Acute kidney injury (AKI) is a devastating condition with major complications including death and, in some cases, progression to chronic kidney disease (CKD). We have previously shown that pulsed ultrasound (pUS) can reduce kidney ischemia-reperfusion injury (IRI) by activating the cholinergic anti-inflammatory pathway. The efficacy of a spleen-targeted pUS regimen in AKI of other etiologies and its long-term impact are unclear. Using a new spleen-targeted US approach, pUS was delivered to male mice 24 h before folic acid (FA), lipopolysaccharide, or bilateral kidney IRI. Mice were monitored and assessed for markers of inflammation, renal function, and kidney fibrosis. When compared with sham, mice that received spleen-targeted pUS had reduced plasma TNFα and blood urea nitrogen (BUN) after sepsis-associated AKI, reduced plasma creatinine, and BUN after kidney IRI and reduced plasma creatinine, BUN, and kidney fibrosis after FA administration. pUS-treated mice displayed reduced myeloid cell infiltration to the kidneys after FA and IRI. In sham-treated mice, markers associated with ongoing maladaptive repair including Sox9, Wnt2, and Wnt4 were increased on day 14 after FA in comparison with pUS-treated mice. These data demonstrate that pulsed ultrasound of the spleen is a novel, safe, and effective therapy for the prevention of AKI of multiple etiologies and the subsequent development of CKD. Findings from this study are critical for advancing human translation of ultrasound as a preventative measure for AKI and CKD.NEW & NOTEWORTHY We developed a safe and effective pulsed ultrasound (pUS) protocol targeting the mouse spleen to block inflammation and reduce AKI of multiple etiologies. By using a model of the AKI to CKD transition, we demonstrated the long-term benefits of pUS. The findings of these studies will be used to advance the human translation of spleen-targeted US as a preventative measure for AKI and CKD.

The findings of our study are significant in several ways: 1) loss of an amino acid chaperone in the proximal tubule is sufficient to cause hypertension, 2) the results in global and proximal tubule-specific collectrin knockout mice support the notion that vascular dysfunction is required for salt sensitivity or that impaired renal tubule function causes hypertension but is not sufficient to cause salt sensitivity, and 3) our study is the first to implicate a role of collectrin in human hypertension.

Acute kidney injury (AKI) is a frequent complication of sepsis and an important cause of morbidity and mortality worldwide. A cornerstone of sepsis-associated AKI is dysregulated inflammation, leading to increased tissue oxidative stress and free radical formation, which leads to multiple forms of cell death. DJ-1 is a peroxiredoxin protein with multiple functions, including its ability to control cellular oxidative stress. Although DJ-1 is expressed prominently by renal tubules, its role in AKI has not been investigated. In the present study, we examined the effect of DJ-1 deficiency in a murine model of endotoxin-induced AKI. Endotoxemia induced greater kidney injury in DJ-1-deficient mice. Furthermore, DJ-1 deficiency increased renal oxidative stress associated with increased renal tubular apoptosis and with expression of death domain-associated protein (DAXX). Similar to the in vivo model, in vitro experiments using a medullary collecting duct cell line (mIMCD3) and cytotoxic serum showed that serum obtained from wild-type mice resulted in increased expression of s100A8/s100A9, DAXX, and apoptosis in DJ-1-deficient mIMCD3 cells. Our findings demonstrate a novel renal protective role for renal tubular DJ-1 during endotoxemia through control of oxidative stress, renal inflammation, and DAXX-dependent apoptosis.

