Grande, Joseph P.

Background. Glomerular size in renal allografts is impacted by donor–recipient factors and response to injury. In serial biopsies of patients with well-functioning grafts, increased glomerular size correlates with better survival. However, no previous study has addressed the association of glomerular size at the time of a for-cause biopsy and clinical/histopathologic markers of injury, or effect on long-term graft outcome. Methods. Two cohorts of kidney transplant recipients enrolled in the Deterioration of Kidney Allograft Function study were evaluated. The prospective cohort (PC, n = 581): patients undergoing first for-cause kidney biopsy 1.7 ± 1.4 (mean ± SD) y posttransplant; and the cross-sectional cohort (CSC, n = 446): patients developing new-onset renal function deterioration 7.7 ± 5.6 y posttransplant. Glomerular planar surface area and diameter were measured on all glomeruli containing a vascular pole. Kidney biopsy was read centrally in a blinded fashion according to the Banff criteria. Results. Glomerular area was significantly higher in the CSC than the PC; time from transplant to indication biopsy was associated with glomerular area in both cohorts (P values ≤ 0.001). Glomerular area was associated with indices of microvascular inflammation (glomerulitis, peritubular capillary infiltrates; P values ≤ 0.001) and segmental glomerulosclerosis (P value < 0.0001). In the CSC, higher glomerular area was associated with higher estimated glomerular filtration rate (P value ≤ 0.001) and increased graft survival after accounting for microvascular inflammation (adjusted hazard ratio = 0.967; 95% confidence interval: 0.948-0.986; hazard ratio in biopsies without evidence of diabetes or antibody mediated rejection = 0.919, 95% confidence interval: 0.856-0.987). Conclusions. Glomerular size is associated with histopathologic features present at the time of indication biopsy and with increased graft survival in the CSC.

Aim The aim of this is to identify the gene expression profiles indicative of reversible versus irreversible injury in experimental (murine) renal artery stenosis (RAS). Method Two‐kidney‐one‐cuff hypertension was established by placement of a polytetrafluoroethylene cuff on the right renal artery (N=18). Sham surgery was performed by manipulating the right renal artery without cuff placement (N=6). To determine potential reversibility, the cuff was removed at 7 days (7D cuff‐off, N=6) and 14 days (14D cuff‐off, N=6) after initial RAS surgery. In a control RAS group, the cuff was left in place for 28 days (28D RAS, N=6). Kidneys were harvested at 28 days for assessment of atrophy and fibrosis. Renal blood perfusion and oxygenation were measured using 16.4T MRI. RNASeq analysis was performed on all the groups at 28 days following initial surgery. Results Following cuff placement, intra‐renal perfusion was reduced by 65% (p<0.0001 vs. baseline). In the 7D cuff‐off group intra‐renal perfusion was restored to baseline values after cuff removal, whereas in the 14D cuff‐off group perfusion was not significantly improved following cuff removal. In the 28D RAS group the reduction in perfusion was maintained throughout the study period. The 7D cuff‐off group had a significantly lower % atrophy (7 ± 2.2%, p=0.0042) and % area of interstitial fibrosis (0.57 ± 0.08 %, p<0.0001) compared to 28D RAS group (atrophy 66 ± 13%, area of fibrosis 7.79 ± 0.89 %). The 14D cuff‐off group had severe atrophy (56 ± 16.6%) and interstitial fibrosis (5.05 ± 1.22%) similar to that seen in 28D RAS group. RNASeq analysis data were normalized to appropriate sham controls in all the groups and was performed at 28 days following initial surgery. Induction of greater than Log2 ratio >2 and p values < 0.00001 were considered significant (following adjustment for multiple comparisons). Fibrosis associated genes were differentially regulated in the 7D cuff‐off compared to the 14D cuff‐off and 28D RAS group (table ). Conclusion We conclude that cuff removal at 7 days is associated with relative preservation of renal structure, indicating that renal injury at this point is reversible. We have identified a number of fibrosis associated genes that are differentially regulated in the 7D cuff‐off group versus 14D cuff‐off group. Further analysis will determine the functional relevance of these differentially regulated genes. Support or Funding Information This project was funded by National Institutes of Health, NIAID R01 AI100911. Values of Log 2 Ratio Genes 7D Cuff‐off vs Sham 14D Cuff‐off vs Sham 28D RAS vs Sham COL1A1 0.96 2.62 2.23 COL1A2 0.79 2.07 1.91 COL3A1 1.26 2.60 2.35 MMP12 1.24 2.85 2.95 MMP14 1.34 2.70 2.55 MMP19 2.89 3.86 4.05 MMP2 1.55 3.49 3.22 MMP7 6.96 9.22 9.59 MTOR −0.46 −1.67 −2.06 SERPINE1 1.25 3.37 2.35 TGFB1i1 0.44 1.46 1.53 TGFB2 0.82 1.83 1.97 TGFB3 0.59 1.91 1.99 TGFBi 0.98 1.88 2.02 KLF15 −0.90 −1.76 −2.60 UBD 4.49 6.23 6.18 TIMP1 2.94 5.30 5.16 TIMP2 0.50 1.46 1.52 TIMP3 −0.35 −1.26 −1.36 HSPB1 0.16 2.00 2.13 HSPB8 1.51 2.26 2.30 CCL2 3.44 4.89 4.31 CCL12 2.88 3.71 4.12 SERPINE1 1.25 3.37 2.35 SERPINE2 1.17 2.39 2.35 SERPINF1 2.02 2.76 3.11 SERPINF2 −0.51 −2.03 −2.71 SERPING1 1.15 1.81 2.09 CLU 1.45 2.54 3.07 STC1 0.45 1.99 2.12 SPP1 1.75 3.06 3.37

