Key PointsUnderstanding how heme proteins/heme induce AKI and whether NAD+-boosting ameliorates heme protein-mediated AKI is clinically relevant.Heme proteins/heme directly decrease kidney NAD+ content and NAD+-boosting is markedly protective in heme protein-mediated AKI.These findings provide new insights in heme protein-mediated AKI and support interest in NAD+-boosting as a therapeutic strategy in AKI.BackgroundUnderstanding how heme proteins and heme induce AKI is clinically relevant from numerous perspectives, and, in this regard, a widely used model of heme protein-mediated AKI (HP-AKI) involves the glycerol model in mice. Using this model, we have previously demonstrated that NAD+ content is decreased in HP-AKI. Because there is significant current interest in NAD+-boosting as a strategy in clinical AKI, we examined the effect of NAD+-boosting in this model.MethodsNAD+-boosting was achieved by the administration of nicotinamide mononucleotide (NMN) in the glycerol model of HP-AKI. The effect of NMN, compared with vehicle, was examined in mice with HP-AKI and with sham AKI at day 1 after HP-AKI was induced.ResultsThe administration of NMN preserved kidney NAD+ content in HP-AKI, and compared with the administration of vehicle in HP-AKI, NMN improved glomerular filtration markers; reduced histologic injury as assessed by tubular necrosis, dilation, and cast formation and by assessment of dystrophic calcification; reduced expression of kidney injury marker 1, a sensitive marker of AKI; preserved mitochondrial ultrastructure and increased expression of proteins that promote mitochondrial integrity; reduced apoptosis as assessed by terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling staining and genes and protein expression that contribute to apoptosis, while increasing expression of an anti-apoptotic gene; reduced expression of several renal injury-related genes; and mitigated the severity of the senescence phenotype, as assessed by multiple markers. Finally, the administration of myoglobin or heme in vivo diminished kidney NAD+ content in mice with intact kidneys.ConclusionsThese findings demonstrate the remarkable protective effects of NAD+-boosting by NMN in HP-AKI as revealed by a multitude of markers relevant to AKI. The reduction in NAD+ content in the kidney in HP-AKI may reflect the direct effects of heme proteins and/or heme. We suggest that these findings support current interest in NAD+-boosting as a therapeutic strategy in clinical AKI.

Angiotensin-converting enzyme 2 (ACE2) exerts countervailing effects on the renin-angiotensin-aldosterone system (RAAS). ACE2 also engages the spike protein of SARS-CoV-2. ACE2 protein has been shown recently to avidly bind heme. We examined the pathobiological relevance of this heme-binding property of ACE2 by using the glycerol-induced model of heme protein-mediated AKI (HP-AKI), which is characterized by increased kidney heme content. We studied the response of ACE2-wildtype (ACE2+/y) and ACE2-deficient (ACE2-/y) mice to HP-AKI and quantitated kidney and cellular content of heme under relevant conditions. ACE2-deficient mice, compared with ACE2-wildtype mice, were significantly protected against HP-AKI as reflected by filtration markers, less histological injury, and less expression of apoptosis and ferroptosis markers. ACE2-deficient mice also evinced lesser kidney heme content and a blunted induction of HO-1. HEK293 ACE2-overexpressing cells, compared with HEK293-native cells, when exposed to heme, retained higher amounts of heme. In HP-AKI, ACE2 expression and activity were reduced, and myoglobin and heme, administered independently, reduced ACE2 expression in the otherwise intact mouse kidney. Finally, with more severe HP-AKI, the protective effect of ACE2 deficiency was attenuated. We conclude that ACE2 deficiency confers protection against HP-AKI. We suggest that this reflects the recently recognized binding of heme to ACE2, such binding serving to facilitate renal entry of heme, a known nephrotoxin. These findings uncover a novel pathway of heme-dependent acute kidney injury. This is the first demonstration of the biological relevance of chemical binding of heme by ACE2. Finally, we identify heme proteins and heme as novel determinants of ACE2 expression.NEW & NOTEWORTHY ACE2 protein binds heme, which we reasoned would promote heme entry into the kidney and, accordingly, heme protein-mediated acute kidney injury. Our findings support this hypothesis. This study is the first to demonstrate the biological relevance of ACE2-heme binding, uncover a new pathway of heme-dependent kidney injury, and identify myoglobin and heme as novel determinants of ACE2 expression. Our study explains why plasma levels of myoglobin and heme predict poor outcomes in patients with COVID-19.