Lupus nephritis is the end organ manifestation of systemic lupus erythematosus. Iron metabolism and its master regulator, hepcidin, are known to regulate cell proliferation and in flammation, but their direct role in the pathophysiology of lupus nephritis remains under - investigated. Exogenous hepcidin reduced the severity of lupus nephritis in MRL/lpr mice, a preclinical model of spontaneous systemic lupus erythematosus without worsening anemia of in flammation. Hepcidin treatment reduced renal iron accumulation, systemic and intrarenal cytokines, and renal immune cell in filtration, independent of glomerular immune complex deposits and circulating autoantibodies. Hepcidin increased renal H-ferritin (a ferroxidase), reduced expression of free iron dependent DNA synthesis enzymes, Ribonucleotide Reductase 1 and 2, and intra-renal macrophage proliferation. These findings were recapitulated in vitro upon treatment of macrophages with hepcidin and murine colony stimulation factor -1. Furthermore, hepcidin-treated macrophages secreted less IL -1 b and IL -6 upon stimulation with the TLR3 agonist polyinosine-polycytidylic acid. Of clinical relevance, hepcidin reduced progression and severity of nephritis in old mice with established systemic autoimmunity and overt proteinuria, highlighting its therapeutic potential. Thus, our findings provide a proof -of -concept that targeting cellular iron metabolism with hepcidin represents a promising therapeutic strategy in lupus nephritis.

Significance Statement GSTM1 encodes a member of a superfamily of antioxidant enzymes, and a highly prevalent GSTM1 deletion variant is associated with kidney disease progression in two human study cohorts. In this study, the authors demonstrate that Gstm1 knockout mice exhibit increased oxidative stress, kidney injury, and inflammation in models of CKD and hypertension, and that Gstm1 loss in the parenchyma but not in bone marrow–derived cells drives renal inflammation. Importantly, consumption of broccoli powder or cruciferous vegetables was protective against kidney disease only in Gstm1 knockout mice, and was observed mainly in the human participants in the Atherosclerosis Risk in Communities Study who were homozygous for GSTM1 deletion. These findings suggest that targeting antioxidant therapy specifically in individuals carrying the GSTM1 deletion variant may be effective in delaying kidney disease progression. Background GSTM1 encodes glutathione S-transferase μ-1 (GSTM1), which belongs to a superfamily of phase 2 antioxidant enzymes. The highly prevalent GSTM1 deletion variant is associated with kidney disease progression in human cohorts: the African American Study of Kidney Disease and Hypertension and the Atherosclerosis Risk in Communities (ARIC) Study. Methods We generated a Gstm1 knockout mouse line to study its role in a CKD model (involving subtotal nephrectomy) and a hypertension model (induced by angiotensin II). We examined the effect of intake of cruciferous vegetables and GSTM1 genotypes on kidney disease in mice as well as in human ARIC study participants. We also examined the importance of superoxide in the mediating pathways and of hematopoietic GSTM1 on renal inflammation. Results Gstm1 knockout mice displayed increased oxidative stress, kidney injury, and inflammation in both models. The central mechanism for kidney injury is likely mediated by oxidative stress, because treatment with Tempol, an superoxide dismutase mimetic, rescued kidney injury in knockout mice without lowering BP. Bone marrow crosstransplantation revealed that Gstm1 deletion in the parenchyma, and not in bone marrow–derived cells, drives renal inflammation. Furthermore, supplementation with cruciferous broccoli powder rich in the precursor to antioxidant-activating sulforaphane significantly ameliorated kidney injury in Gstm1 knockout, but not wild-type mice. Similarly, among humans (ARIC study participants), high consumption of cruciferous vegetables was associated with fewer kidney failure events compared with low consumption, but this association was observed primarily in participants homozygous for the GSTM1 deletion variant. Conclusions Our data support a role for the GSTM1 enzyme in the modulation of oxidative stress, inflammation, and protective metabolites in CKD.

