Distinct developmental reprogramming footprint of macrophages during acute kidney injury across species.

Acute kidney injury (AKI) is a common finding in hospitalized patients, particularly those who are critically ill. The development of AKI is associated with several adverse outcomes including mortality, morbidity, progression to chronic kidney disease, and an increase in healthcare expenditure. Despite the well-established negative impact of AKI and rigorous efforts to better define, identify, and implement targeted therapies, the overall approach to the treatment of AKI continues to principally encompass supportive measures. This enduring challenge is primarily due to the heterogeneous nature of insults that activate many independent and overlapping molecular pathways. Consequently, it is evident that the identification of common mechanisms that mediate the pathogenesis of AKI, independent of etiology and engaged pathophysiological pathways, is of paramount importance and could lead to the identification of novel therapeutic targets. To better distinguish the commonly modulated mechanisms of AKI, we explored the transcriptional characteristics of human kidney biopsies from patients with acute tubular necrosis (ATN), and acute interstitial nephritis (AIN) using a NanoString inflammation panel. Subsequently, we used publicly available single-cell transcriptional resources to better interpret the generated transcriptional findings. Our findings identify robust acute kidney injury (AKI-induced) developmental reprogramming of macrophages (MΦ) with the expansion of C1Q+, CD163+ MΦ that is independent of the etiology of AKI and conserved across mouse and human species. These results would expand the current understanding of the pathophysiology of AKI and potentially offer novel targets for additional studies to enhance the translational transition of AKI research.NEW & NOTEWORTHY Our findings identify robust acute kidney injury (AKI)-induced developmental reprogramming of macrophages (MΦ) with the expansion of C1Q+, CD163+ MΦ that is independent of the etiology of AKI and conserved across mouse and human species.

Cognate antigen-independent differentiation of resident memory T cells in chronic kidney disease.

Resident memory T cells (TRMs), which are memory T cells that are retained locally within tissues, have recently been described as antigen-specific frontline defenders against pathogens in barrier and nonbarrier epithelial tissues. They have also been noted for perpetuating chronic inflammation. The conditions responsible for TRM differentiation are still poorly understood, and their contributions, if any, to sterile models of chronic kidney disease (CKD) remain a mystery. In this study, we subjected male C57BL/6J mice and OT-1 transgenic mice to five consecutive days of 2 mg/kg aristolochic acid (AA) injections intraperitoneally to induce CKD or saline injections as a control. We evaluated their kidney immune profiles at 2 wk, 6 wk, and 6 mo after treatment. We identified a substantial population of TRMs in the kidneys of mice with AA-induced CKD. Flow cytometry of injured kidneys showed T cells bearing TRM surface markers and single-cell (sc) RNA sequencing revealed these cells as expressing well-known TRM transcription factors and receptors responsible for TRM differentiation and maintenance. Although kidney TRMs expressed Cd44, a marker of antigen experience and T cell activation, their derivation was independent of cognate antigen-T cell receptor interactions, as the kidneys of transgenic OT-1 mice still harbored considerable proportions of TRMs after injury. Our results suggest a nonantigen-specific or antigen-independent mechanism capable of generating TRMs in the kidney and highlight the need to better understand TRMs and their involvement in CKD.NEW & NOTEWORTHY Resident memory T cells (TRMs) differentiate and are retained within the kidneys of mice with aristolochic acid (AA)-induced chronic kidney disease (CKD). Here, we characterized this kidney TRM population and demonstrated TRM derivation in the kidneys of OT-1 transgenic mice with AA-induced CKD. A better understanding of TRMs and the processes by which they can differentiate independent of antigen may help our understanding of the interactions between the immune system and kidneys.

HSP90, a Common Therapeutic Target for Suppressing Skin Injury Caused by Exposure to Chemically Diverse Classes of Blistering Agents.

