HIF-1α, HMOX1, and BID were higher, while the levels of PRKAA2 were lower in the Pi-treated group than those in the control group. However, the addition of ferrostatin-1 (a selective ferroptosis inhibitor) significantly reversed the above changes. Therefore, IL-6, HIF-1α, HMOX1, BID, and PRKAA2 are potential key genes closely associated with ferroptosis in CAVD. Further work is required to explore the underlying ferroptosis-related molecular mechanisms and provide possible therapeutic targets for CAVD.

Glomerular endothelial cell (GEC) dysfunction occurs in diabetic kidney disease (DKD) and generally precedes albuminuria. We recently reported that hedgehog interacting protein (Hhip), highly expressed in GECs, contributes to DKD development in diabetic mice. Here, we hypothesized that urinary Hhip (uHhip) could identify early DKD; we tested uHhip in mice and humans with diabetes (DM). In both type 1 (Akita) and type 2 (db/db) DM mice, uHhip is elevated prior to the development of albuminuria, while non-DM controls excrete minimal amount of uHhip. In 87 type 2 DM patients and 39 healthy controls, the uHhip/creatinine (Cr) ratio provides a significant discrimination between non-DM and DM groups; 0 [0-69.5] in non-DM, 9.9 [1.7-39.5] in normoalbuminuric DM, 167.7 [95.7-558.7] in microalbuminuric DM, and 207.9 [0-957.2] in macroalbuminuric DM (median [IQR] ng/mmol, P < 0.0001). The log-uHhip/Cr is positively correlated with urine albumin/Cr ratio (UACR) (spearman correlation coefficient 0.47, P < 0.0001). The log-uHhip/Cr is also associated with eGFR, pulse pressure, and urinary cytokines (IL-1β, IL-6, IL-8, and TGFβ1) independent of UACR. By immunostaining, Hhip is localized in glomeruli and tubules, and is increased in human DM kidneys compared with non-DM kidneys. TGFβ1 shares the similar staining pattern as Hhip in human DM kidneys. Thus, uHhip appears to be a novel indicator of diabetic GEC injury and is elevated in early DKD before the development of microalbuminuria in mice and humans. Clinical value for detecting early DKD warrants further investigation.

We investigated the molecular mechanism(s) by which insulin prevents Bcl2-modifying factor (Bmf)-induced renal proximal tubular cell (RPTC) apoptosis and loss in diabetic mice. Transgenic mice (Tg) mice specifically overexpressing human BMF in RPTCs and non-Tg littermates were studied at 10 to 20 weeks of age. Non-diabetic littermates, diabetic Akita mice +/− insulin implant, Akita Tg mice specifically overexpressing heterogeneous nuclear ribonucleoprotein F (hnRNP F) in their RPTCs and immortalized rat renal proximal tubular cells (IRPTCs) were also studied. BMF-Tg mice exhibited higher systolic blood pressure, urinary albumin/creatinine ratio, RPTC apoptosis and urinary RPTCs than non-Tg mice. Insulin treatment in Akita mice and Akita mice overexpressing hnRNP F suppressed Bmf expression and RPTC apoptosis. In hyperinsulinemic-euglycemic wild type mice, renal Bmf expression was down-regulated with up-regulation of hnRNP F. In vitro, insulin inhibited high glucose-stimulation of Bmf expression, predominantly via p44/42 mitogen-activated protein kinase (MAPK) signaling. Transfection of p44/42 MAPK or hnRNP F small interfering RNA (siRNA) prevented insulin inhibition of Bmf expression. HnRNP F inhibited Bmf transcription via hnRNP F-responsive element in the Bmf promoter. Our results demonstrate that hnRNP F suppression of Bmf transcription is an important mechanism by which insulin protects RPTCs from apoptosis in diabetes.