Key PointsThe rat arteriovenous fistula (AVF) model exhibits marked upregulation of p16Ink4a and p21Cip1 and multiple markers of senescence.Fisetin, an established vasoprotective senolytic agent, when administered for 3 weeks, increases AVF blood flow and AVF outward remodeling.Heme is shown to be a novel prosenescence metabolite, and when chronically administered, it decreases AVF blood flow.BackgroundMaturational failure of dialysis arteriovenous fistulas (AVFs) not uncommonly occurs and is of considerable and timely importance. Our prior studies demonstrate that senescence, a phenotypic process that promotes vascular and other diseases, occurs in the murine AVF. In this study, we examined whether senescence also occurs in the rat AVF model and the effect of compounds that inhibit or accelerate senescence.MethodsThe rat AVF was created in the femoral vessels by an end vein-side artery anastomosis. In the AVF, we assessed the expression of critical drivers of senescence, specifically, the cell cycle inhibitors p16Ink4a and p21Cip1, and such indices of a senescence phenotype as senescence-associated beta-galactosidase (SA-beta-gal) activity, SA-beta-gal staining, and a senescence-associated secretory phenotype. We examined the effects of compounds that retard or accelerate senescence on AVF blood flow.ResultsThe AVF evinced upregulation of p16Ink4a and p21Cip1 when assessed 3 days after AVF creation. The AVF also demonstrated increased SA-beta-gal activity in the artery and vein; staining for SA-beta-gal in the AVF artery, anastomosis, and vein; and a prominent senescence-associated secretory phenotype. Fisetin, an established senolytic that is protective in other models of vascular injury, when administered for 3 weeks, increased AVF blood flow and outward remodeling. Hemin, when administered for 3 weeks, decreased AVF blood flow. We demonstrate that hemin is a novel inducer of a senescence phenotype in endothelial cells, as reflected by several senescence indices. However, when administered relatively acutely (for 5 days), hemin increased AVF blood flow by heme oxygenase-dependent mechanisms because the latter was entirely prevented by a competitive inhibitor of heme oxygenase activity.ConclusionsThe rat AVF exhibits senescence within 3 days of its creation. Chronic administration of a senolytic compound (fisetin) increases AVF blood flow, whereas chronic administration of a prosenescence compound (hemin) decreases AVF blood flow.

IntroductionThe immunomodulatory properties of mesenchymal stromal cells (MSC) have been well-characterized in in-vitro and in-vivo models. We have previously shown that liver MSC (L-MSC) are superior inhibitors of T-cell activation/proliferation, NK cell cytolytic function, and macrophage activation compared to adipose (A-MSC) and bone marrow MSC (BM-MSC) in-vitro.MethodTo test these observations in-vivo, we infused these types of MSC into mice with unilateral renal artery stenosis (RAS), an established model of kidney inflammation. Unilateral RAS was induced via laparotomy in 11-week-old, male 129-S1 mice under general anesthesia. Control mice had sham operations. Human L-MSC, AMSC, and BM-MSC (5x105 cells each) or PBS vehicle were injected intra-arterially 2 weeks after surgery. Kidney morphology was studied 2 weeks after infusion using micro-MRI imaging. Renal inflammation, apoptosis, fibrosis, and MSC retention were studied ex-vivo utilizing western blot, immunofluorescence, and immunohistological analyses.ResultsThe stenotic kidney volume was smaller in all RAS mice, confirming significant injury, and was improved by infusion of all MSC types. All MSC-infused groups had lower levels of plasma renin and proteinuria compared to untreated RAS. Serum creatinine improved in micetreated with BM- and L-MSC. All types of MSC located to and were retained within the stenotic kidneys, but L-MSC retention was significantly higher than A- and BM-MSC. While all groups of MSC-treated mice displayed reduced overall inflammation and macrophage counts, L-MSC showed superior potency in-vivo at localizing to the site of inflammation and inducing M2 (reparative) macrophage polarization to reduce inflammatory changes.DiscussionThese in-vivo findings extend our in-vitro studies and suggest that L-MSC possess unique anti-inflammatory properties that may play a role in liver-induced tolerance and lend further support to their use as therapeutic agents for diseases with underlying inflammatory pathophysiology.

