Grande, Joseph P.

Aim The aim of this study was to define the functional correlates of reversibilitiy following restoration of blood flow in the murine 2K1C model of renovascular hypertension. Method : Renal artery stenosis surgery (RAS) was performed by placement of a polytetrafluoroethylene cuff on the right renal artery (N=18). Sham surgery was performed without cuff placement (N=6). In two groups the cuff was removed at 7 days (7D cuff‐off, N=6) and 14 days (14D cuff‐off, N=6) after surgery. In the RAS group, cuff was left in place for 28 days (28D RAS, N=6). Kidneys were harvested at 28 days for assessment of histology and microvessel density (MVD). Renal blood perfusion and oxygenation were measured using 16.4T MRI. Blood pressure measurements were done using tail cuff method. Results In 7D cuff‐off group, baseline intra‐renal perfusion (891±142.8 ml/100g/min) was significantly reduced (311±73.4 ml/100g/min, p<0.0001) following cuff placement. Perfusion was restored to normal level (810±81.2 ml/100g/min) at 28 days following cuff removal at 7 days after RAS. In 14D cuff‐off group, baseline intra‐renal perfusion (846±74.5 ml/100g/min) was also significantly reduced (304±45.9 ml/100g/min, p=0.0002) following cuff placement with non‐significant improvement (579±112.9 ml/100g/min) at 28 days after cuff removal at 14 days after RAS. Baseline intra‐renal perfusion (905±54.9 ml/100g/min) in the 28D RAS group was significantly reduced (455±56.4 ml/100g/min, p<0.0034) following cuff placement and the reduction was maintained throughout the study period (478±22.2 ml/100g/min). 7D cuff‐off group (142±8.2 mg) showed no significant difference in kidney weights measured at 28 days compared to the sham group (154±4.8 mg), but was significantly higher compared to the 28D RAS group (67±10.5 mg, p=0.0010). The kidney weights of the 14D cuff‐off group (86±18.3 mg) measured at 28 days was not significantly different from the 28D RAS group. The 7D cuff‐off group had a significantly lower % atrophy (7±2.2%, p=0.0042) and higher MVD count (152±6.5, p=0.0045) compared to 28D RAS group (% atrophy 66±13%, MVD count 78±15.2). The 14D cuff‐off group had severe atrophy (56±16.6%) and reduced MVD count (94±20.1) similar to that seen in 28D RAS group. 50% of mice that developed hypoxia at day 3 had an improvement in their oxygenation after cuff removal at day 7, whereas, mice undergoing cuff removal at day 14 and 28 day ras mice remained hypoxic. Conclusion Based on these results, we conclude that the renal architecture and microvessel density are preserved when the cuff is removed no longer than 7 days post surgery. We Identified that 2 weeks after cuff placement is the “point of no return” in the renal injury in murine RAS model due to their reduction in microvessel density. Studies to identify biomarkers associated with irreversible renal injury in this model are currently being performed. Support or Funding Information NIH AI 100911 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The objective of this study was to define a role for chemokine generation by parenchymal cells versus circulating inflammatory cells in the initiation of renal damage in the 2K1C model of renovascular hypertension. 129 SV mice were subjected to renal artery stenosis (RAS) by placement of a polytetraethylene cuff on the right renal artery. Systolic blood pressure increased within 2 days of surgery. The cuffed kidney developed tubular atrophy, whereas the contralateral kidney developed compensatory enlargement. Immunohistochemical staining of the cuffed kidney revealed less than 2 CD3+ T cells per 200x field, and rare F4/80+ macrophages or CD20+ B cells in the first 2 weeks following RAS, with progressive increases in macrophages and T cells throughout the 6‐week observation period. Despite the relative paucity of infiltrating inflammatory cells, there was a dramatic increase in chemokine generation within the first week following RAS, as assessed by qPCR analysis of renal cortical homogenates, with 25‐fold induction of MCP‐1, 47‐fold induction of RANTES, and 15‐fold induction of MIP‐2. Chemokine generation continued to increase throughout the 6 weeks, during which circulating inflammatory cells infiltrated the cuffed kidney. Increased expression of MCP‐1 was detected in tubular epithelial cells within 1 week of RAS. We conclude that chemokine generation precedes inflammatory cell influx in RAS.