Background It remains unclear whether triglyceride-glucose (TyG) index, a surrogate marker of insulin resistance, is prospectively associated with incident peripheral arterial disease (PAD). Methods We included 12,320 Atherosclerosis Risk in Communities Study participants (aged 54.3 ± 5.7 years) free of a history of PAD at baseline (visit 1: 1987–1989). The TyG index was determined using ln (fasting triglycerides [mg/dL] × fasting glucose [mg/dL]/2), and measured at 5 visits between 1987 and 2013. Incident PAD was defined as the first hospitalization with PAD diagnosis or a new onset of measured ABI Results Over a median follow-up of 23 years, 1300 participants developed PAD. After adjustment for traditional PAD risk factors, each 1-SD (0.58) increase in TyG index was associated with an 11.9% higher risk of incident PAD [hazard ratio, 1.119 (95% CI, 1.049–1.195)]. Results were similar when individuals were categorized by TyG index quartiles [hazard ratio, 1.239 (95% CI, 1.028–1.492); comparing extreme quartiles]. Four distinct trajectories of stable TyG indexes at various levels along the follow-up duration were identified [low (22.2%), moderate (43.2%), high (27.5%), and very high (7.1%) trajectory groups]. Compared with those with a TyG index trajectory at a low level, those participants with TyG index trajectories at high and very high levels had an even greater risk of future incident PAD [odds ratio (95%CI): 1.404 (1.132–1.740) and 1.742 (1.294–2.344), respectively] after multivariate adjustments for traditional PAD risk factors. Conclusions Higher TyG index is independently associated with an increased risk of incident PAD. Long-term trajectories of TyG index help identify individuals at a higher risk of PAD who deserve specific preventive and therapeutic approaches. Trial registration: Clinical trial registration number: The ARIC trial was registered at clinicaltrials.gov as NCT00005131.

The angiotensin II receptor type 2 (ATAge- and sex-matched offspring of non-diabetic and diabetic dams of wild-type (WT) and ATBody weight was progressively lower in female compared with male offspring throughout the lifespan. Female but not male offspring from diabetic ATAT

Supplemental Digital Content is available in the text., Objective: To investigate whether low-carbohydrate diets (LCDs) were associated with coronary artery calcium (CAC) progression. Approach and Results: We included the participants who completed computed tomography assessment of baseline CAC in 2000 to 2001 (year 15) and follow-up (year 20 or 25) and food frequency questionnaire (years 0, 7, and 20) in the CARDIA study (Coronary Artery Risk Development in Young Adults). CAC progression was defined as CAC >0 at follow-up among participants with baseline CAC of 0 and an annualized change of 10 or percent change of ≥10% for those with 0

Zinc is considered protective against atherosclerosis; however, the association between dietary zinc intake and cardiovascular disease remains debated. We investigated whether dietary zinc intake was associated with coronary artery calcium (CAC) progression in the Multi-Ethnic Study of Atherosclerosis (MESA).This analysis included 5186 participants aged 61.9 ± 10.2 years (48.8% men; 41.3% white, 25.0% black, 21.6% Hispanic, and 12.1% Chinese American) from the MESA. Dietary zinc intake was assessed by a self-administered, 120-item food frequency questionnaire at baseline (2000-2002). Baseline and follow-up CAC were measured by computed tomography. CAC progression was defined as CAC 0 at follow-up for participants with CAC = 0 at baseline; and an annualized change of 10 or percent change of ≥ 10% for those with 0 CAC 100 or CAC ≥ 100 at baseline, respectively.Dietary zinc intake was 8.4 ± 4.5 mg/day and 2537 (48.9%) of the included participants had CAC at baseline. Over a median follow-up of 3.4 years (25th-75th percentiles = 2.0-9.1 years), 2704 (52.1%) participants had CAC progression. In the fully adjusted model, higher dietary zinc was associated with a lower risk of CAC progression in both men (hazard ratio [HR] 0.697, 95% confidence interval [CI] 0.553-0.878; p = 0.002) and women (HR 0.675; 95% CI 0.496-0.919; p = 0.012, both comparing extreme groups). Furthermore, such an inverse association was attributable to dietary zinc intake from non-red meat (p 0.05), rather than red meat sources (p 0.05).In this multiethnic population free of clinically apparent cardiovascular disease, higher dietary zinc intake from non-red meat sources was independently associated with a lower risk of CAC progression.The MESA trial was registered at clinicaltrials.gov as NCT00005487.

