A Reproducible Mouse Model of Moderate CKD With Early Manifestations of Osteoblastic Transition of Cardiovascular System.

Chronic kidney disease (CKD) is a significant public health challenge with a substantial associated risk of mortality, morbidity, and health care expenditure. Culprits that lead to development and progression of CKD are multifaceted and heterogenous in nature. This notion underscores the need for diversification of animal models to investigate its pathophysiology, related complications, and to subsequently enable discovery of novel therapeutics. Importantly, animal models that could recapitulate complications of CKD in both genders are desperately needed. Cardiovascular disease is the most common cause of death in CKD patients that may be due in part to high prevalence of vascular calcification (VC). Using DBA/2 mice that are susceptible to development of VC, we sought to investigate the feasibility and reproducibility of a unilateral ischemia-reperfusion model followed by contralateral nephrectomy (UIRI/Nx) to induce CKD and its related complications in female and male mice. Our results demonstrate that irrespective of gender, mice faithfully displayed complications of moderate CKD following UIRI/Nx as evidenced by significant rise in serum creatinine, albuminuria, higher degree of collagen deposition, elevated expression of classic fibrotic markers, higher circulating levels of FGF-23, PTH and hepcidin. Moreover, we corroborate the osteoblastic transition of aortic smooth muscle cells and cardiomyocytes based on higher levels of osteoblastic markers namely, Cbfa-1, osteopontin, osteocalcin, and osterix. Our data confirms a viable, and consistent model of moderate CKD and its associated complications in both male and female mice. Furthermore, early evidence of osteoblastic transition of cardiovascular system in this model confirms its suitability for studying and implementing potential preventive and/or therapeutic approaches that are urgently needed in this field.

Expanded renal lymphatics improve recovery following kidney injury.

Acute kidney injury (AKI) is a major cause of patient mortality and a major risk multiplier for the progression to chronic kidney disease (CKD). The mechanism of the AKI to CKD transition is complex but is likely mediated by the extent and length of the inflammatory response following the initial injury. Lymphatic vessels help to maintain tissue homeostasis through fluid, macromolecule, and immune modulation. Increased lymphatic growth, or lymphangiogenesis, often occurs during inflammation and plays a role in acute and chronic disease processes. What roles renal lymphatics and lymphangiogenesis play in AKI recovery and CKD progression remains largely unknown. To determine if the increased lymphatic density is protective in the response to kidney injury, we utilized a transgenic mouse model with inducible, kidney-specific overexpression of the lymphangiogenic protein vascular endothelial growth factor-D to expand renal lymphatics. "KidVD" mouse kidneys were injured using inducible podocyte apoptosis and proteinuria (POD-ATTAC) or bilateral ischemia reperfusion. In the acute injury phase of both models, KidVD mice demonstrated a similar loss of function measured by serum creatinine and glomerular filtration rate compared to their littermates. While the initial inflammatory response was similar, KidVD mice demonstrated a shift toward more CD4+ and fewer CD8+ T cells in the kidney. Reduced collagen deposition and improved functional recovery over time was also identified in KidVD mice. In KidVD-POD-ATTAC mice, an increased number of podocytes were counted at 28 days post-injury. These data demonstrate that increased lymphatic density prior to injury alters the injury recovery response and affords protection from CKD progression.

VEGFR3 tyrosine kinase inhibition aggravates cisplatin nephrotoxicity.

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity.NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

Quantitative 3-dimensional imaging and tissue cytometry reveals lymphatic expansion in acute kidney injury.