Vesicants such as arsenicals and mustards produce highly painful cutaneous inflammatory and blistering responses, hence developed as chemical weapons during World War I/II. Here, using lewisite and sulfur mustard surrogates, namely phenylarsine oxide (PAO) and 2-chloroethyl ethyl sulfide (CEES), respectively, we defined a common underlying mechanism of toxic action by these two distinct classes of vesicants. Murine skin exposure to these chemicals causes tissue destruction characterized by increase in skin bifold thickness, Draize score, infiltration of inflammatory cells, and apoptosis of epidermal and dermal cells. RNA sequencing analysis identified ∼346 inflammatory genes that were commonly altered by both PAO and CEES, along with the identification of cytokine signaling activation as the top canonical pathway. Activation of several proinflammatory genes and pathways is associated with phosphorylation-dependent activation of heat shock protein 90α (p-HSP90α). Topical treatment with known HSP90 inhibitors SNX-5422 and IPI-504 post PAO or CEES skin challenge significantly attenuated skin damage including reduction in overall skin injury and clinical scores. In addition, highly upregulated inflammatory genes Saa3, Cxcl1, Ccl7, IL-6, Nlrp3, Csf3, Chil3, etc. by both PAO and CEES were significantly diminished by treatment with HSP90 inhibitors. These drugs not only reduced PAO- or CEES-induced p-HSP90α expression but also its client proteins NLRP3 and pP38 and the expression of their target inflammatory genes. Our data confirm a critical role of HSP90 as a shared underlying molecular target of toxicity by these two distinct vesicants and provide an effective and novel medical countermeasure to suppress vesicant-induced skin injury. SIGNIFICANCE STATEMENT: Development of effective and novel mechanism-based antidotes that can simultaneously block cutaneous toxic manifestations of distinct vesicants is important and urgently needed. Due to difficulties in determining the exact nature of onsite chemical exposure, a potent drug that can suppress widespread cutaneous damage may find great utility. Thus, this study identified HSP90 as a common molecular regulator of cutaneous inflammation and injury by two distinct warfare vesicants, arsenicals and mustards, and HSP90 inhibitors afford significant protection against skin damage.

Cutaneous Arsenical Exposure Induces Distinct Metabolic Transcriptional Alterations of Kidney Cells.

Arsenicals are deadly chemical warfare agents that primarily cause death through systemic capillary fluid leakage and hypovolemic shock. Arsenical exposure is also known to cause acute kidney injury, a condition that contributes to arsenical-associated death due to the necessity of the kidney in maintaining whole-body fluid homeostasis. Because of the global health risk that arsenicals pose, a nuanced understanding of how arsenical exposure can lead to kidney injury is needed. We used a nontargeted transcriptional approach to evaluate the effects of cutaneous exposure to phenylarsine oxide, a common arsenical, in a murine model. Here we identified an upregulation of metabolic pathways such as fatty acid oxidation, fatty acid biosynthesis, and peroxisome proliferator-activated receptor (PPAR)-α signaling in proximal tubule epithelial cell and endothelial cell clusters. We also revealed highly upregulated genes such as Zbtb16, Cyp4a14, and Pdk4, which are involved in metabolism and metabolic switching and may serve as future therapeutic targets. The ability of arsenicals to inhibit enzymes such as pyruvate dehydrogenase has been previously described in vitro. This, along with our own data, led us to conclude that arsenical-induced acute kidney injury may be due to a metabolic impairment in proximal tubule and endothelial cells and that ameliorating these metabolic effects may lead to the development of life-saving therapies. SIGNIFICANCE STATEMENT: In this study, we demonstrate that cutaneous arsenical exposure leads to a transcriptional shift enhancing fatty acid metabolism in kidney cells, indicating that metabolic alterations might mechanistically link topical arsenical exposure to acute kidney injury. Targeting metabolic pathways may generate promising novel therapeutic approaches in combating arsenical-induced acute kidney injury.

Heme Oxygenases in Cardiovascular Health and Disease.

Heme oxygenases are composed of two isozymes, Hmox1 and Hmox2, that catalyze the degradation of heme to carbon monoxide (CO), ferrous iron, and biliverdin, the latter of which is subsequently converted to bilirubin. While initially considered to be waste products, CO and biliverdin/bilirubin have been shown over the last 20 years to modulate key cellular processes, such as inflammation, cell proliferation, and apoptosis, as well as antioxidant defense. This shift in paradigm has led to the importance of heme oxygenases and their products in cell physiology now being well accepted. The identification of the two human cases thus far of heme oxygenase deficiency and the generation of mice deficient in Hmox1 or Hmox2 have reiterated a role for these enzymes in both normal cell function and disease pathogenesis, especially in the context of cardiovascular disease. This review covers the current knowledge on the function of both Hmox1 and Hmox2 at both a cellular and tissue level in the cardiovascular system. Initially, the roles of heme oxygenases in vascular health and the regulation of processes central to vascular diseases are outlined, followed by an evaluation of the role(s) of Hmox1 and Hmox2 in various diseases such as atherosclerosis, intimal hyperplasia, myocardial infarction, and angiogenesis. Finally, the therapeutic potential of heme oxygenases and their products are examined in a cardiovascular disease context, with a focus on how the knowledge we have gained on these enzymes may be capitalized in future clinical studies.