Aims SGLT2 inhibitors have been proposed as an adjunct to insulin therapy for glycemic control in type 1 diabetes (T1D) patients. However, concern has been raised due to an increase in renin–angiotensin-system (RAS) activity reported in a clinical trial in which an SGLT2 inhibitor was added while insulin dose was reduced in T1D patients. We previously reported that insulin inhibits intrarenal angiotensinogen (Agt) gene transcription and RAS activation. We hypothesized that insulin, rather than SGLT2 inhibition might regulate the intrarenal RAS. Methods We compared RAS activity in non-diabetic wild type mice, Akita mice (T1D model) and Akita mice treated with insulin or the SGLT2 inhibitor canagliflozin. Results Treatment of Akita mice with insulin or canagliflozin produced similar reductions in blood glucose, whereas insulin, but not canagliflozin, reduced elevated systolic blood pressure. Akita mice exhibited increased renal Agt mRNA/protein expression, which was attenuated by insulin, but not by canagliflozin. Furthermore, insulin was more effective than canagliflozin in lowering kidney weight and albuminuria. Conclusions Insulin, but not canagliflozin, lowers intrarenal RAS activity in Akita mice. Our findings can be of potential clinical importance, especially for T1D patients who are not on RAS inhibitors at the time of adding SGLT2 inhibitors.

We investigated whether renal hedgehog interacting protein (Hhip) expression contributes to the progression of diabetic nephropathy (DN) and studied its related mechanism(s) in vivo and in vitro. Here, we show that Hhip expression is highly elevated in glomerular endothelial cells of adult type 1 diabetic (T1D) Akita and T2D db/db mouse kidneys as compared to non-diabetic control littermates. Hyperglycemia enhances reactive oxygen species (ROS) generation via NADPH oxidase 4 (Nox4) activation and stimulates renal Hhip gene expression, and that elevated renal Hhip gene expression subsequently activates the TGFβ1- Smad2/3 cascade and promotes endothelial to mesenchymal transition associated with endothelial cell fibrosis/apoptosis in vivo and in vitro. Furthermore, kidneys of low-dose streptozotocin-induced diabetic heterozygous Hhip deficient (Hhip+/−) mice displayed a normal albumin/creatinine ratio with fewer features of DN (glomerulosclerosis/fibrosis and podocyte apoptosis/loss) and less evidence of renal compensation (glomerular hypertrophy and hyperfiltration) as compared to diabetic wild type controls (Hhip+/+). Thus, our studies demonstrated that renal Hhip expression is associated with nephropathy development in diabetes and that hyperglycemia-induced renal Hhip expression may mediate glomerular endothelial fibrosis and apoptosis in diabetes, a novel finding.

Angiotensin II type 2 receptor (AT2 R) deficiency in AT2 R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT2 R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT2 RKO mice were used to assess glomerulogenesis, while wild-type and AT2 RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT2 R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT2 R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT2 R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase-3 and p53-related apoptotic processes resulting in podocyte loss, or activates TGFβ1-Smad2/3 cascades and α-SMA expression to transform differentiated podocytes to undifferentiated podocyte-derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT2 R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Oxidative stress induces endogenous antioxidants via nuclear factor erythroid 2–related factor 2 (Nrf2), potentially preventing tissue injury. We investigated whether insulin affects renal Nrf2 expression in type 1 diabetes (T1D) and studied its underlying mechanism. Insulin normalized hyperglycemia, hypertension, oxidative stress, and renal injury; inhibited renal Nrf2 and angiotensinogen (Agt) gene expression; and upregulated heterogeneous nuclear ribonucleoprotein F and K (hnRNP F and hnRNP K) expression in Akita mice with T1D. In immortalized rat renal proximal tubular cells, insulin suppressed Nrf2 and Agt but stimulated hnRNP F and hnRNP K gene transcription in high glucose via p44/42 mitogen-activated protein kinase signaling. Transfection with small interfering RNAs of p44/42 MAPK, hnRNP F, or hnRNP K blocked insulin inhibition of Nrf2 gene transcription. Insulin curbed Nrf2 promoter activity via a specific DNA-responsive element that binds hnRNP F/K, and hnRNP F/K overexpression curtailed Nrf2 promoter activity. In hyperinsulinemic-euglycemic mice, renal Nrf2 and Agt expression was downregulated, whereas hnRNP F/K expression was upregulated. Thus, the beneficial actions of insulin in diabetic nephropathy appear to be mediated, in part, by suppressing renal Nrf2 and Agt gene transcription and preventing Nrf2 stimulation of Agt expression via hnRNP F/K. These findings identify hnRNP F/K and Nrf2 as potential therapeutic targets in diabetes., Our present study identifies an inhibitory action of insulin on renal Nrf2 gene transcription via a putative IRE in the Nrf2 gene promoter that binds hnRNP F/K.