The lymphatic system plays an integral role in physiology and has recently been identified as a key player in disease progression. Tissue injury stimulates lymphatic expansion, or lymphangiogenesis (LA), though its precise role in disease processes remains unclear. LA is associated with inflammation, which is a key component of acute kidney injury (AKI), for which there are no approved therapies. While LA research has gained traction in the last decade, there exists a significant lack of understanding of this process in the kidney. Though innovative studies have elucidated markers and models with which to study LA, the field is still evolving with ways to visualize lymphatics in vivo. Prospero-related homeobox-1 (Prox-1) is the master regulator of LA and determines lymphatic cell fate through its action on vascular endothelial growth factor receptor expression. Here, we investigate the consequences of AKI on the abundance and distribution of lymphatic endothelial cells using Prox1-tdTomato reporter mice (ProxTom) coupled with large-scale three-dimensional quantitative imaging and tissue cytometry (3DTC). Using these technologies, we describe the spatial dynamics of lymphatic vasculature in quiescence and post-AKI. We also describe the use of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) as a marker of lymphatic vessels using 3DTC in the absence of the ProxTom reporter mice as an alternative approach. The use of 3DTC for lymphatic research presents a new avenue with which to study the origin and distribution of renal lymphatic vessels. These findings will enhance our understanding of renal lymphatic function during injury and could inform the development of novel therapeutics for intervention in AKI.

Lipoxygenase inhibitor ML351 dysregulated an innate inflammatory response leading to impaired cardiac repair in acute heart failure.

The presistent increase of 12/15 lipoxygenase enzyme activity is correlated with uncontrolled inflammation, leading to organ dysfunction. ML351, a potent 12/15 lipoxygenase (12/15LOX) inhibitor, was reported to reduce infarct size and inflammation in a murine ischemic stroke model. In the presented work, we have applied three complementary experimental approaches, in-vitro, ex-vivo, and in-vivo, to determine whether pharmacological inhibition of 12/15LOX could dampen the inflammatory response in adult mice after Kdo2-Lipid A (KLA) as an endotoxin stimulator or post myocardial infarction (MI). Male C57BL/6 (8-12 weeks) mice were subjected to permanent coronary ligation thereby inducing acute heart failure (MI-d1 and MI-d5) for in-vivo studies. 12/15LOX antagonist ML351 (50 mg/kg) was subcutaneously injected 2 h post-MI, while MI-controls received saline. For ex-vivo experiments, ML351 (25 mg/kg) was injected as bolus after 5 min of inflammatory stimulus (KLA 1 µg/g) injection. Peritoneal macrophages (PMɸ) were harvested after 4 h post KLA. For in-vitro studies, PMɸ were treated with KLA (100 ng/mL), ML351 (10 µM), or KLA + ML351 for 4 h, and inflammatory response was evaluated. In-vivo, 5LOX expression was reduced after ML351 administration, inducing a compensatory increase of 12LOX that sensitized PMɸ toward a proinflammatory state. This was marked by higher inflammatory cytokines and dysregulation of the splenocardiac axis post-MI. ML351 treatment increased CD11b+ and Ly6Chigh populations in spleen and Ly6G+ population in heart, with a decrease in F4/80+ macrophage population at MI-d1. In-vitro results indicated that ML351 suppressed initiation of inflammation while ex-vivo results suggested ML351 overactivated inflammation consequently delaying the resolution process. Collectively, in-vitro, ex-vivo, and in-vivo results indicated that pharmacological blockade of lipoxygenases using ML351 impaired initiation of inflammation thereby dysregulated acute immune response in cardiac repair.

Expression of lactate dehydrogenase A and B isoforms in the mouse kidney.

Cellular metabolic rates in the kidney are critical for maintaining normal renal function. In a hypoxic milieu, cells rely on glycolysis to meet energy needs, resulting in the generation of pyruvate and NADH. In the absence of oxidative phosphorylation, the continuation of glycolysis is dependent on the regeneration of NAD+ from NADH accompanied by the fermentation of pyruvate to lactate. This reaction is catalyzed by lactate dehydrogenase (LDH) isoform A (LDHA), whereas LDH isoform B (LDHB) catalyzes the opposite reaction. LDH is widely used as a potential injury marker as it is released from damaged cells into the urine and serum; however, the precise isoform-specific cellular localization of the enzyme along the nephron has not been characterized. By combining immunohistochemistry results and single-cell RNA-sequencing data on healthy mouse kidneys, we identified that LDHA is primarily expressed in proximal segments, whereas LDHB is expressed in the distal parts of the nephron. In vitro experiments in mouse and human renal proximal tubule cells showed an increase in LDHA following hypoxia with no change in LDHB. Using immunofluorescence, we observed that the overall expression of both LDHA and LDHB proteins decreased following renal ischemia-reperfusion injury as well as in the adenine-diet-induced model of chronic kidney disease. Single-nucleus RNA-sequencing analyses of kidneys following ischemia-reperfusion injury revealed a significant decline in the number of cells expressing detectable levels of Ldha and Ldhb; however, cells that were positive showed increased average expression postinjury, which subsided during the recovery phase. These data provide information on the cell-specific expression of LDHA and LDHB in the normal kidney as well as following acute and chronic kidney disease.NEW & NOTEWORTHY Cellular release of lactate dehydrogenase (LDH) is being used as an injury marker; however, the exact localization of LDH within the nephron remains unclear. We show that LDH isoform A is expressed proximally, whereas isoform B is expressed distally. Both subunit expressions were significantly altered in models of acute kidney injury and chronic kidney disease. Our study provides new insights into basal and postinjury renal lactate metabolism.