Despite proven efficacy, ARB therapy reduces glomerular filtration in patients with unilateral renal artery stenosis (RAS). The objective of this study was to determine whether ARB exacerbates renal injury in 2K1C hypertension. RAS was established in mice by placement of a cuff on the right renal artery, which reduces blood flow by 70%. Both Hydralazine (H) and Valsartan (ARB) reduced blood pressure to a similar extent. RAS‐mediated cardiac hypertrophy (heart weight 134% sham (S) p<0.001) was prevented by ARB (102% S, p=ns) but not H (116% S, p=0.03); cardiac fibrosis was prevented in both ARB and H. Although ARB did not exacerbate renal atrophy (91.4 ± 2.1% atrophy vehicle (V) vs 75.3 ± 9.5% ARB, p=ns), H reduced renal atrophy (91.4 ± 2.1% V vs 28.4 ± 13.5% H, p<0.01). The contralateral kidney underwent compensatory hyperplasia (123% S; p<0.001), which was blunted with ARB or H treatment. No significant histopathologic alterations were observed in the contralateral kidneys of any group. RAS induced proteinuria in V mice. Urine protein excretion in ARB RAS mice was reduced to levels observed in S mice; H did not significantly alter urine protein excretion. Severe renal atrophy was observed in the cuffed kidneys of V mice, which was not exacerbated by ARB. However, ARB reduced proteinuria to baseline levels, indicating that ARB may protect overall renal function in unilateral RAS. These studies were supported by NIH grant P01 HL85307.

Recent studies have implicated CCR2 ‐ CCL2 signaling in macrophage influx and development of chronic renal disease in human and experimental renal artery stenosis (RAS). We sought to test the hypothesis that development of chronic renal disease would be abrogated in CCR2 knockout (KO) mice. We established RAS in CCR2 KO and WT mice (n=10) through placement of a cuff on the right renal artery We have shown that the cuff reproducibly reduces blood flow by 65% to the kidney. Sham mice underwent manipulation of the right renal artery without cuff placement. Both CCR2 KO and WT mice developed hypertension, which persisted throughout the 4 week study. There was no difference in renal atrophy between CCR2 KO (53+//‐14.6% cortical surface area) and WT (61.2%+/‐14%) mice. Furthermore, there were no significant differences in F4/80+ macrophage infiltration (12.78+/‐4.9% surface area for CCR2 KO mice and 13.13+/‐ 3.1% for WT mice) or CD3+ T cell infiltration. Infiltrating macrophages were predominantly iNOS+ M1 phenotype; few arginase+ cells were observed in any group. Urine protein excretion was similar in CCR2 KO and WT mice. CCL was induced to a similar extent in CCR2 KO and WT mice. Furthermore, no differences in CCL5, CCL7, CCR5, CCR1, or iNOS expression was observed between CCR2 KO and WT mice. We conclude that macrophage infiltration and chronic renal damage occurs in mice with deletion of CCR2. Future studies will identify signaling pathway(s) responsible for macrophage influx and chronic renal damage in this model. Grant Funding Source : AI‐100911

We have previously demonstrated that, in vitro, c‐AMP specific PDE3 inhibitors block mitogenesis of mesangial cells (Am J Physiol Renal Physiol, 2004) and PDE4 inhibitors block cytokine stimulated MCP‐1 expression (Am J Physiol Cell Physiol, 2005). The in vivo relevance of these findings has not been previously established. Mice were treated with the nonselective cAMP PDE inhibitor pentoxifylline (PTX) prior to bilateral clamping of the renal arteries for 30 minutes, followed by reperfusion for 1 and 4 days. PTX blunted the increase in BUN and creatinine after 1 day (−38% and −43%, respectively, p<0.05), and after 4 days (−80% and −44%, respectively, p<0.05). PTX reduced the extent of tubular necrosis (1 day: 39.13% vs. 52.5%; 4 days: 2.5% vs. 23.33%, p<0.05, p=NS. % cortical area in PTX vs. IR). PTX reduced PCNA expression 4 days after IR, as assessed by Western blot (−43%, p<0.05), and reduced caspase 3 activity one day after IR (−43%, p<0.05). PTX markedly reduced expression of MMP10 and Cox‐2 one day after IR (26 and 4 folds, respectively, p<0.05), which are induced in response to acute inflammation. PTX significantly reduced cyclin F, cyclin A2 and Ki67 expression 4 days after IR (124, 7 and 9 folds, respectively, p<0.05), which are induced in response to proliferation. We conclude that PTX ameliorates IR injury through c‐AMP mediated reduction in proliferative and inflammatory responses to acute injury.