Abstract Background Lupus nephritis (LN) is an end-organ complication of Systemic lupus erythematosus and is more common in premenopausal women. Hepcidin, the master regulator of iron homeostasis, modulates inflammation and is negatively regulated by estrogen. Therefore, we hypothesized that exogenous hepcidin may reduce the severity and delay the onset of LN. Methods Pre-nephritic 8-week-old or nephritic 16-week-old MRL/lpr female mice were injected Hepcidin (50ug, i.p) or vehicle twice a week and markers of renal injury and inflammation were examined at 18 and 20 weeks of age. The direct effect of hepcidin on macrophages was studied in-vitro. Results Hepcidin reduced intrarenal iron accumulation, and increased H-ferritin. This was associated with reduced renal inflammation and immune cell infiltration, which collectively mitigated microalbuminuria and tubular injury, independent of immune complex deposits and autoantibodies. The increase in H-ferritin, was associated with a reduced number of renal Ki-67+-F4/80+ macrophages. In-vitro, hepcidin induced H-ferritin in macrophages and reduced labile (Fe2+) iron. H-ferritinhi macrophages proliferated less upon Mcsf-1 stimulation and secreted less IL-1b, and IL-6 upon TLR-3 activation. Hepcidin reduced microalbuminuria when administered to nephritic, 16-week-old mice without worsening lupus-associated anemia. Conclusions We have identified that hepcidin targets iron homeostasis and reduces cardinal pathogenic features of LN. Importantly, Hepcidin treatment ameliorates kidney disease in mice with established proteinuria. Thus, our data highlight that targeting cellular iron metabolism with hepcidin represents a promising and a new therapeutic strategy in LN.

Hypertension affects over 1 billion people worldwide and increases the risk for heart failure, stroke, and chronic kidney disease. Despite high prevalence and devastating impact, its etiology still remains poorly understood for most hypertensive cases. Rcn2, which encodes reticulocalbin 2, is a candidate gene for atherosclerosis that we have previously reported in mice. Here, we identified Rcn2 as a novel regulator of blood pressure in mice. Rcn2 was abundantly expressed in the endothelium and adventitia of normal arteries and was dramatically upregulated in the medial layer of the artery undergoing structural remodeling. Deletion of Rcn2 lowered basal blood pressure and attenuated ANG II-induced hypertension in C57BL/6 mice. siRNA knockdown of Rcn2 dramatically increased production of the nitric oxide (NO) breakdown products nitrite and nitrate by endothelial cells but not by smooth muscle cells. Isolated carotid arteries from Rcn2-/- mice showed an increased sensitivity to the ACh-induced NO-mediated relaxant response compared with arteries of Rcn2+/+ mice. Analysis of a recent meta-data set showed associations of genetic variants near RCN2 with blood pressure in humans. These data suggest that Rcn2 regulates blood pressure and contributes to hypertension through actions on endothelial NO synthase.

The renin-angiotensin system tightly controls aldosterone synthesis. Dysregulation is evident in hypertension (primary aldosteronism), low renin, and resistant hypertension) but also can exist in normotension. Whether chronic, mild aldosterone autonomy can elicit hypertension remains untested. Previously, we reported that global genetic deletion of 2 pore-domain TWIK-relative acid-sensitive potassium channels, TASK-1 and TASK-3, from mice produces striking aldosterone excess, low renin, and hypertension. Here, we deleted TASK-1 and TASK-3 channels selectively from zona glomerulosa cells and generated a model of mild aldosterone autonomy with attendant hypertension that is aldosterone-driven and Ang II (angiotensin II)-independent. This study shows that a zona glomerulosa-specific channel defect can produce mild autonomous hyperaldosteronism sufficient to cause chronic blood pressure elevation.