Renal Inflammation and Fibrosis: A Double-edged Sword.

Renal tissue injury initiates inflammatory and fibrotic processes that occur to promote regeneration and repair. After renal injury, damaged tissue releases cytokines and chemokines, which stimulate activation and infiltration of inflammatory cells to the kidney. Normal tissue repair processes occur simultaneously with activation of myofibroblasts, collagen deposition, and wound healing responses; however, prolonged activation of pro-inflammatory and pro-fibrotic cell types causes excess extracellular matrix deposition. This review focuses on the physiological and pathophysiological roles of specialized cell types, cytokines/chemokines, and growth factors, and their implications in recovery or exacerbation of acute kidney injury.

Dynamic signature of lymphangiogenesis during acute kidney injury and chronic kidney disease.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are interconnected syndromes with significant attributable morbidity and mortality. The disturbing trend of increasing incidence and prevalence of these clinical disorders highlights the urgent need for better understanding of the underlying mechanisms that are involved in pathogenesis of these conditions. Lymphangiogenesis and its involvement in various inflammatory conditions is increasingly recognized while its role in AKI and CKD remains to be fully elucidated. Here, we studied lymphangiogenesis in three models of kidney injury. Our results demonstrate that the main ligands for lymphangiogenesis, VEGF-C and VEGF-D, are abundantly present in tubules at baseline conditions and the expression pattern of these ligands is significantly altered following injury. In addition, we show that both of these ligands increase in serum and urine post-injury and suggest that such increment may serve as novel urinary biomarkers of AKI as well as in progression of kidney disease. We also provide evidence that irrespective of the nature of initial insult, lymphangiogenic pathways are rapidly and robustly induced as evidenced by higher expression of lymphatic markers within the kidney.

Ferritin Light Chain Confers Protection Against Sepsis-Induced Inflammation and Organ Injury.

Despite the prevalence and recognition of its detrimental impact, clinical complications of sepsis remain a major challenge. Here, we investigated the effects of myeloid ferritin heavy chain (FtH) in regulating the pathogenic sequelae of sepsis. We demonstrate that deletion of myeloid FtH leads to protection against lipopolysaccharide-induced endotoxemia and cecal ligation and puncture (CLP)-induced model of sepsis as evidenced by reduced cytokine levels, multi-organ dysfunction and mortality. We identified that such protection is predominantly mediated by the compensatory increase in circulating ferritin (ferritin light chain; FtL) in the absence of myeloid FtH. Our in vitro and in vivo studies indicate that prior exposure to ferritin light chain restrains an otherwise dysregulated response to infection. These findings are mediated by an inhibitory action of FtL on NF-κB activation, a key signaling pathway that is implicated in the pathogenesis of sepsis. We further identified that LPS mediated activation of MAPK pathways, specifically, JNK, and ERK were also reduced with FtL pre-treatment. Taken together, our findings elucidate a crucial immunomodulatory function for circulating ferritin that challenges the traditional view of this protein as a mere marker of body iron stores. Accordingly, these findings will stimulate investigations to the adaptive nature of this protein in diverse clinical settings.

Resident macrophages reprogram toward a developmental state after acute kidney injury.