KLFs (Kruppel‐like factors) comprise a family of over 17 distinct transcription factors that have been shown to regulate cell metabolism and renal function, including podocyte differentiation and maintenance of the glomerular filtration barrier. In non‐renal systems, KLF11 has been shown to regulate fibrosis. However, no previous studies have established a potential role for KLF11 in renal pathophysiology. We sought to test the hypothesis that KLF11 regulates renal fibrosis in unilateral ureteral obstruction, a well‐established murine model of renal fibrosis. Mice with homozygous deletion of the KLF11 gene (KO) and wild‐type (WT) mice were subjected to unilateral ureteral obstruction (UUO; n=8 per group); sham controls (n=4 per group) were subjected to manipulation of the ureter without ligation. Kidneys were harvested at 9 days following surgery,. Semiquantitative histopathologic analysis of interstitial fibrosis, tubular atrophy, and interstitial inflammation was performed in a blinded fashion. Analysis of gene expression was performed on renal cortex using Pathway Detect RNA arrays and by RNASeq. Histopathologic analysis of sham controls revealed no significant alterations. However, expression of several elements of the TGF‐beta signaling pathway was significantly higher in KLF11 KO sham mice, compared to WT mice, including TGF‐beta receptors type I and type II, Smad2, Smad 7, Bmpr1B, and Bmpr2R, and the KLF‐related transcription factor Sp1 (all p<0.05). In mice subjected to UUO, interstitial fibrosis, tubular atrophy, and interstitial inflammation were significantly greater in KLF11 KO mice compared to WT mice. Interstitial inflammation was associated with influx of alternatively activated macrophages, as assessed by quantitative analysis of CD206+ infiltrating cells (p<0.05); no significant increase in CD3+ T cell numbers were noted. Expression of several members of the TGF‐beta signaling pathway were significantly increased in KLF11 KO UUO mice, compared to WT UUO mice, including TGF‐beta 1, TGF‐beta‐2, and TGF‐beta3, TGF‐beta receptors type I and type II, and Smad 1,2,3,4, and 5 (all p<0.05). Conversely, expression of the transcription factor JunB was markedly increased in WT UUO mice compared to WT sham mice (12.2 ‐fold; p<0.0001). However, JunB expression was markedly decreased in KLF11 KO mice (both sham and UUO). Although MMP expression was not significantly different in KO versus WT mice, TIMP3 expression was higher in KO mice compared to WT mice. In conclusion, we found that renal damage is exacerbated in KLF11 KO mice subjected to UUO, compared to WT UUO mice and is associated with influx of potentially pro‐fibrotic, alternatively activated M2 macrophages. KLF11 appears to be a critical regulator of TGF‐beta signaling, as KLF11 deficiency is associated with increased expression of TGF‐beta and signaling intermediates of this fibrogenic pathway.

