Joan C. Krepinsky

BACKGROUND: Essential hypertension, characterized by vascular dysfunction, remains a leading modifiable cause of death globally. Perivascular adipose tissue (PVAT), which normally reduces vasoconstriction, becomes contractile in hypertension due to increased reactive oxygen species (ROS). Brown PVAT has emerged as a protective regulator of vascular tone. Follistatin, an activin antagonist, induces adipose tissue browning via AMPK (AMP-activated protein kinase). We recently reported improved blood pressure and vascular function by follistatin in the spontaneously hypertensive rat (SHR) model of essential hypertension. Here, we investigate whether follistatin reduces ROS and induces browning in SHR PVAT to restore PVAT-mediated vascular function. METHODS: SHRs were treated with vehicle, follistatin, or hydralazine for 8 weeks. Mesenteric white and thoracic brown PVAT from SHR and normotensive Wistar-Kyoto mice were used, with wire myography used to assess vascular function. PVAT nitric oxide, ROS, and browning markers were assessed via immunohistochemistry. PVAT was treated ex vivo to assess mechanisms of browning. Unbiased proteomic analysis of PVAT was performed using liquid chromatography-mass spectrometry. RESULTS: SHR PVAT dysfunction, manifesting as a procontractile effect, was inhibited by follistatin through reducing ROS, enhancing NO bioavailability, and promoting browning. These effects were mediated by activin neutralization and AMPK phosphorylation. Hydralazine-induced blood pressure reduction did not replicate follistatin effects, suggesting vascular benefits of follistatin are mediated by direct PVAT modulation. Proteomic analysis revealed a shift in proteome towards a normal state, upregulating processes associated with adipose browning. CONCLUSIONS: Follistatin restores PVAT-mediated vascular relaxation via ROS reduction, activin neutralization, and AMPK-dependent browning, positioning the potential for PVAT as a therapeutic target for vascular dysfunction in essential hypertension.

Aims/hypothesis Diabetic kidney disease (DKD), a prevalent complication of diabetes, is the leading cause of chronic kidney disease globally. The current standard of care cannot halt disease progression and thus new therapeutic targets are needed. We previously showed that the metalloprotease ADAM17 mediates the profibrotic response to high glucose in kidney mesangial cells. Its upregulation in high glucose conditions augments its profibrotic effects. Here we investigate regulation of the Adam17 promoter region −2304/−1567, previously shown to be glucose responsive, for which regulatory factors have not yet been identified. Methods Adam17 promoter regulation, cell surface translocation and activation were assessed in primary rat mesangial cells using standard molecular biology techniques. Type 1 diabetes was induced in mice using streptozocin and kidney function and development of fibrosis were assessed after 24 weeks. Human and mouse kidneys were immunostained for LASP1. Results In rat mesangial cells, the LIM and SH3 protein 1 (LASP1) was identified as a regulator of the Adam17 promoter in response to high glucose by mass spectrometry of nuclear lysate proteins binding to the −2304/−1567 promoter region. Knockdown of LASP1 prevented glucose-induced Adam17 promoter activation and transcript and protein upregulation. LASP1 nuclear localisation and regulation of Adam17 promoter activity in high glucose required phosphorylation of LASP1 on S146 by protein kinase A, but not protein kinase G, and Y171 phosphorylation by Src kinase. LASP1 also regulated glucose-induced ADAM17 cell surface localisation and activation, dependent on its phosphorylation by Src and interaction with focal adhesion kinase. Profibrotic responses to glucose were inhibited by LASP1 downregulation. In vivo, LASP1 expression was increased in the kidneys of type 1 diabetic mice and in kidneys of patients with DKD. Mice with Lasp1 knockout showed attenuated development of DKD. Conclusions/interpretation LASP1 regulates the synthesis and activation of ADAM17 in mesangial cells and is required for the profibrotic response to high glucose. Its deletion protects against DKD in mice. Targeting LASP1 may have therapeutic value as an indirect method of ADAM17 inhibition to inhibit fibrosis in DKD. Graphical Abstract