Overt dysregulation of the renin-angiotensin-aldosterone system (primary aldosteronism: APA and IHA) has long been known for its deleterious cardiovascular consequences. Recently, however, subclinical mild hyperaldosteronism was recognized as a significant risk factor for hypertension, and may represent an early stage in the disease spectrum. Previously, we developed mouse models of hyperaldosteronism by globally deleting subunits of the TWIK-related acid sensitive potassium channel (TASK). Global TASK-1 (Kcnk3) and TASK-3 (Kcnk9) KO mice markedly overproduce Ang II-independent (autonomous) aldosterone, that is not suppressed by high dietary sodium and is accompanied by low renin and elevated blood pressure. Here, we report a novel mouse model of mild hyperaldosteronism produced by the zona glomerulosa (zG)-specific deletion of TASK channels. To generate trigenic Cyp11b2Cre/+ :: Kcnk3f/f :: Kcnk9f/f mice (zG-TASK-KO), the Cyp11b2Cre/+ strain that expresses Cre-recombinase under the aldosterone synthase promoter was crossed with the Kcnk3f/f :: Kcnk9f/f strain. We confirmed restriction of Cre-expression to the zG layer by qRT-PCR and immunohistochemistry using a Cre-dependent reporter mouse (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J ). Despite normal plasma renin, zG-TASK-KO mice displayed a mild increase in 24-hr. urinary aldosterone excretion (11.8±0.8 KO vs 9.3±0.6 controls; ng/mg-aldosterone/creatinine, p<0.05) that was much less than the frank increase observed in global TASK-KO mice (42.6±6.9 ng/mg, aldosterone/creatinine, p<0.05). Hyperaldosteronism of zG-TASK-KO mice failed to normalize with AT1R antagonism (candesartan) or salt suppression (4% Na-diet). Twenty-four-hour blood pressure of conscious, freely behaving zG-TASK-KO mice was increased compared to control mice (systolic: 133.3±1.8 mmHg KO vs 122.7±1.2 mmHg controls, p<0.05) and failed to normalize with candesartan, but corrected with MR antagonism (spironolactone). This study demonstrates that a zG-specific channel defect can produce mild autonomous hyperaldosteronism sufficient to cause chronic aldosterone-driven and Angiotensin II-independent blood pressure elevation.

Chronic kidney disease (CKD) affects ~10% of the global population, with considerable ethnic differences in prevalence and aetiology. We assemble genome-wide association studies of estimated glomerular filtration rate (eGFR), a measure of kidney function that defines CKD, in 312,468 individuals of diverse ancestry. We identify 127 distinct association signals with homogeneous effects on eGFR across ancestries and enrichment in genomic annotations including kidney-specific histone modifications. Fine-mapping reveals 40 high-confidence variants driving eGFR associations and highlights putative causal genes with cell-type specific expression in glomerulus, and in proximal and distal nephron. Mendelian randomisation supports causal effects of eGFR on overall and cause-specific CKD, kidney stone formation, diastolic blood pressure and hypertension. These results define novel molecular mechanisms and putative causal genes for eGFR, offering insight into clinical outcomes and routes to CKD treatment development.

Background: Acute kidney injury (AKI) portends worse prognosis following sepsis, with limited available interventions. Host iron acquisition by pathogens and systemic inflammatory response are key events in the pathogenesis of sepsis. In sepsis, hepcidin induces iron sequestration to limit iron availability to pathogens. Hepcidin is also known to limit inflammation. Since its role in pathophysiology of sepsis-associated AKI is unknown, we investigated the effect of exogenous hepcidin in endotoxin- and peritonitis-induced pathology and AKI. Methods: C57BL/6 mice were treated with saline or 50-100 µg of hepcidin, pre- and post-LPS injection, or cecal ligation and puncture (CLP, model of peritonitis). Splenectomized mice were challenged with LPS, with and without hepcidin. Mice were euthanized at 24 h after LPS injection and at different time points after CLP. Systemic inflammation and renal injury markers were assessed. Direct effect of hepcidin on renal tubular and endothelial cells was evaluated using endotoxin-induced cytotoxic serum. Role of heavy chain ferritin (H-ferritin) in mediating hepcidin-induced anti-inflammatory effect on LPS stimulated macrophages was evaluated with siRNA studies. Results: Twenty-four hours pretreatment with hepcidin significantly reduced LPS-induced AKI. Hepcidin ameliorated LPS-induced increase in serum TNFα and renal Cox-2, and prevented loss in PGC1α and cytochrome c oxidase activity. This was associated with reduced glomerular injury and preserved mitochondrial structure. Hepcidin did not exert direct protection on the renal parenchymal cells but reduced endotoxin-induced serum cytotoxicity to mitigate renal injury. Splenectomy reduced LPS-induced early inflammation and AKI, independent of hepcidin, indicating the importance of systemic inflammation. Higher splenic H-ferritin in hepcidin-treated animals was associated with reduced splenocytes apoptosis and inflammation. Hepcidin reduced LPS-induced IL-6 secretion in macrophages in H-ferritin dependent manner. Hepcidin significantly reduced CLP-induced AKI, and mortality (20% hepcidin treated vs 80% PBS treated). Importantly hepcidin reduced bacteremia and AKI even when administered after onset of sepsis. Conclusion: We demonstrate a protective role of hepcidin in endotoxin- and peritonitis-induced pathologies and AKI, exerted primarily through its anti-inflammatory effects, and antibacterial property. Macrophage H-ferritin plays an important role in hepcidin-mediated protection against endotoxin-induced inflammation. We uncover a novel prophylactic and therapeutic role of hepcidin in sepsis-associated bacteremia, AKI, and mortality.