Fenfluramine provides clinically meaningful reduction in frequency of drop seizures in patients with Lennox-Gastaut syndrome: Interim analysis of an open-label extension study.

OBJECTIVE: This study was undertaken to evaluate the long-term safety and effectiveness of fenfluramine in patients with Lennox-Gastaut syndrome (LGS). METHODS: Eligible patients with LGS who completed a 14-week phase 3 randomized clinical trial enrolled in an open-label extension (OLE; NCT03355209). All patients were initially started on .2 mg/kg/day fenfluramine and after 1 month were titrated by effectiveness and tolerability, which were assessed at 3-month intervals. The protocol-specified treatment duration was 12 months, but COVID-19-related delays resulted in 142 patients completing their final visit after 12 months. RESULTS: As of October 19, 2020, 247 patients were enrolled in the OLE. Mean age was 14.3 ± 7.6 years (79 [32%] adults) and median fenfluramine treatment duration was 364 days; 88.3% of patients received 2-4 concomitant antiseizure medications. Median percentage change in monthly drop seizure frequency was -28.6% over the entire OLE (n = 241) and -50.5% at Month 15 (n = 142, p < .0001); 75 of 241 patients (31.1%) experienced ≥50% reduction in drop seizure frequency. Median percentage change in nondrop seizure frequency was -45.9% (n = 192, p = .0038). Generalized tonic-clonic seizures (GTCS) and tonic seizures were most responsive to treatment, with median reductions over the entire OLE of 48.8% (p < .0001, n = 106) and 35.8% (p < .0001, n = 186), respectively. A total of 37.6% (95% confidence interval [CI] = 31.4%-44.1%, n = 237) of investigators and 35.2% of caregivers (95% CI = 29.1%-41.8%, n = 230) rated patients as Much Improved/Very Much Improved on the Clinical Global Impression of Improvement scale. The most frequent treatment-emergent adverse events were decreased appetite (16.2%) and fatigue (13.4%). No cases of valvular heart disease (VHD) or pulmonary arterial hypertension (PAH) were observed. SIGNIFICANCE: Patients with LGS experienced sustained reductions in drop seizure frequency on fenfluramine treatment, with a particularly robust reduction in frequency of GTCS, the key risk factor for sudden unexpected death in epilepsy. Fenfluramine was generally well tolerated; VHD or PAH was not observed long-term. Fenfluramine may provide an important long-term treatment option for LGS.

Fenfluramine in the treatment of Dravet syndrome: Results of a third randomized, placebo-controlled clinical trial.

OBJECTIVE: This study was undertaken to assess the safety and efficacy of fenfluramine in the treatment of convulsive seizures in patients with Dravet syndrome. METHODS: This multicenter, randomized, double-blind, placebo-controlled, parallel-group, phase 3 clinical trial enrolled patients with Dravet syndrome, aged 2-18 years with poorly controlled convulsive seizures, provided they were not also receiving stiripentol. Eligible patients who had ≥6 convulsive seizures during the 6-week baseline period were randomized to placebo, fenfluramine .2 mg/kg/day, or fenfluramine .7 mg/kg/day (1:1:1 ratio) administered orally (maximum dose = 26 mg/day). Doses were titrated over 2 weeks and maintained for an additional 12 weeks. The primary endpoint was a comparison of the monthly convulsive seizure frequency (MCSF) during baseline and during the combined titration-maintenance period in patients given fenfluramine .7 mg/kg/day versus patients given placebo. RESULTS: A total of 169 patients were screened, and 143 were randomized to treatment. Mean age was 9.3 ± 4.7 years (±SD), 51% were male, and median baseline MCSF in the three groups ranged 12.7-18.0 per 28 days. Patients treated with fenfluramine .7 mg/kg/day demonstrated a 64.8% (95% confidence interval = 51.8%-74.2%) greater reduction in MCSF compared with placebo (p < .0001). Following fenfluramine .7 mg/kg/day, 72.9% of patients had a ≥50% reduction in MCSF compared with 6.3% in the placebo group (p < .0001). The median longest seizure-free interval was 30 days in the fenfluramine .7 mg/kg/day group compared with 10 days in the placebo group (p < .0001). The most common adverse events (>15% in any group) were decreased appetite, somnolence, pyrexia, and decreased blood glucose. All occurred in higher frequency in fenfluramine groups than placebo. No evidence of valvular heart disease or pulmonary artery hypertension was detected. SIGNIFICANCE: The results of this third phase 3 clinical trial provide further evidence of the magnitude and durability of the antiseizure response of fenfluramine in children with Dravet syndrome.