Background: Essential hypertension, characterized by vascular dysfunction, remains a leading modifiable cause of death globally. Perivascular adipose tissue (PVAT), which normally reduces vasoconstriction, becomes less anticontractile in hypertension due at least in part to increased reactive oxygen species (ROS). Brown PVAT has emerged as a protective regulator of vascular tone. Follistatin, an activin antagonist, induces browning of peripheral adipose tissue depots via AMPK. We recently reported improved blood pressure, vascular function and reduced vascular ROS by follistatin in the spontaneously hypertensive rat (SHR) model of essential hypertension. Here, we investigate whether follistatin reduces ROS and induces browning in SHR PVAT to restore PVAT function. Methods: SHR were treated with vehicle, follistatin or hydralazine for 8 weeks. Mesenteric white and thoracic brown PVAT from SHR and normotensive Wistar-Kyoto were utilized, with wire myography used to assess vascular function. Nitric oxide (NO) and nitrite were measured in PVAT using DAF-2 FM fluorescence or Griess reagent, respectively. PVAT ROS and browning markers were assessed biochemically and via immunohistochemistry. PVAT was treated ex vivo for 3 days to assess mechanisms of browning. Unbiased proteomic analysis of PVAT was performed using liquid chromatography-mass spectrometry. Results: SHR PVAT dysfunction, manifest as a pro-contractile effect, was inhibited by follistatin through reducing ROS, enhancing NO bioavailability and inducing browning. Activin neutralization and AMPK phosphorylation mediated the beneficial effect of follistatin on SHR PVAT. Hydralazine-induced blood pressure reduction did not replicate follistatin effects, suggesting vascular benefits of follistatin are mediated by direct PVAT modulation. Proteomic analysis showed that follistatin shifts PVAT proteome towards normal state, upregulating processes associated with adipose tissue browning. Conclusion: Follistatin restores PVAT-mediated vascular relaxation via ROS reduction, activin neutralization and AMPK-dependent browning, positioning the potential for PVAT as a therapeutic target for vascular dysfunction in essential hypertension.

We recently showed that cell surface translocation of the endoplasmic reticulum–resident protein GRP78, when bound by activated α 2-macroglobulin (α2M*), induces pro-fibrotic responses in glomerular mesangial cells in response to high glucose and regulates activation of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1), implicating a pathogenic role in glomerulosclerosis. Interstitial fibrosis, largely mediated by proximal tubular epithelial cells (PTEC) and renal fibroblasts, develops later in kidney disease and correlates with functional decline. Here we investigated whether interstitial fibrosis was mediated by cell surface GRP78 (csGRP78)/α2M*. High glucose and TGF-β1 increased csGRP78 and α2M* in PTEC and renal fibroblasts, and their inhibition prevented fibrotic protein production. Interestingly, for TGF-β1, this depended on inhibition of noncanonical signaling through YAP/TAZ, with Smad3 activation unaffected. In vivo, type 1 diabetic Akita mice overexpressing TGF-β1 were treated with either a neutralizing antibody for csGRP78 (C38) or α2M* (Fα2M) or an inhibitory peptide blocking csGRP78/α2M* interaction, and mice with unilateral ureteral obstruction were treated with Fα2M or inhibitory peptide. Consistently, inhibition by antibody or peptide attenuated fibrosis and pro-fibrotic signaling. These findings show an important role for csGRP78/α2M* in mediating tubulointerstitial fibrosis in both diabetic and nondiabetic kidney disease and support their inhibition as a potential antifibrotic therapeutic intervention.

Glomerular extracellular matrix protein accumulation, mediated largely by mesangial cells(MC), is a defining feature of diabetic kidney disease(DKD). Previously we showed that TGFβ1, a profibrotic cytokine in kidney fibrosis, inhibits expression of the antifibrotic follistatin through induction of microRNA-299a-5p. Whether this microRNA contributes to DKD is unknown. We show that microRNA-299a-5p is increased in mouse and human diabetic kidneys, and by high glucose in primary MC. Overexpression of microRNA-299a-5p in MC increased basal ECM protein production. Conversely, microRNA-299a-5p inhibition prevented the glucose-induced profibrotic response. Bioinformatics screening revealed that cripto-1 is also a target of microRNA-299a-5p. Induction of microRNA-299a-5p by high glucose mediated the MC fibrotic response by inhibiting follistatin and cripto-1 which led to increased activin A and TGFβ1 signaling. In vivo, microRNA-299a-5p inhibition reduced clinical markers of DKD, and was associated with increased expression of follistatin and cripto-1. Thus, microRNA-299a-5p is an important mediator of glucose-induced profibrotic responses in diabetic kidneys.

