Grande, Joseph P.

Renal artery stenosis (RAS) is a common public health problem, affecting up to 7% of individuals over 65 years of age. Optimal therapy for patients with RAS is controversial, as recent studies have indicated that revascularization via stenting fails to improve renal function or patient survival. Medical therapy is associated with high morbidity and mortality, primarily due to cardiovascular disease. However, morphologic correlates for reversibility following restoration of blood flow have not been adequately defined. The purpose of this study was to standardize an experimental model for the restoration of blood flow, employing a well characterized murine RAS model. Total N=10 C57B/6J male mice were used. RAS was induced on the right renal artery by putting a polytetrafluoroethylene cuff (N=6) and sham surgery was performed without cuff placement (N=4). To determine the feasibly of restoring the blood flow to the stenotic kidney, cuff was taken off at day 7 following the RAS surgery. Blood pressure measurements were done using tail cuff method at baseline, before taking cuff off and then every week until harvest. MRI images were captured using an Avance DRX 700WB spectrometer with a vertical 16.4 T wide bore magnet connected with the imaging tools at baseline, 3 days after putting the cuff on or sham surgery, and at 3 weeks after cuff off. The animals were harvested at day 28 following surgery and kidney weights are obtained. Baseline renal perfusion (1018±41.6 ml/100g/min) was significantly reduced (634.7±141.3 ml/100g/min, p=0.01) following cuff placement. Perfusion was restored to normal (955±59.8 ml/100g/min) at 28 days following cuff removal at 7 days after RAS. Similarly, volume of the stenotic kidney was significantly reduced (at day 3 after RAS 172±8.6 mm 3 ) compared to baseline (206.3±4.4 mm 3 , p=0.0007). There was no significant difference in perfusion and volume after cuff off compared to baseline and sham groups. There was no significant difference in kidney weight between cuff‐off (167± 4.7 mg) and sham groups (163±9.95 mg). Also, there was no difference in heart weight between cuff off (150±3.7mg) and sham groups (148±6.7mg). We conclude that this model will provide the basis for determining the latest time point associated with recovery of renal structure and potential histopathologic markers that predict preservation of renal structure following cuff removal. Also, this model would provide the basis for determining efficacy of additional therapies to target inflammation, etc. to widen the “therapeutic window” during which restoration of renal blood flow is beneficial. Support or Funding Information This project is funded by National Institutes of health, NIAID R01 AI100911.

CX3CR1 is a chemokine receptor widely expressed on inflammatory cells which recognizes CX3CL1 (fractalkine) a membrane bound chemokine that can function as an adhesion receptor. Blockade of CX3CR1 has been shown to reduce inflammation and chronic renal injury in a variety of models. We sought to determine whether CX3CR1 deficient mice are protected from chronic renal injury in experimental renal artery stenosis (RAS). RAS was established in CX3CR1 knockout (KO) and wild‐type C57BLK/6 mice (WT) through placement of a cuff on the right renal artery. Sham mice underwent manipulation of the right renal artery without placement of a cuff. As assessed by MRI, mean flow reduction was 65.3% in the WT mice and 66.5% in the KO mice (p=ns) subjected to RAS. Both KO and WT mice developed hypertension, which persisted throughout the 4 week study. KO and WT mice developed a similar degree of cardiac hypertrophy (166 mg WT, 172 mg KO, p=ns; 117 mg sham). Stenotic kidney weights were reduced to a similar extent in KO and WT mice (sham 148 mg, WT 83 mg, KO 53 mg) with compensatory hypertrophy of the contralateral kidney (sham 136 mg, WT 170 mg, KO 206 mg, p=0.0016 KO vs WT). The stenotic kidney of both KO and WT mice developed severe atrophy, characterized by flattening and simplification of tubular epithelium and influx of interstitial T cells and macrophages (32.8 % cortical surface area for WT and 76.4% cortical surface area for KO, p=0.01). The contralateral kidney was without significant histopathologic abnormality. We conclude that development of severe atrophy is not prevented in CX3CR1 KO mice, indicating that alternative pathways contribute to chronic renal damage in this model. Support or Funding Information This work is supported by NIH Grant R01 AI100911.