We reported previously that overexpression of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in renal proximal tubular cells (RPTCs) suppresses angiotensinogen (Agt) expression, and attenuates systemic hypertension and renal injury in diabetic Hnrnpf-transgenic (Tg) mice. We thus hypothesized that deletion of Hnrnpf in the renal proximal tubules (RPT) of mice would worsen systemic hypertension and kidney injury, perhaps revealing novel mechanism(s). Tubule-specific Hnrnpf knockout (KO) mice were generated by crossbreeding Pax8-Cre mice with floxed Hnrnpf mice on a C57BL/6 background. Both male and female KO mice exhibited elevated systolic blood pressure, increased urinary albumin/creatinine ratio, tubulo-interstitial fibrosis and glycosuria without changes in blood glucose or glomerular filtration rate compared with control littermates. However, glycosuria disappeared in male KO mice at the age of 12 weeks, while female KO mice had persistent glycosuria. Agt expression was elevated, whereas sodium-glucose co-transporter 2 (Sglt2) expression was down-regulated in RPTs of both male and female KO mice as compared to control littermates. In vitro, KO of HNRNPF in human RPTCs (HK-2) by CRISPR gRNA up-regulated AGT and down-regulated SGLT2 expression. The Sglt2 inhibitor canagliflozin treatment had no effect on Agt and Sglt2 expression in HK-2 and in RPTCs of wild-type mice but induced glycosuria. Our results demonstrate that Hnrnpf plays a role in the development of hypertension and glycosuria through modulation of renal Agt and Sglt2 expression in mice, respectively.

Hedgehog interacting protein (Hhip) is essential for islet formation and beta-cell proliferation during pancreatic development; abnormally elevated Hhip expression has been linked to human pancreatitis. Here, we investigate the role of Hhip in modulating insulin secretion in adult Hhip mice (Hhip +/− vs. Hhip+/+) fed high fat diets (HFD). Both sexes of HFD-Hhip +/+ mice developed impaired glucose intolerance, that was only ameliorated in male HFD-Hhip +/− mice that had high levels of circulating plasma insulin, but not in female HFD-Hhip +/− mice. HFD stimulated Hhip gene expression, mainly in beta cells. Male HFD-Hhip +/+ mice had more large islets in which insulin content was reduced; islet architecture was disordered; and markers of oxidative stress (8-OHdG and Nox 2) were increased. In contrast, male HFD-Hhip +/− mice had more small islets with increased beta cell proliferation, enhanced GSIS, less oxidative stress and preserved islet integrity. In vitro, recombinant Hhip increased Nox2 and NADPH activity and decreased insulin-positive beta cells. siRNA-Hhip increased GSIS and abolished the stimulation of sodium palmitate (PA)-BSA on Nox2 gene expression. We conclude that pancreatic Hhip gene inhibits insulin secretion by altering islet integrity and promoting Nox2 gene expression in beta cells in response to HDF-mediated beta cell dysfunction, a novel finding.

We investigated the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in renin-angiotensin system (RAS) gene expression in renal proximal tubule cells (RPTCs) and in the development of systemic hypertension and kidney injury in diabetic Akita mice. We used adult male Akita Nrf2 knockout mice and Akita mice treated with trigonelline (an Nrf2 inhibitor) or oltipraz (an Nrf2 activator). We also examined rat immortalized RPTCs (IRPTCs) stably transfected with control plasmids or plasmids containing rat angiotensinogen (Agt), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme-2 (Ace2), or angiotensin 1-7 (Ang 1-7) receptor (MasR) gene promoters. Genetic deletion of Nrf2 or pharmacological inhibition of Nrf2 in Akita mice attenuated hypertension, renal injury, tubulointerstitial fibrosis, and the urinary albumin/creatinine ratio. Furthermore, loss of Nrf2 upregulated RPTC Ace2 and MasR expression, increased urinary Ang 1-7 levels, and downregulated expression of Agt, ACE, and profibrotic genes in Akita mice. In cultured IRPTCs, Nrf2 small interfering RNA transfection or trigonelline treatment prevented high glucose stimulation of Nrf2 nuclear translocation, Agt, and ACE transcription with augmentation of Ace2 and MasR transcription, which was reversed by oltipraz. These data identify a mechanism, Nrf2-mediated stimulation of intrarenal RAS gene expression, by which chronic hyperglycemia induces hypertension and renal injury in diabetes.