Background:Mesangial expansion (ME) is an understudied histologic lesion in renal allografts. The current Banff mm score is not reproducible and may miss important ME features. The study aimed to improve the quantification of ME using morphometry, assess changes over time, and determine its association with allograft loss.Methods:We studied ME in 1-y and 5-y surveillance biopsies in 835 kidney transplants performed between January 2000 and December 2013. ME was assessed using the Banff mm score by a central pathologist and by morphometry. We derived 3 different morphometric measures: (1) %ME mm (%glomeruli with ME in ≥2 lobules, like Banff mm); (2) %MEany (%glomeruli with any ME lesion); and (3) %ME area (sum of all ME areas/all glomerular tuft areas). Unadjusted and adjusted Cox models assessed the risk of death-censored allograft loss.Results:From 1- to 5-y biopsies, the mean Banff mm score increased from 0.18 to 0.34, whereas %ME mm increased from 2.5% to 13.3%. Banff mm score had modest correlations with morphometric ME measures. Moderate-severe %ME mm was present in 20.1% of 5-y biopsies, whereas only 6.6% of Banff mm scores were. In general, higher ME on both 1- and 5-y biopsies was associated with a deceased donor, older recipient age, recipient diabetes/obesity (present in >50% of severely affected biopsies), higher hemoglobin A1c at 5 y posttransplant, and recurrent kidney disease. Higher ME on 5-y biopsies was associated with delayed graft function. A higher Banff mm score at 1-y biopsy and morphometry ME measures at 5-y biopsy were associated with rejection during the first year posttransplant. Morphometric ME measures were associated with allograft loss independent of Banff scores and all clinical characteristics, including kidney function and recurrent disease. The model with %MEany had the highest c-statistic (0.872).Conclusions:Banff mm score underestimates the pervasiveness of ME in 5-y biopsies. ME is common and associated with alloimmune and nonalloimmune causes of graft loss.

Background and Objective: Biopsy stands as the gold standard for kidney transplant assessment, yet its invasive nature restricts frequent use. Shear wave elastography (SWE) is emerging as a promising alternative for kidney transplant monitoring. A parametric study involving 12 biopsy data sets categorized by standard biopsy scores (3 with normal histology, 3 with interstitial inflammation (i), 3 with interstitial fibrosis (ci), and 3 with tubular atrophy (ct)), was conducted to evaluate the interdependence between microstructural variations triggered by chronic allograft rejection and corresponding alterations in SWE measurements. Methods: Heterogeneous shear wave motion simulations from segmented kidney cortex sections were performed employing the staggered-grid finite difference (SGFD) method. The SGFD method allows the mechanical properties to be defined on a pixel -basis for shear wave motion simulation. Segmentation techniques enabled the isolation of four histological constituents: glomeruli, tubules, interstitium, and fluid. Baseline ex vivo Kelvin-Voigt mechanical properties for each constituent were drawn from established literature. The parametric evaluation was then performed by altering the baseline values individually. Shear wave velocity dispersion curves were measured with the generalized Stockwell transform in conjunction with slant frequency-wavenumber analysis (GST-SFK) algorithm. By fitting the curve within the 100-400 Hz range to the Kelvin-Voigt model, the rheological parameters, shear elasticity (mu 1) and viscosity (mu 2), were estimated. A time-to-peak algorithm was used to estimate the group velocity. The resultant in silico models emulated the heterogeneity of kidney cortex within the shear wave speed (SWS) reconstructions. Results: The presence of inflammation showed considerable spatial composition disparities compared to normal cases, featuring a 23 % increase in interstitial area and a 19 % increase in glomerular area. Concomitantly, there was a reduction of 12 % and 47 % in tubular and fluid areas, respectively. Consequently, mechanical changes induced by inflammation predominate in terms of rheological differentiation, evidenced by increased elasticity and viscosity. Mild tubular atrophy showed significant elevation in group velocity and mu 1. Conversely, mild and moderate fibrosis exhibited negligible alterations across all parameters, compatible with relatively limited morphological impact. Conclusions: This proposed model holds promise in enabling patient-specific simulations of the kidney cortex, thus facilitating exploration into how pathologies altering cortical morphology correlates to modifications in SWE-derived rheological measurements. We demonstrated that inflammation caused substantial changes in measured mechanical properties.