Abstract Background and Aims Kruppel-like factors (KLFs) comprise a family of zinc-finger transcription factors that play a critical role in development, proliferation, and regeneration following injury. There are over 17 members of this family; recent studies have shown that KLF family members regulate podocyte differentiation, preservation of the glomerular filtration barrier, and regulation of mitochondrial function. However, a role for KLF11 in renal pathophysiology has not been previously established. Method Wild-type (WT) and KLF11 knockout (KO) mice were subjected to unilateral ureteric obstruction (UUO), a well-established model of renal inflammation and fibrosis; controls included mice subjected to manipulation of the ureter without ligation. Kidneys were harvested after 9 days (n=8 animals per group). Semiquantitative histopathologic analysis of renal atrophy, fibrosis, and inflammation was performed in a blinded fashion. Gene expression analysis was performed on renal cortex employing the Pathway Detect RNA array and RNASeq. Results In UUO, renal atrophy was more severe in KLF11 KO mice than WT mice (p&lt;0.001). Deposition of collagen, as assessed by quantitative analysis of Sirus Red stained sections, was greater in KLF11 KO mice, compared to WT mice subjected to UUO; COL3A1 expression was also increased (p&lt;0.05). Atrophy was associated with an increase in F4/80+ (p&lt;0.01) and CD206+ macrophages (p&lt;0.05), but not CD3+ T cells in KLF11 KO vs. WT mice. Induction of CC chemokines, including CCL2, CCL5, CCL7, CCL12, and CCL2 as well as CCR2 was significantly higher in KLF11 KO versus WT mice subjected to UUO (all p&lt;0.001). Expression of NF-kB (p&lt;0.01) and TNF alpha (p&lt;0.01), but not IL-1 beta, IL-6, or IL-10 were significantly higher in KLF11 KO than WT mice with UUO. Expression of TGF-beta 1, Smad2, and Smad3 were also higher in KLF11 KO mice than WT mice with UUO (p&lt;0.05). Conclusion Renal injury in UUO is exacerbated in KLF11 KO mice, compared to WT mice. Injury is associated with increased macrophage influx and production of pro-inflammatory chemokines. Future studies will determine how KLF11 deficiency directs transcription of pro-inflammatory and pro-fibrotic genes.

Abstract Key Points The healthy kidney prominently expresses angiotensin-converting enzyme 2 (ACE2) mRNA, protein, and activity, with ACE2 protein abundantly displayed in proximal tubules. Renal ACE2 expression decreases after ischemic AKI but is induced by LPS in a heme oxygenase-1-dependent manner. ACE2 induction/angiotensin (1–7) and angiotensin (1–7) may offer therapeutic approaches for AKI without or with coronavirus disease 2019, respectively. Background The actions of angiotensin-converting enzyme 2 (ACE2) oppose those of the renin-angiotensin-aldosterone system. ACE2 may be a cytoprotectant in some tissues. This study examined ACE2 expression in models of AKI. Methods ACE2 mRNA and protein expression and ACE2 activity were assessed in murine ischemic AKI. Renal ACE2 mRNA expression was evaluated in LPS-induced AKI in wild-type (C57BL/6J) mice, in heme oxygenase-1 +/+ and heme oxygenase-1 −/− mice, and after unilateral ureteral obstruction (UUO) in wild-type mice. The effect of sex and age on renal ACE2 protein expression was also assessed. Results In ischemic AKI, ACE2 mRNA and protein expression and ACE2 activity were reduced as compared with such indices in the intact kidney. In ischemic AKI, ACE2, which, in health, is prominently expressed in the tubular epithelium, especially proximal tubules, is decreased in expression in these segments. Decreased ACE2 expression in AKI did not reflect reduced GFR, because ACE2 mRNA expression was unaltered after UUO. LPS induced renal ACE2 mRNA expression in wild-type mice, but this effect did not occur in heme oxygenase-1–deficient mice. In ischemic and LPS-induced AKI, renal expression of the Mas receptor was increased. In the intact kidney, renal ACE2 protein expression decreased in female mice as compared with male mice, but was unaltered with age. Conclusion We conclude that renal ACE2 expression is decreased in ischemic AKI, characterized by decreased GFR and abundant cell death, but is upregulated in LPS-induced AKI, an effect requiring heme oxygenase-1. Determining the significance of ACE2 expression in experimental AKI merits further study. We suggest that understanding the mechanism underlying ACE2 downregulation in AKI may offer insights relevant to COVID-19: ACE2 expression is downregulated after ACE2 mediates SARS-CoV-2 cellular entry; such downregulation is proinflammatory; and AKI commonly occurs and determines outcomes in COVID-19.