In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2 −/− mice compared with HO-2 +/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin −/− mice compared with hemopexin +/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs. NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.

Heme oxygenase (HO) activity is exhibited by inducible (HO-1) and constitutive (HO-2) proteins. HO-1 protects against ischemic and nephrotoxic acute kidney injury (AKI). We have previously demonstrated that HO-2 protects against heme protein-induced AKI. The present study examined whether HO-2 is protective in ischemic AKI. Renal ischemia was imposed on young and aged HO-2+/+ and HO-2−/− mice. On days 1 and 2 after renal ischemia, there were no significant differences in renal function between young male HO-2+/+ and HO-2−/− mice, between young female HO-2+/+ and HO-2−/− mice, or between aged female HO-2+/+ and HO-2−/− mice. However, in aged male mice, HO-2 deficiency worsened renal function on days 1 and 2 after ischemic AKI, and, on day 2 after ischemia, such deficiency augmented upregulation of injury-related genes and worsened histological injury. Renal HO activity was markedly decreased in unstressed aged male HO-2−/− mice and remained so after ischemia, despite exaggerated HO-1 induction in HO-2−/− mice after ischemia. Such exacerbation of deficiency of HO-2 protein and HO activity may reflect phosphorylated STAT3, as activation of this proinflammatory transcription factor was accentuated early after ischemia in aged male HO-2−/− mice. This exacerbation may not reflect impaired induction of nephroprotectant genes, since the induction of HO-1, sirtuin 1, and β-catenin was accentuated in aged male HO-2−/− mice after ischemia. We conclude that aged male mice are hypersensitive to ischemic AKI and that HO-2 mitigates such sensitivity. We speculate that this protective effect of HO-2 may be mediated, at least in part, by suppression of phosphorylated STAT3-dependent signaling.

Rationale: Acute kidney injury (AKI) has a high prevalence and mortality in critically ill patients. It is also a powerful risk factor for heart failure incidence driven by hemodynamic changes and neurohormonal activation. However, no drugs have been approved by the Food and Drug Administration. Endogenous pGC-A (particulate guanylyl cyclase A receptor) activators were reported to preserve renal function and improve mortality in AKI patients, although hypotension accompanied by pGC-A activators have limited their therapeutic potential. Objective: We investigated the therapeutic potential of a nonhypotensive pGC-A activator/designer natriuretic peptide, CRRL269, in a short-term, large animal model of ischemia-induced AKI and also investigated the potential of uCNP (urinary C-type natriuretic peptide) as a biomarker for AKI. Methods and Results: We first showed that CRRL269 stimulated cGMP generation, suppressed plasma angiotensin II, and reduced cardiac filling pressures without lowering blood pressure in the AKI canine model. We also demonstrated that CRRL269 preserved glomerular filtration rate, increased renal blood flow, and promoted diuresis and natriuresis. Further, CRRL269 reduced kidney injury and apoptosis as evidenced by ex vivo histology and tissue apoptosis analysis. We also showed, compared with native pGC-A activators, that CRRL269 is a more potent inhibitor of apoptosis in renal cells and induced less decreases in intracellular Ca 2+ concentration in vascular smooth muscle cells. The renal antiapoptotic effects were at least mediated by cGMP/PKG pathway. Further, CRRL269 inhibited proapoptotic genes expression using a polymerase chain reaction gene array. Additionally, we demonstrated that AKI increased uCNP levels. Conclusions: Our study supports developing CRRL269 as a novel renocardiac protective agent for AKI treatment.