Glutathione- S -transferase μ-1 ( GSTM1 ) enzyme belongs to the superfamily of phase II antioxidant glutathione- S -transferases (GSTs) that are downstream targets of the Nrf2 antioxidant transcription factor. In humans, a common GSTM1 gene deletion variant, the null allele GSTM1(0) , results in decreased or complete absence of GSTM1 enzymatic activity and is associated with higher levels of oxidative stress. We reported that GSTM1(0) is associated with accelerated kidney disease progression in the African Americans Study of Kidney Disease (AASK) participants. To understand the direct impact of GSTM1 deficiency on renal inflammation and oxidative stress, we generated Gstm1 deficient mice (KO) to determine their response to angiotensin II, delivered @ 1000 ng/kg/min for 4 weeks via mini-osmotic pump. Blood pressure (BP) was measured by radiotelemetery. Kidney histopathology was assessed by a renal pathologist blinded to genotype and experimental conditions. Renal leukocyte populations were analyzed quantitatively by flow cytometry. Gstm1 KO mice had significantly higher levels of baseline systolic BP (SBP) and ~ 17 mmHg higher SBP after 4 weeks of Ang II-HTN, compared to WT mice. Gstm1 KO mice have increased renal superoxide levels by nearly 3 folds - independent of activation of Nox2 and Nox4 NADPH oxidases or alteration in superoxide dismutase - and worse kidney injury. These changes were associated with significantly increased renal expression of genes involved in inflammation - chemokine ligand 1 (CXCL-1), monocyte chemotactic protein 1 (MCP-1), Interleukin-1β (IL-1β) and IL-6 ( P <0.05). By flow cytometry, Gstm1 KO mice displayed ~ two fold increases in all leukocyte populations in the kidney, with the exception of the numbers of B cells that were not significantly different between groups. This increased renal inflammation was attenuated by dietary administration of broccoli powder that is rich in sulforaphane, a phytochemical that increases Nrf2 expression, independent of BP. In Ang II-hypertension, loss of GSTM1 enzyme may be deleterious by augmenting oxidative stress and inflammation in the kidney. Stimulation of the Nrf2 pathway in hypertension may be a novel therapeutic approach to prevent kidney disease progression in GSTM1 deficiency.