Background. Delayed graft function (DGF) of a kidney transplant results in increased cost and complexity of management. For clinical care or a DGF trial, it would be ideal to accurately predict individual DGF risk and provide preemptive treatment. A calculator developed by Irish et al has been useful for predicting population but not individual risk. Methods. We analyzed the Irish calculator (IC) in the DeKAF prospective cohort (incidence of DGF = 20.4%) and investigated potential improvements. Results. We found that the predictive performance of the calculator in those meeting Irish inclusion criteria was comparable with that reported by Irish et al. For cohorts excluded by Irish: (a) in pump-perfused kidneys, the IC overestimated DGF risk; (b) in simultaneous pancreas kidney transplants, the DGF risk was exceptionally low. For all 3 cohorts, there was considerable overlap in IC scores between those with and those without DGF. Using a modified definition of DGF—excluding those with single dialysis in the first 24 h posttransplant—we found that the calculator had similar performance as with the traditional DGF definition. Studying whether DGF prediction could be improved, we found that recipient cardiovascular disease was strongly associated with DGF even after accounting for IC-predicted risk. Conclusions. The IC can be a useful population guide for predicting DGF in the population for which it was intended but has limited scope in expanded populations (SPK, pump) and for individual risk prediction. DGF risk prediction can be improved by inclusion of recipient cardiovascular disease.

Significance Statement Approximately 20% of patients with lupus nephritis, the most common renal manifestation of systemic lupus erythematosus, show membranous lupus nephritis on kidney biopsy, and nearly 10% eventually develop ESKD. Recently, two proteins, Exostosin 1 and Exostosin 2 (EXT1/EXT2), were shown to be present in a subset of membranous lupus nephritis kidney biopsy specimens. In an examination of 374 membranous lupus nephritis kidney biopsy specimens, the authors found 32.6% to be EXT1/EXT2-positive. Kidney biopsy specimens from patients with EXT1/EXT2-positive membranous lupus nephritis showed less chronicity features (glomerulosclerosis, interstitial fibrosis, and tubular atrophy) compared with those from EXT1/EXT2-negative patients. EXT1/EXT2-negative patients were also more likely to reach ESKD than EXT1/EXT2-positive patients. These findings suggest that the presence of EXT1/EXT2 is favorable, and that EXT1/EXT2-positive patients have better renal outcomes compared with EXT1/EXT2-negative patients. Background In patients with secondary (autoimmune) membranous nephropathy, two novel proteins, Exostosin 1 and Exostosin 2 (EXT1/EXT2), are potential disease antigens, biomarkers, or both. In this study, we validate the EXT1/EXT2 findings in a large cohort of membranous lupus nephritis. Methods We conducted a retrospective cohort study of patients with membranous lupus nephritis, and performed immunohistochemistry studies on the kidney biopsy specimens against EXT1 and EXT2. Clinicopathologic features and outcomes of EXT1/EXT2-positive versus EXT1/EXT2-negative patients were compared. Results Our study cohort included 374 biopsy-proven membranous lupus nephritis cases, of which 122 (32.6%) were EXT1/EXT2-positive and 252 (67.4%) were EXT1/EXT2-negative. EXT1/EXT2-positive patients were significantly younger ( P =0.01), had significantly lower serum creatinine levels ( P =0.02), were significantly more likely to present with proteinuria ≥3.5 g/24 h ( P =0.009), and had significantly less chronicity features (glomerulosclerosis, P =0.001 or interstitial fibrosis and tubular atrophy, P <0.001) on kidney biopsy. Clinical follow-up data were available for 160 patients, of which 64 (40%) biopsy results were EXT1/EXT2-positive and 96 (60%) were EXT1/EXT2-negative. The proportion of patients with class 3/4 lupus nephritis coexisting with membranous lupus nephritis was not different between the EXT1/EXT2-positive and EXT1/EXT2-negative groups (25.0% versus 32.3%; P= 0.32). The patients who were EXT1/EXT2-negative evolved to ESKD faster and more frequently compared with EXT1/EXT2-positive patients (18.8% versus 3.1%; P =0.003). Conclusions The prevalence of EXT1/EXT2 positivity was 32.6% in our cohort of membranous lupus nephritis. Compared with EXT1/EXT2-negative membranous lupus nephritis, EXT1/EXT2-positive disease appears to represent a subgroup with favorable kidney biopsy findings with respect to chronicity indices. Cases of membranous lupus nephritis that are EXT1/EXT2-negative are more likely to progress to ESKD compared with those that are EXT1/EXT2-positive.