Introduction: The cytokine activin A is emerging as an important regulator of kidney fibrosis. Its expression, negligible in normal kidney, is significantly increased in various fibrotic kidney diseases. TGFβ1 is a cytokine belonging to the same family, which is well established to be a central mediator of kidney fibrosis. Although targeting TGFβ1 therapeutically is not feasible due to its homeostatic roles, we previously showed that activin A is upregulated by, and mediates the profibrotic effects of, TGFβ1.Methods: We investigated the transcriptional regulation of activin A by TGFβ1 in primary kidney mesangial cells (MC). Cells were transfected with a luciferase reporter construct containing the activin A promoter or a series of deletion constructs. Guided by MatInspector, key TGFβ1-responsive consensus elements were identified.Results: TGFβ1 increased transcription of the activin A subunit inhba. Using a series of deletion constructs of the inhba promoter, we identified a critical regulatory region located 350bp from the transcription start site that is responsive to TGFβ1. Analysis of this region for transcription factor regulatory elements, coupled with mutation analyses and transcription factor downregulation with siRNA, showed that Stat5 and FoxP1, but not Sox9, regulate inhba transcription by TGFβ1. Interestingly, although no consensus binding site in this region was identified for Smad3, a well-established mediator of TGFβ1 signaling, both a Smad3 inhibitor and use of MC isolated from Smad3 knockout kidneys, showed its requirement for the TGFβ1 response. We further identified a CT microsatellite just upstream of 350bp which suppressed promoter activity.Conclusion: These findings provide insight into potential therapeutic targets for activin A targeting and attenuation of kidney fibrosis.

Glomerular extracellular matrix protein accumulation, mediated largely by mesangial cells, is a defining feature of early diabetic kidney disease. Previously we showed that TGFβ1, a profibrotic cytokine with a well-defined pathogenic role in kidney fibrosis, inhibits expression of the antifibrotic follistatin through induction of microRNA-299a-5p. Whether this microRNA contributes to diabetic kidney disease is unknown. We show that microRNA-299a-5p is increased in mouse and human diabetic kidneys, and by high glucose in primary mesangial cells. Overexpression of microRNA-299a-5p in mesangial cells increased basal extracellular matrix protein production. Conversely, microRNA-299a-5p inhibition prevented the glucose-induced profibrotic response. Bioinformatics screening revealed that cripto-1 is also a target of microRNA-299a-5p. It is known that follistatin and cripto-1 inhibit activin A and TGFβ1 respectively. Induction of microRNA-299a-5p by high glucose mediated the mesangial cell fibrotic response by inhibiting expression of both follistatin and cripto-1 which led to increased activin A and TGFβ1 signaling. In vivo , microRNA-299a-5p inhibition reduced albuminuria, glomerular hypertrophy, loss of podocyte nephrin and extracellular matrix production, and this was associated with increased expression of follistatin and cripto-1. Thus, microRNA-299a-5p is an important mediator of glucose-induced profibrotic responses in mesangial cells and diabetic kidneys. Its inhibition may be a potential novel therapy.

The endoplasmic reticulum (ER) has crucial roles in metabolically active cells, including protein translation, protein folding and quality control, lipid biosynthesis, and calcium homeostasis. Adverse metabolic conditions or pathogenic genetic variants that cause misfolding and accumulation of proteins within the ER of kidney cells initiate an injurious process known as ER stress that contributes to kidney disease and its cardiovascular complications. Initiation of ER stress activates the unfolded protein response (UPR), a cellular defence mechanism that functions to restore ER homeostasis. However, severe or chronic ER stress rewires the UPR to activate deleterious pathways that exacerbate inflammation, apoptosis and fibrosis, resulting in kidney injury. This insidious crosstalk between ER stress, UPR activation, oxidative stress and inflammation forms a vicious cycle that drives kidney disease and vascular damage. Furthermore, genetic variants that disrupt protein-folding mechanisms trigger ER stress, as evidenced in autosomal-dominant tubulointerstitial kidney disease and Fabry disease. Emerging therapeutic strategies that enhance protein-folding capacity and reduce the burden of ER stress have shown promising results in kidney diseases. Thus, integrating knowledge of how genetic variants cause protein misfolding and ER stress into clinical practice will enhance treatment strategies and potentially improve outcomes for various kidney diseases and their vascular complications.

Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin (A2M) is increased in DKD and is a critical regulator of the fibrotic response in glomerular mesangial cells (MC), an initial site of injury during DKD development. How A2M is regulated by high glucose (HG) has not yet been elucidated and is the focus of this investigation. Using serial deletions of the full A2M promoter, we identified the −405 bp region as HG-responsive in MC. Site-directed mutagenesis, siRNA, and ChIP studies showed that the transcription factor, nuclear factor of activated T cells 5 (NFAT5), regulated A2M promoter activity and protein expression in response to HG. Forkhead box P1 (FOXP1) served as a cooperative binding partner for NFAT5, required for A2M upregulation. Lastly, we showed that Smad3, known for its role in kidney fibrosis, regulated A2M promoter activity and protein production independently of HG. The importance of NFAT5, FOXP1, and Smad3 in A2M regulation was confirmed in ex vivo studies using isolated glomeruli. In conclusion, Smad3 is required for basal and HG-induced A2M expression, while NFAT5 and FOXP1 cooperatively regulate increased A2M transcription in response to HG. Inhibition of NFAT5/FOXP1 will be further evaluated as a potential therapeutic strategy to inhibit A2M production and attenuate profibrotic signaling in DKD.