Pulmonary pathogenesis in a murine model of inhaled arsenical exposure.

Arsenic trioxide (ATO), an inorganic arsenical, is a toxic environmental contaminant. It is also a widely used chemical with industrial and medicinal uses. Significant public health risk exists from its intentional or accidental exposure. The pulmonary pathology of acute high dose exposure is not well defined. We developed and characterized a murine model of a single inhaled exposure to ATO, which was evaluated 24 h post-exposure. ATO caused hypoxemia as demonstrated by arterial blood-gas measurements. ATO administration caused disruption of alveolar-capillary membrane as shown by increase in total protein and IgM in the bronchoalveolar lavage fluid (BALF) supernatant and an onset of pulmonary edema. BALF of ATO-exposed mice had increased HMGB1, a damage-associated molecular pattern (DAMP) molecule, and differential cell counts revealed increased neutrophils. BALF supernatant also showed an increase in protein levels of eotaxin/CCL-11 and MCP-3/CCL-7 and a reduction in IL-10, IL-19, IFN-γ, and IL-2. In the lung of ATO-exposed mice, increased protein levels of G-CSF, CXCL-5, and CCL-11 were noted. Increased mRNA levels of TNF-a, and CCL2 in ATO-challenged lungs further supported an inflammatory pathogenesis. Neutrophils were increased in the blood of ATO-exposed animals. Pulmonary function was also evaluated using flexiVent. Consistent with an acute lung injury phenotype, respiratory and lung elastance showed significant increase in ATO-exposed mice. PV loops showed a downward shift and a decrease in inspiratory capacity in the ATO mice. Flow-volume curves showed a decrease in FEV0.1 and FEF50. These results demonstrate that inhaled ATO leads to pulmonary damage and characteristic dysfunctions resembling ARDS in humans.

A Comprehensive Immune Cell Atlas of Cystic Kidney Disease Reveals the Involvement of Adaptive Immune Cells in Injury-Mediated Cyst Progression in Mice.

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45+ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Iron-rich coal fly ash-polydopamine-silver nanocomposite (IRCFA-PDA-Ag NPs): tailored material for remediation of methylene blue dye from aqueous solution.

Water pollution has become one of the most acute environmental problems. One of the pollutants coming to water bodies from industries are dyes, which are harmful to human health, living organisms, and the esthetic appearance of water. Most dyes are toxic, carcinogenic, rarely biodegradable, and highly soluble in water. Therefore, industrial wastewater treatment has become important. Adsorption technique of removal of dyes from water is simple, efficient, and inexpensive as compared to other techniques. Adsorption efficiency depends on the type and surface area of adsorbents. Iron-rich coal fly ash (IRCFA)-Polydopamine (PDA)@ Silver (Ag) nanocomposite was prepared by separating the iron-rich part (IRCFA) from coal fly ash and coated with polydopamine. IRCFA was mixed with 10 mM tris buffer solution containing 1 g dopamine. The prepared IRCFA-PDA was added to an aqueous solution of silver nitrate, heated at 60 °C, and then 30 mL of flower waste extract was added to this solution. Solid IRCFA-PDA@Ag was obtained, and the prepared nanocomposite was used for the removal of methylene blue (MB) dye from water. The nanocomposite used was prepared by a cost-effective method and has high reusability, separability, and fast regeneration ability. The mechanism of removal of MB dye has been discussed in detail.

Floral waste synthesized silver nanoparticles as sensor for Cr (VI) ion detection.