Acute kidney injury (AKI) is a devastating clinical condition affecting at least two-thirds of critically ill patients, and, among these patients, it is associated with a greater than 60% risk of mortality. Kidney mononuclear phagocytes (MPs) are implicated in pathogenesis and healing in mouse models of AKI and, thus, have been the subject of investigation as potential targets for clinical intervention. We have determined that, after injury, F4/80hi-expressing kidney-resident macrophages (KRMs) are a distinct cellular subpopulation that does not differentiate from nonresident infiltrating MPs. However, if KRMs are depleted using polyinosinic/polycytidylic acid (poly I:C), they can be reconstituted from bone marrow-derived precursors. Further, KRMs lack major histocompatibility complex class II (MHCII) expression before P7 but upregulate it over the next 14 days. This MHCII- KRM phenotype reappears after injury. RNA sequencing shows that injury causes transcriptional reprogramming of KRMs such that they more closely resemble that found at P7. KRMs after injury are also enriched in Wingless-type MMTV integration site family (Wnt) signaling, indicating that a pathway vital for mouse and human kidney development is active. These data indicate that mechanisms involved in kidney development may be functioning after injury in KRMs.

Paradigm shift - Metabolic transformation of docosahexaenoic and eicosapentaenoic acids to bioactives exemplify the promise of fatty acid drug discovery.

Fatty acid drug discovery (FADD) is defined as the identification of novel, specialized bioactive mediators that are derived from fatty acids and have precise pharmacological/therapeutic potential. A number of reports indicate that dietary intake of omega-3 fatty acids and limited intake of omega-6 promotes overall health benefits. In 1929, Burr and Burr indicated the significant role of essential fatty acids for survival and functional health of many organs. In reference to specific dietary benefits of differential omega-3 fatty acids, docosahexaenoic and eicosapentaenoic acids (DHA and EPA) are transformed to monohydroxy, dihydroxy, trihydroxy, and other complex mediators during infection, injury, and exercise to resolve inflammation. The presented FADD approach describes the metabolic transformation of DHA and EPA in response to injury, infection, and exercise to govern uncontrolled inflammation. Metabolic transformation of DHA and EPA into a number of pro-resolving molecules exemplifies a novel, inexpensive approach compared to traditional, expensive drug discovery. DHA and EPA have been recommended for prevention of cardiovascular disease since 1970. Therefore, the FADD approach is relevant to cardiovascular disease and resolution of inflammation in many injury models. Future research demands identification of novel action targets, receptors for biomolecules, mechanism(s), and drug-interactions with resolvins in order to maintain homeostasis.

Aging dysregulates D- and E-series resolvins to modulate cardiosplenic and cardiorenal network following myocardial infarction.

Post-myocardial infarction (MI), overactive inflammation is the hallmark of aging, however, the mechanism is unclear. We hypothesized that excess influx of omega 6 fatty acids may impair resolution, thus impacting the cardiosplenic and cardiorenal network post-MI. Young and aging mice were fed on standard lab chow (LC) and excess fatty acid (safflower oil; SO)-enriched diet for 2 months and were then subjected to MI surgery. Despite similar infarct areas and left ventricle (LV) dysfunction post-MI, splenic mass spectrometry data revealed higher levels of arachidonic acid (AA) derived pro-inflammatory metabolites in young-SO, but minimal formation of docosanoids, D- and E- series resolvins in SO-fed aged mice. The aged mice receiving excess intake of fatty acids exhibit; 1) decreased lipoxygenases (5-,12-, and 15) in the infarcted LV; 2) lower levels of 14HDHA, RvD1, RvD5, protectin D1, 7(S)maresin1, 8-,11-,18-HEPE and RvE3 with high levels of tetranor-12-HETEs; 3) dual population of macrophages (CD11blow/F480high and CD11bhigh/F480high) with increased pro-inflammatory (CD11bp+F4/80+Ly6Chi) phenotype and; 4) increased kidney injury marker NGAL with increased expression of TNF-α and IL-1ß indicating MI-induced non-resolving response compared with LC-group. Thus, excess fatty acid intake magnifies the post-MI chemokine signaling and inflames the cardiosplenic and cardiorenal network towards a non-resolving microenvironment in aging.