Abstract Bacterial superantigens (BSAg) are produced by Staphylococcus aureus and Streptococcus pyogenes. BSAg bind directly to the MHC class II molecules without undergoing any processing and subsequently activate T cells in an antigen non-specific manner by interacting with the TCR Vβ region. BSAg have been implicated primarily in acute diseases such as food poisoning, toxic shock syndrome, etc. Since carrier strains of S. aureus and S. pyogenes can elaborate superantigens, there is a distinct possibility of chronic exposure to low doses of BSAg and it is imperative to study its outcome. As BSAg bind more avidly to human (HLA) than to murine (H-2) MHC class II, we delivered low concentrations (total of 10 μg/mouse) of BSAg to HLA-DR3 [staphylococcal enterotoxin (SE) B] or HLA-DQ8 [streptococcal pyrogenic exotoxin (SPE) A] transgenic mice through subcutaneously implanted miniosmotic pumps for 7 days. Continuous exposure to low concentrations of BSAg resulted in a significant protracted expansion of CD4+ T cells expressing TCR Vβ8 (for SEB); Vβ8 and Vβ14 (for SPEA) with a concomitant decrease in percentage of CD8+ T cells expressing these TCR. T cells bearing TCR Vβ6 showed an inverse correlation (slight decrease in CD4+ and increase in CD8+ compartments with SEB and SPEA). Histological examinations of major organs showed intense perivascular infiltration in lungs, kidneys and liver along with inflammatory infiltrates in the cardiac valvular endothelium in mice bearing pumps delivering BSAg but not PBS. In conclusion, we have shown the immunological outcome of chronic exposure to low levels of BSAg using the HLA class II transgenic mouse model.

Abstract Renal artery stenosis is common in the elderly population; biomarkers that predict response to restoration of blood flow have not been previously established. Methods We have previously shown in murine model that cuff removal 7 days (7D Cuff-off) after initial placement on the renal artery results in reversible injury whereas cuff removal 14 days (14D Cuff-off) after placement results in irreversible renal injury, when assessed at 28 days after initial surgery. Positive and negative controls consisted of cuff placement for 28 days (28D) and sham surgeries respectively. We performed RNASeq on whole renal cortex to identify innate immune pathways that correlate with reversible versus irreversible injury. Data were normalized to appropriate sham controls in all the groups. Induction of greater than log2 ratio > 2 and p < 0.00001 were considered significant (following adjustment for multiple comparisons). Results As assessed by RNASeq, progression of renal disease (14D Cuff-off & 28D mice) was associated with persistent upregulation of genes associated with T cell signaling and macrophage polarization compared to reversible injury (7D Cuff off). Upregulated genes included but not limited to: CCL5, CCR5, CCL2, CXCL1, CXCL9, CXCL10, CXCR3, TLR2, KLF6, CD74, CD4, CD14, CD44, CD83, CD84, IL1R1, IL1B, SPP1, AIF1, SLC7A2, TRAF1, S100A4, SLC9A9, CRIP1, CLEC7A, CLEC10A, EGR2, MGL2, SOCS2, CTLA2B, and FCGR1. Conclusion We have identified genes associated with T cell and macrophage activation that were down regulated in the 7D Cuff-off versus 14D Cuff-off and 28D mice; future studies in isolated T cells and macrophages will determine whether these genes represents biomarker(s) of renal disease progression in 2K-1C renovascular hypertension.

Abstract Atherosclerotic renal artery sclerosis (RAS) is a significant public health concern, affecting 7% of the population over 65 years old and conveying an increased risk for cardiovascular as well as renal disease. Recent reports have failed to show a benefit of renal artery stenting to treat RAS. We tested the hypothesis that mesenchymal stem cells (MSC) slow the progression of renal injury by reducing inflammation. MSCs were isolated and cultured from murine adipocyte tissue and injected via the carotid artery into the animals 4 weeks after inducing RAS, a time at which the stenotic kidney has undergone severe atrophy. Conjugated microsphere labeling and flow cytometry confirmed that the MSCs migrate to the stenotic kidney. Half of the treatment group demonstrated a 90% reduction in atrophy and significantly lower level of regulatory T cell (Treg) infiltration while the other half demonstrated no change when compared to vehicle treated RAS mice. Array analysis showed a significant down-regulation of NOS2 (M1 macrophages), RORc (Th 17 cells), and CCR4 (associated with M1 macrophages) within those mice with low renal atrophy. The results suggest that under certain conditions, MSCs have a possible effect on the inflammatory environment present in RAS mice. Future studies will determine whether intervention prior to development of severe atrophy will increase therapeutic efficacy of MSC.