In humans, a common deletion variant of the GSTM1 gene, the GSTM1(0) null allele, results in decreased GSTM1 enzymatic activity and is associated with higher levels of oxidative stress. We reported that GSTM1(0) is associated with increased risks of chronic kidney disease (CKD) progression and death in the African Americans Study of Kidney Disease. To establish a causal relationship between GSTM1 deficiency and CKD progression, we deleted Gstm1 in the mouse, and determined its effect in the sub-total nephrectomy (Nx) model of CKD. After the 8 th week after Nx, 4 out of 8 Gstm1 knockout (KO) mice began to die, whereas wild-type (WT) mice had 100% survival by the study endpoint at 13 th week. Remaining KO mice had significantly higher plasma creatinine (p < 0.05), suggestive of lower GFR. Due to early mortality in KO mice after the 8th week after Nx, we phenotyped mice at the 8 th week (n=6 each). KO mice had significantly higher 1) mean systolic blood pressure (by radiotelemetry): KO 157.2 ± 5, WT 141.1 ± 2.4 mmHg, 2) urine albumin/creatinine ratio (μg/mg): KO 748.4 ± 187.9, WT 235.1 ± 24.6, and 3) renal superoxide (O2·-) levels (counts/min/mg dry tissue): KO 249.7 ± 63; WT 74.5 ± 14.7; P ≤ 0.02. Hydrogen peroxide levels were not different. Renal mRNA levels of the superoxide dismutase isoforms SOD1, SOD2, and SOD3 were significantly lower in KO mice (KO 7.78 ± 0.047, 0.446 ± 0.062, 0.18 ± 0.03; WT 10.64 ± 0.43, 2.432 ± 0.386, 1.188 ± 0.124, respectively, P ≤ 0.003. Total kidney pathology score (KO 18.7 ± 1.5; WT 8 ± 0.6; P <0.0001) showed that KO mice had worse kidney injury, with severe glomerulosclerosis, proteinaceous casts, and chronic inflammation. Both glomerular surface area (SA)/total non-infarcted kidney area (KO 3.347 ± 0.301; WT 2.286 ± 0.268; P = 0.02) and mesangial SA/glomerular SA (KO 35.62 ± 2.219; WT 23.6 ± 2.569; P = 0.005) were significantly increased in KO mice. GSTM1 has also been shown to suppress ASK1 - the apoptosis signal-regulating kinase 1 that induces apoptosis via activation of caspase-3. Consistent with activated ASK1 pathway, KO mice had significantly higher renal TUNEL staining and caspase-3 protein levels (KO 3.594 ± 0.327; WT 2.511 ± 0.208; P = 0.01). In conclusion, in CKD, loss of GSTM1 leads to deleterious outcome associated with increased O2·- levels and apoptosis.

There is emerging evidence that extracellular vesicles (EVs) may be novel bio-markers and bio-activators in the pathogenesis of HTN. We hypothesize that the phenotypes of EVs from the circulation and kidney are altered in Angiotensin II (AII) induced HTN and normalize with anti-hypertensive treatment (Rx). AII was delivered via osmotic mini pumps (500 ng/kg/min) alone, or with candesartan (C) @ 10 mg/kg/day, or with hydralazine, hydrochlorothiazide, reserpine (HHR) @ 30/10/0.2 mg/kg/day in drinking water for 2 weeks to 129S6 mice (n = 5 each). Systolic blood pressure (SBP) was measured daily for 2 weeks by tail-cuff manometry. After 2 weeks, mice were euthanized and citrated blood and kidneys collected. Kidney EVs (KEVs) were isolated after incubation with Collagenase type A and subsequent DC. Both circulating EVs and KEVs were isolated through differential centrifugation and characterized by imaging flow cytometry (AmnisImage-StreamX Mark II) using PECAM-1 (CD31), leukocyte (CD45), platelet (CD41) and endothelial (CD105) markers. AII treated mice had significantly higher SBP (mmHg) (162.05 ± 7.20) than normotensive controls ((N) 120.25 ± 7.12), p=0.02. SBP was similarly reduced with C and HHR (134.12 ± 6.15 and 122.45 ± 5.20, respectively, p=NS), and significantly lower than AII, p=0.0026 and =0.03, respectively). Circulating leukocyte derived EVs (LEVs, CD31+/CD45+) increased after 2 weeks in AII treated mice compared to N (AII 2.01E+0.3, N 2.04E+0.4 particles/mL; p=0.0130). This effect was not observed with endothelial and platelet derived EVs. Numbers of circulating LEVs correlated significantly with SBP [(r 2 =0.675); p=0.0029]. Circulating LEVs were reduced after equal normalization of SBP, though no difference in EVs was observed between Rx with C and HHR. CD45+ KEVs in AII treated mice were significantly increased compared to N (AII 7.52E+05 vs N 4.79E+04; p= 0.0257). KEVs also correlated significantly with SBP [(r 2 =0.682); p=0.0032]. In conclusion, circulating and kidney derived EVs may play a role in the immune response in HTN and may be influenced by blood pressure threshold, independent of AT 1 receptor activation. Further studies are needed to confirm and to characterize specific subtype(s) of LEVs and dissect their functional role.