Background Understanding the pathogenetic basis for AKI involves the study of ischemic and nephrotoxic models of AKI, the latter including heme protein-mediated AKI (HP-AKI). Recently, interest has grown regarding the role of senescence as a mechanism of kidney injury, including AKI. We examined whether senescence occurs in HP-AKI and potential inducers of and the role of a key driver of senescence, namely, p16Ink4a, in HP-AKI. Methods The long-established murine glycerol model of HP-AKI was used, and indices of senescence were examined. To evaluate the interaction of heme and p16Ink4a expression, murine models of genetic deficiency of hemopexin (HPX) and heme oxygenase-1 (HO-1) were used. To determine the involvement of p16Ink4a in HP-AKI, the population of p16Ink4a-expressing cells was reduced using the INK-ATTAC model. Results Using multiple indices, a senescence phenotype appears in the kidney within hours after the induction of HP-AKI. This phenotype includes significant upregulation of p16Ink4a. p16Ink4a is upregulated in the kidney after the individual administration of myoglobin, hemoglobin, and heme, as well as in renal epithelial cells exposed to heme in vitro. Genetic deficiencies of HPX and HO-1, which, independently, are expected to increase heme content in the kidney, exaggerate induction of p16Ink4a in the kidney and exacerbate HP-AKI, the latter shown in the present studies involving HPX-/- mice and in previous studies involving HO-1(-/-) mice. Finally, reduction in the population of p16Ink4a-expressing cells in the kidney improves renal function in HP-AKI even within 24 hours. Conclusions The pathogenesis of HP-AKI involves senescence and the induction of p16Ink4a, the latter driven, in part, by hemoglobin, myoglobin, and heme.

BACKGROUND:Contraction of interstitial fibrosis/tubular atrophy (IFTA) may cause %IFTA to under-represent the severity of nephron loss. Higher density of IFTA foci is an important predictor of progressive chronic kidney disease in native kidneys independent of %IFTA. METHODS:We studied kidney transplant recipients transplanted between 2000-2013 who had a 5-year surveillance kidney biopsy and subsequent follow-up. Banff ci score (interstitial fibrosis) was obtained from the pathology reports. After digitizing the biopsies, we traced cortex area and each distinct IFTA focus on a single trichrome-stained section. Percent IFTA area and IFTA foci density (count of IFTA foci/cortex area) were calculated. Cox models assessed the risk of death-censored graft failure after the 5-year biopsy with Banff ci score, morphometric %IFTA, and IFTA foci density. RESULTS:There were 58 death-censored allograft failures among 835 kidney recipients during the 5 years of follow-up. Biopsies from grafts that failed had higher mean Banff ci score (1.5 vs 0.7, p<0.0001), %IFTA (22.2% vs 7.0%, p<0.0001), and IFTA foci density (1.3 vs. 0.4 per mm2, p<0.001). After adjusting for other Banff scores or clinical variables, Banff ci did not correlate with allograft failure, but both higher %IFTA (HR=1.56, p<0.0001) and higher IFTA foci density (HR=2.34, p<0.001) did. All but 4 allograft failures by 10 years had biopsies in the top quartile of either %IFTA or IFTA foci density at 5 years. A model using just these two morphometric measures without clinical characteristics resulted in a c-statistic of 0.891 with respect to allograft failure. CONCLUSIONS:Morphometric characterization of IFTA foci density is a strong predictor of death censored allograft failure not captured in current Banff classification for grading of kidney fibrosis.

Abstract The homologous proteins p68 and p72 are members of the DEAD box family of RNA helicases. Here, we show that expression of both of these helicases strongly increases during the polyp→adenoma→adenocarcinoma transition in the colon. Furthermore, p68 and p72 form complexes with β-catenin and promote the ability of β-catenin to activate gene transcription. Conversely, simultaneous knockdown of p68 and p72 leads to reduced expression of the β-catenin–regulated genes, c-Myc, cyclin D1, c-jun, and fra-1, all of which are proto-oncogenes. Moreover, transcription of the cell cycle inhibitor p21WAF1/CIP1, whose expression is suppressed by c-Myc, is enhanced on p68/p72 knockdown. Thus, p68/p72 may contribute to colon cancer formation by directly up-regulating proto-oncogenes and indirectly by down-regulating the growth suppressor p21WAF1/CIP1. Accordingly, knockdown of p68 and p72 in colon cancer cells inhibits their proliferation and diminishes their ability to form tumors in vivo. Altogether, these results suggest that p68/p72 overexpression is not only a potential marker of colon cancer but is also causally linked to this disease. Therefore, p68 and p72 may be novel targets in the combat against colon cancer. [Cancer Res 2007;67(16):7572–8]

Supplementary Legends for Figures 1-4, Tables 1-2 from Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81

Supplementary Table 1 from Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81