Abstract Renovascular hypertension (RVH) affects up to 7% of individuals over 65 years of age. We have previously demonstrated that induction of oxidative stress is an early event in the development of RVH that leads to development of inflammation and renal atrophy. NLRP3 is one of the most well-known sensors of oxidative stress. However, the role of NLRP3 in the development of RVH and subsequent end-organ damage is not well understood. We therefore sought to test the hypothesis that inhibition of NLRP3 activation protects the heart and the kidneys of mice subjected to RVH. In accordance with our previous observations, wild type (WT) mice subjected to RVH developed 26±6% of renal atrophy within 1 week following cuff placement that progressed to almost 100% by 4 weeks. In contrast, although NLRP3-/- mice (KO) subjected to RVH developed similar degree of atrophy to their age-matched WT in the first week (29 ± 17 vs. 39 ± 20), the atrophy did not progress when evaluated at 4 weeks (29 ± 13 vs. 78 ± 10, p=0.013). Histologic evaluation of renal cortex of KO mice showed no significant interstitial infiltration with CD3+ T cells or F4/80+ macrophages. In addition, KO mice developed no cardiac hypertrophy, no cardiac fibrosis, or aortic lesion despite similar elevation in systolic blood pressure to the WT mice. We propose that NLRP3 activation plays a critical role in the development of renal atrophy in mice subjected to RVH and represent a potential therapeutic target.

Abstract Renal artery stenosis is a common finding in patients with cardiovascular disease. Recent studies have provided evidence that innate immunity plays a critical role in the development of chronic renal disease in renovascular hypertension. We have previously demonstrated that interstitial infiltration of lymphocytes is an early event in the development of renal atrophy in the 2K1C model of renovascular hypertension. We therefore sought to test the hypothesis that lymphocytes play a role in the development of progressive atrophy/fibrosis in this model. We subjected mice to renal artery stenosis through placement of a polytetrafluoroethylene cuff on the right renal artery. In accordance with our previous observations, wild type mice subjected to RAS developed renal atrophy within 2 weeks following placement of the cuff (70.5 +/- 11.9%). Chemokine expression in renal cortex of the stenotic kidney reflected predominantly a Th1 response (STAT1, STAT4, TBX21); CCL2, CCL17, CCL12, and TNF mRNA expression were also upregulated, but not IL-6 and IL-10. In striking contrast to wild type mice, Rag1 mice developed significantly less atrophy (23.1 +/- 10.4, p<0.0037 vs wild type). Unlike wild type mice, histologic evaluation of renal cortex of Rag1 mice showed no significant interstitial infiltration with CD3+ T cells or F4/80+ macrophages. We propose that T cell-mediated inflammation plays a critical role in the development of renal atrophy in 2K1C mice.

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

AbstractBackgroundComputer‐assisted scoring is gaining prominence in the evaluation of renal histology; however, much of the focus has been on identifying larger objects such as glomeruli. Total inflammation impacts graft outcome, and its quantification requires tools to identify objects at the cellular level or smaller. The goal of the current study was to use CD45 stained slides coupled with image analysis tools to quantify the amount of non‐glomerular inflammation within the cortex.MethodsSixty renal transplant whole slide images were used for digital image analysis. Multiple thresholding methods using pixel intensity and object size were used to identify inflammation in the cortex. Additionally, convolutional neural networks were used to separate glomeruli from other objects in the cortex. This combined measure of inflammation was then correlated with rescored Banff total inflammation classification and outcomes.ResultsIdentification of glomeruli on biopsies had high fidelity (mean pixelwise dice coefficient of .858). Continuous total inflammation scores correlated well with Banff rescoring (maximum Pearson correlation .824). A separate set of thresholds resulted in a significant correlation with alloimmune graft loss.ConclusionsAutomated scoring of inflammation showed a high correlation with Banff scoring. Digital image analysis provides a powerful tool for analysis of renal pathology, not only because it is reproducible and can be automated, but also because it provides much more granular data for studies.