Aims/hypothesis We investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-β1 signalling, TGF-β1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes. Methods Adult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied. Results Overexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-β1, TGF-β receptor II [Tgf-βrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1–7 receptor [MasR]) expression, and normalised urinary Ang 1–7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-β1 and Tgf-βrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-β1 signalling and TGF-β1 inhibition of Ace-2 gene expression. Conclusions/interpretation These data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-β1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes. Electronic supplementary material The online version of this article (doi:10.1007/s00125-015-3700-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

This study investigated the impact of catalase (Cat) overexpression in renal proximal tubule cells (RPTCs) on nuclear factor erythroid 2–related factor 2 (Nrf2) stimulation of angiotensinogen (Agt) gene expression and the development of hypertension and renal injury in diabetic Akita transgenic mice. Additionally, adult male mice were treated with the Nrf2 activator oltipraz with or without the inhibitor trigonelline. Rat RPTCs, stably transfected with plasmid containing either rat Agt or Nrf2 gene promoter, were also studied. Cat overexpression normalized systolic BP, attenuated renal injury, and inhibited RPTC Nrf2, Agt, and heme oxygenase-1 (HO-1) gene expression in Akita Cat transgenic mice compared with Akita mice. In vitro, high glucose level, hydrogen peroxide, and oltipraz stimulated Nrf2 and Agt gene expression; these changes were blocked by trigonelline, small interfering RNAs of Nrf2, antioxidants, or pharmacological inhibitors of nuclear factor-κB and p38 mitogen-activated protein kinase. The deletion of Nrf2-responsive elements in the rat Agt gene promoter abolished the stimulatory effect of oltipraz. Oltipraz administration also augmented Agt, HO-1, and Nrf2 gene expression in mouse RPTCs and was reversed by trigonelline. These data identify a novel mechanism, Nrf2-mediated stimulation of intrarenal Agt gene expression and activation of the renin-angiotensin system, by which hyperglycemia induces hypertension and renal injury in diabetic mice.

We hypothesised that maternal diabetes impairs kidney formation in offspring via augmented expression of hedgehog interacting protein (HHIP). Our gene-array results were performed in neonatal kidneys from our murine model of maternal diabetes and indicated that Hhip expression was significantly modulated by maternal diabetes.We systematically examined the functional role of HHIP in kidney formation in our murine maternal diabetes model and elucidated the potential mechanisms related to dysnephrogenesis in vitro.The kidneys of the offspring of diabetic dams, compared with those of the offspring of control non-diabetic dams, showed retardation of development--small kidneys and less ureteric bud (UB) branching morphogenesis. Augmented HHIP expression was observed in the offspring of diabetic dams, initially localised to differentiated metanephric mesenchyme and UB epithelium and subsequently in maturing glomerular endothelial and tubulointerstitial cells. The heightened HHIP targeting TGF-β1 signalling was associated with dysmorphogenesis. In vitro, HHIP overexpression decreased sonic hedgehog and paired box gene 2 proteins (SHH and PAX2, respectively) and increased transcriptional nuclear factor-kappa B (NFκB, p50/p65), phosphorylation of p53, and TGF-β1 expression. In contrast, overexpression of PAX2 inhibited HHIP and NFκB and activated SHH, N-myc and p27(Kip1) expression. Moreover, high glucose stimulated HHIP expression, and then targeted TGF-β1 signalling. Thus, PAX2, via a negative autocrine feedback mechanism, attenuated the stimulatory effect of high glucose on HHIP expression.Maternal diabetes modulates kidney formation in young progeny mediated, at least in part, via augmented HHIP expression.