Abstract Adipose tissue inflammation and dysfunction are associated with obesity‐related insulin resistance and diabetes, but mechanisms underlying this relationship are unclear. Although senescent cells accumulate in adipose tissue of obese humans and rodents, a direct pathogenic role for these cells in the development of diabetes remains to be demonstrated. Here, we show that reducing senescent cell burden in obese mice, either by activating drug‐inducible “suicide” genes driven by the p16 Ink4a promoter or by treatment with senolytic agents, alleviates metabolic and adipose tissue dysfunction. These senolytic interventions improved glucose tolerance, enhanced insulin sensitivity, lowered circulating inflammatory mediators, and promoted adipogenesis in obese mice. Elimination of senescent cells also prevented the migration of transplanted monocytes into intra‐abdominal adipose tissue and reduced the number of macrophages in this tissue. In addition, microalbuminuria, renal podocyte function, and cardiac diastolic function improved with senolytic therapy. Our results implicate cellular senescence as a causal factor in obesity‐related inflammation and metabolic derangements and show that emerging senolytic agents hold promise for treating obesity‐related metabolic dysfunction and its complications.

ABSTRACT Dimethylarginine dimethylaminohydrolases (DDAHs) are known to degrade asymmetric dimethylarginine, an endogenous inhibitor of NOS, and maintain vascular homeostasis; however, the regulatory pathways of DDAHs remain unclear. In this study, we aimed to define the role of transmembrane glycoprotein neuropilin‐1 (NRP1) in the expression of DDAHs and investigate the potential roles of NRP1 in regulation of blood pressure. Short hairpin RNA–mediated knockdown of NRP1 reduced the level and mRNA stability of DDAH1 but not DDAH2 in HUVECs, whereas overexpression of NRP1 increased the mRNA stability of DDAH1. Meanwhile, mesenteric arteries and lung vascular endothelial cells of tamoxifen‐inducible endothelial cell–specific NRP1 knockout mice exhibited decreased expression of DDAH1 and slightly increased expression of DDAH2. Mechanistically, the regulation of NRP1 on DDAH1 expression is mediated by a posttranscriptional mechanism involving miR‐219‐5p in HUVECs. Although the endothelial cell–specific NRP1 knockout mice did not exhibit any significant change in blood pressure at the basal level, they were more sensitive to low‐dose angiotensin II infusion–induced increases in blood pressure. Our results show that NRP1 is required for full expression of DDAH1 in endothelial cells and that NRP1 contributes to protection from low‐dose angiotensin II–induced increases in blood pressure.—Wang, Y., Wang, E., Zhang, Y., Madamsetty, V. S., Ji, B., Radisky, D. C., Grande, J. P., Misra, S., Mukhopadhyay, D. Neuropilin‐1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II–induced hypertension. FASEB J. 33, 494–500 (2019). www.fasebj.org

There is no therapy that promotes maturation and functionality of a dialysis arteriovenous fistula (AVF). The search for such therapies largely relies on evaluation of vascular responses and putative therapies in experimental AVFs. We studied an AVF in mice with chronic kidney disease (CKD). We demonstrate numerous stressors in the vein of the AVF-CKD group, including pathological shear, mitogenic, inflammatory, and hypoxia-reoxygenation stress. Because stress promotes premature senescence, we examined whether senescence is induced in the vein of the AVF-CKD model. We demonstrate a senescence phenotype in the AVF-CKD model, as indicated by increased expression of p16Ink4a, p21Cip1, and p53 and expected changes for certain senescence-associated microRNAs. RNA-sequencing analysis demonstrated differential expression of ~10,000 genes, including upregulation of proinflammatory and proliferative genes, in the vein of the AVF-CKD group. The vein in the AVF-CKD group exhibited telomere erosion and increased senescence-associated β-galactosidase activity and staining. Senescence was induced in the artery of the AVF-CKD group and in the vein of the AVF without CKD. Finally, given the rapidly rising clinical interest in senolytics, we provide proof of concept of senolytics as a therapeutic approach by demonstrating that senolytics decrease p16Ink4a expression in the AVF-CKD model. This study introduces a novel concept underlying the basis for maturational and functional failure in human dialysis AVFs and identifies a new target for senolytic therapy.