Background: Endometriosis is a debilitating disease typically characterized by prolific fibrotic scarring. Earlier we reported downregulation of two transcription factors belonging TGF-& beta;R signaling pathway Sp/Kruppel-like factor 11 (KLF11) and 10 (KLF10) in human endometriosis lesions. Here we investigated the role of these nuclear factors and immunity in the scaring fibrosis associated with endometriosis. Methods: We used a well characterized experimental mouse model of endometriosis. WT, KLF10 or KLF11 deficient mice were compared. The lesions were evaluated histologically, fibrosis was quantified with Masons' Trichome staining, immune-infiltrates were quantified by immunohistochemistry, peritoneal adhesions were score, gene expression was evaluated by bulk RNA sequencing. Results: Intense fibrotic reactions and large changes in gene expression were detected in KLF11 deficient implants associated with squamous metaplasia of the ectopic endometrium, as compared to KLF10 deficient or WT implants. Fibrosis was mitigated with pharmacologic agents that blocked histone acetylation or TGF-& beta;R signaling or with genetic deficiency for SMAD3. The lesions were richly infiltrated with T-cells, regulatory T-cells, and innate immune cells. Fibrosis was exacerbated when implants expressed ectopic genes implicating autoimmunity as a major factor contributing to the scaring fibrosis. Conclusions: Our findings identify KLF11 and TGF-& beta;R signaling as cell intrinsic mechanisms and autoimmune responses as cell extrinsic mechanisms of scaring fibrosis in ectopic endometrium lesions. General significance: Immunological factors associated with inflammation and tissue repair drive scaring fibrosis in experimental endometriosis, providing the rationale for immune therapy of endometriosis.

SIGNIFICANCE STATEMENT:Glomerular volume, ischemic glomeruli, and global glomerulosclerosis are not consistently assessed on kidney transplant biopsies. The authors evaluated morphometric measures of glomerular volume, the percentage of global glomerulosclerosis, and the percentage of ischemic glomeruli and assessed changes in these measures over time to determine whether such changes predict late allograft failure. All three features increased from transplant to five-year biopsy. Kidneys with smaller glomeruli at 5 years had more global glomerulosclerosis and a higher percentage of ischemic-appearing glomeruli. Smaller glomeruli and increasing percentages of global glomerulosclerosis and ischemic glomeruli at 5 years predicted allograft failure. Only increased percentage of ischemic glomeruli predicted allograft failure at 5 years independent of all Banff scores. Glomerular changes reflect pathologic processes that predicted allograft loss; measuring them quantitatively might enhance the current Banff system and provide biomarkers for intervention trials.BACKGROUND:Histology can provide insight into the biology of renal allograft loss. However, studies are lacking that use quantitative morphometry to simultaneously assess changes in mean glomerular volume and in the percentages of globally sclerosed glomeruli (GSG) and ischemic-appearing glomeruli in surveillance biopsies over time to determine whether such changes are correlated with late graft failure.METHODS:We used digital scans of surveillance biopsies (at implantation and at 1 and 5 years after transplantation) to morphometrically quantify glomerular volume and the percentages of GSG and ischemic-appearing glomeruli in a cohort of 835 kidney transplants. Cox proportional hazards models assessed the risk of allograft failure with these three glomerular features.RESULTS:From implantation to 5 years, mean glomerular volume increased by nearly 30% (from 2.8×10 6 to 3.6×10 6 µm 3 ), mean percentage of GSG increased from 3.2% to 13.2%, and mean percentage of ischemic-appearing glomeruli increased from 0.8% to 9.5%. Higher percentages of GSG and ischemic-appearing glomeruli at 5-year biopsy predicted allograft loss. The three glomerular features at 5-year biopsy were related; the percentage of GSG and the percentage of ischemic glomeruli were positively correlated, and both were inversely correlated to glomerular volume. At 5 years, only 5.3% of biopsies had ≥40% ischemic glomeruli, but 45% of these grafts failed (versus 11.6% for <40% ischemic glomeruli). Higher Banff scores were more common with increasing percentages of GSG and ischemia, but at 5 years, only the percentage of ischemic glomeruli added to predictive models adjusted for Banff scores.CONCLUSIONS:Glomerular changes reflect important pathologic processes that predict graft loss. Measuring glomerular changes quantitatively on surveillance biopsies, especially the proportion of ischemic-appearing glomeruli, may enhance the current Banff system and be a useful surrogate end point for clinical intervention trials.PODCAST:This article contains a podcast at.

Supplementary Figure 1 from Involvement of RNA Helicases p68 and p72 in Colon Cancer

Croatt, Anthony J.; Grande, Joseph P.; Ackerman, Allan W.; Singh, Raman D.; Nath, Karl A. Author Information