The presence of heavy metal ions, particularly Cr (VI) in water, is a serious environmental concern. There is a need to develop low-cost and efficient methods for sensing and removing Cr (IV) ions selectively. In this paper, floral waste (FW) extract is used for the synthesis of Ag NPs for sensing Cr (VI) ions in an aqueous solution. Silver nanoparticles (Ag-NPs) were characterized using spectroscopic (UV-visible and FTIR), X-ray diffraction, TEM, and SEM techniques. UV-visible spectroscopic studies revealed that in the presence of Cr (VI) ions, there is an abrupt change in the λmax value of Ag NPs in aqueous solution, indicating that the synthesized Ag-NP is an excellent sensor for the spectroscopical detection of Cr (VI) ions with a low detection limit of 0.5 ppm. The method is fast, economical, simple, and efficient.

Estimating Vitamin D threshold for the Indian population: Delving into the actual disease burden.

Background: Based on the current guidelines in practice, a vast majority of the healthy Indian population is vitamin D deficient. Since the serum 25 hydroxycholecalciferol (25HCC) levels are affected by race and skin pigmentation, the normal range of vitamin D may differ in the Indians compared to the Western population. This study attempted to determine a population-specific threshold for 25 HCC levels associated with adequate bone health and calcium and phosphate homeostasis in healthy Indians. Methods: Subjects aged 20-50 years were included in the study. The exclusion criteria were obesity, chronic renal disease, liver failure, diabetes mellitus, thyroid disorders, a recent history of fracture, constant joint pain, and postmenopausal status. In addition, participants on prescribed medication such as glucocorticoids, anticonvulsants, or antifungals, as well as vitamin D and calcium supplementation, were also excluded.Blood samples were analyzed for serum calcium, phosphate, alkaline phosphatase, 25HCC, 1,25dihydroxycholecalciferol, parathyroid hormone (PTH), procollagen type-I N propeptide, and C-terminal telopeptide of type 1 collagen.Locally estimated smoothing scatter plot (LOESS) curves and Spearman correlation were utilized to study the correlation of all the biochemical parameters with 25 HCC to achieve thresholds. Results: The study consisted of 270 healthy participants, out of which 97.8% were found to have vitamin D levels below 30 ng/ml. In addition, 8.8% had raised PTH, and 1.85% had hypocalcemia. Furthermore, 1.48% had raised serum alkaline phosphatase and hypophosphatemia, respectively. A weak inverse correlation was seen between 25 HCC and PTH (rs = -0.437, p < 0.001), as well as alkaline phosphatase (rs = -0.1475, p = 0.015), while a weak positive correlation was seen with serum phosphate (rs = 0.128, p = 0.047). Conclusion: For a healthy Indian population, the reference range of 25 HCC is much lower, and the lower limit of normal is approximately 13.5 ng/ml. This study indicates that vitamin D insufficiency in this population starts at 25 HCC values of 13.5 ng/ml and deficiency at 7 ng/ml.

Development of BRD4 inhibitors as anti-inflammatory agents and antidotes for arsenicals.

Arsenicals belong to the class of chemical warfare agents known as vesicants, which are highly reactive, toxic and cause robust inflammatory response. Cutaneous exposure to arsenicals causes a wide range of systemic organ damage, beginning with cutaneous injuries, and later manifest multi-organ damage and death. Thus, the development of suitable antidotes that can effectively block injury following exposure to these agents is of great importance. Bromodomain 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) family, plays crucial role in regulating transcription of inflammatory, proliferation and cell cycle genes. In this context, the development of potent small molecule inhibitors of BRD4 could serve as potential antidotes for arsenicals. Herein, we describe the synthesis and biological evaluation of a series of compounds.

Renal control of disease tolerance to malaria.

Malaria, the disease caused by Plasmodium spp. infection, remains a major global cause of morbidity and mortality. Host protection from malaria relies on immune-driven resistance mechanisms that kill Plasmodium However, these mechanisms are not sufficient per se to avoid the development of severe forms of disease. This is accomplished instead via the establishment of disease tolerance to malaria, a defense strategy that does not target Plasmodium directly. Here we demonstrate that the establishment of disease tolerance to malaria relies on a tissue damage-control mechanism that operates specifically in renal proximal tubule epithelial cells (RPTEC). This protective response relies on the induction of heme oxygenase-1 (HMOX1; HO-1) and ferritin H chain (FTH) via a mechanism that involves the transcription-factor nuclear-factor E2-related factor-2 (NRF2). As it accumulates in plasma and urine during the blood stage of Plasmodium infection, labile heme is detoxified in RPTEC by HO-1 and FTH, preventing the development of acute kidney injury, a clinical hallmark of severe malaria.

Kidney resident macrophages in the rat have minimal turnover and replacement by blood monocytes.

Kidney resident macrophages (KRMs) are involved in maintaining renal homeostasis and in controlling the pathological outcome of acute kidney injury and cystic kidney disease in mice. In adult mice, KRMs maintain their population through self-renewal with little or no input from the peripheral blood. Despite recent data suggesting that a transcriptionally similar population of KRM-like cells is present across species, the idea that they are self-renewing and minimally dependent on peripheral blood input in other species has yet to be proven due to the lack of an appropriate model and cross-species expression markers. In this study, we used our recently identified cross-species KRM cell surface markers and parabiosis surgery in inbred Lewis rats to determine if rat KRMs are maintained independent of peripheral blood input, similar to their mouse counterparts. Flow cytometry analysis indicated that parabiosis surgery in the rat results in the establishment of chimerism of T/B cells, neutrophils, and monocyte-derived infiltrating macrophages in the blood, spleen, and kidney 3 wk after parabiosis surgery. Analysis of KRMs using the cell surface markers CD81 and C1q indicated that these cells have minimal chimerism and, therefore, receive little input from the peripheral blood. These data indicate that KRM properties are conserved in at least two different species.NEW & NOTEWORTHY In this report, we performed parabiosis surgery on inbred Lewis rats and showed that rat kidney resident macrophages (KRMs), identified using our novel cross-species markers, are minimally dependent on peripheral blood input. Thus, for the first time, to our knowledge, we confirm that a hallmark of mouse KRMs is also present in KRMs isolated from another species.

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.

UAB-UCSD O'Brien Center for Acute Kidney Injury Research.

Acute kidney injury (AKI) remains a significant clinical problem through its diverse etiologies, the challenges of robust measurements of injury and recovery, and its progression to chronic kidney disease (CKD). Bridging the gap in our knowledge of this disorder requires bringing together not only the technical resources for research but also the investigators currently endeavoring to expand our knowledge and those who might bring novel ideas and expertise to this important challenge. The University of Alabama at Birmingham-University of California-San Diego O'Brien Center for Acute Kidney Injury Research brings together technical expertise and programmatic and educational efforts to advance our knowledge in these diverse issues and the required infrastructure to develop areas of novel exploration. Since its inception in 2008, this O'Brien Center has grown its impact by providing state-of-the-art resources in clinical and preclinical modeling of AKI, a bioanalytical core that facilitates measurement of critical biomarkers, including serum creatinine via LC-MS/MS among others, and a biostatistical resource that assists from design to analysis. Through these core resources and with additional educational efforts, our center has grown its investigator base to include >200 members from 51 institutions. Importantly, this center has translated its pilot and catalyst funding program with a $37 return per dollar invested. Over 500 publications have resulted from the support provided with a relative citation ratio of 2.18 ± 0.12 (iCite). Through its efforts, this disease-centric O'Brien Center is providing the infrastructure and focus to help the development of the next generation of researchers in the basic and clinical science of AKI. This center creates the promise of the application at the bedside of the advances in AKI made by current and future investigators.

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.

Association of cystic fibrosis transmembrane conductance regulator with epithelial sodium channel subunits carrying Liddle's syndrome mutations.

The association of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) in the pathophysiology of cystic fibrosis (CF) is controversial. Previously, we demonstrated a close physical association between wild-type (WT) CFTR and WT ENaC. We have also shown that the F508del CFTR fails to associate with ENaC unless the mutant protein is rescued pharmacologically or by low temperature. In this study, we present the evidence for a direct physical association between WT CFTR and ENaC subunits carrying Liddle's syndrome mutations. We show that all three ENaC subunits bearing Liddle's syndrome mutations (both point mutations and the complete truncation of the carboxy terminus), could be coimmunoprecipitated with WT CFTR. The biochemical studies were complemented by fluorescence lifetime imaging microscopy (FLIM), a distance-dependent approach that monitors protein-protein interactions between fluorescently labeled molecules. Our measurements revealed significantly increased fluorescence resonance energy transfer between CFTR and all tested ENaC combinations as compared with controls (ECFP and EYFP cotransfected cells). Our findings are consistent with the notion that CFTR and ENaC are within reach of each other even in the setting of Liddle's syndrome mutations, suggestive of a direct intermolecular interaction between these two proteins.

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.