Murine Kidney Research Articles

FOXI1 Promotes Expression of V‐ATPase and GPR116 in M‐1 Cells

G protein‐coupled receptors (GPCRs) are a diverse family of integral membrane proteins that have significant roles in numerous physiological systems. We previously reported that GPR116 (ADGRF5), an adhesion‐class GPCR, is a critical regulator of vacuolar‐type H+‐ATPase (V‐ATPase) surface expression in A‐type intercalated cells (AICs) in mouse kidney cortical collecting ducts. The V‐ATPase is a multi‐subunit proton pump that localizes to the plasma membrane in specialized acid‐secreting epithelial cells. FOXI1 is a transcription factor that regulates V‐ATPase expression in ICs and other mitochondria‐rich cells. Recently, FOXI1 was identified as a key regulator of CFTR‐rich pulmonary ionocytes which express both V‐ATPase and GPR116. Therefore, we hypothesized that FOXI1 is a transcription factor in AICs that upregulates V‐ATPase as well as GPR116. To test this hypothesis, we cloned FOXI1 from whole mouse kidney into a pME18s expression vector (pFOXI1). Transfection with pFOXI1 significantly increased GPR116 expression (qPCR) in M‐1 mouse cortical collecting duct cells (n=3, ΔCt ±SEM: untransfected = 13.8±0.6, pFoxi1 = 10.7±0.2, p<0.05), but not in HEK293T cells (n=3, not significant). RNA‐scope hybridization on murine kidney sections and pFOXI1‐transfected M‐1 cells revealed colocalization of FOXI1 and GPR116 mRNA in the same cell. Treating pFOXI1‐transfected M‐1 cells with a synthetic agonist peptide for GPR116 results in an increase of [Ca2+]iin a subset of cells (n=3, ΔF340/380 ±SEM: pFoxi1 = 0.296±0.017). Internal calcium mobilization is never seen with the agonist peptide in untransfected control cells, indicating that pFOXI1 is sufficient for the production of physiologically responsive GPR116 on the cell surface (n=3, ΔF340/380±SEM: untransfected = 0 ±0.0009). Moreover, only M‐1 cells transfected with pFOXI1 express GPR110 (ADGRF1), an adhesion‐GPCR with unknown function in the kidney, as well as three V‐ATPase subunits, ATP6V1B1, ATP6V1G3, ATP6V0D2, indicating a larger transcriptional program initiated by pFOXI1 (n=3, untransfected: Ct ~ 40 for all genes, pFoxi1 ΔCt±SEM: ADGRF1 = 13.2±0.21, ATP6V1B1 = 11.4±0.3, ATP6V1G3 = 10±0.1, ATP6V0D2 = 12.1±0.03). Additionally, these pFOXI1 cells also upregulate the expression of SLC26A4 (pendrin), a known marker of B‐type intercalated cells, suggesting that FOXI1‐driven regulation is upstream of AIC fate determination (n=3, ΔCt ± SEM: untransfected = 19.3±0.7, pFoxi1 = 16.1±0.2, p<0.01). These data reveal that FOXI1 upregulates both GPR116 and V‐ATPase in M‐1 cells, generating IC‐like cells. Finally, our study suggests that GPR116 may be a universal and genetically‐coded regulator of V‐ATPase surface expression in FOXI1‐positive cells.

Current kidney function parameters overestimate kidney tissue repair in reversible experimental kidney disease

Although underlying mechanisms and the clinical course of kidney disease progression are well described, less is known about potential disease reversibility. Therefore, to analyze kidney recovery, we adapted a commonly used murine chronic kidney disease (CKD) model of 2,8- dihydroxyadenine (2,8-DHA) crystal-induced nephropathy to study disease recovery and efficacy of disease-modifying interventions. The recovery phase after CKD was characterized by improved kidney function after two weeks which remained stable thereafter. By contrast, even after eight weeks recovery, tubular injury and inflammation were only partially reduced, and fibrosis persisted. Deep-learning-based histologic analysis of 8,604 glomeruli and 596,614 tubular cross sections revealed numerous tubules had undergone either prominent dilation or complete atrophy, leading to atubular glomeruli and irreversible nephron loss. We confirmed these findings in a second CKD model, reversible unilateral ureteral obstruction, in which a rapid improvement of glomerular filtration rate during recovery also did not reflect the permanent histologic kidney injury. In 2,8-DHA nephropathy, increased drinking volume was highly effective in disease prevention. However, in therapeutic approaches, high fluid intake was only effective in moderate but not severe CKD and established tissue injury was again poorly reflective of kidney function parameters. The injury was particularly localized in the medulla, which is often not analyzed. Thus, recovery after crystal- or obstruction-induced CKD is characterized by ongoing tissue injury, fibrosis, and nephron loss, but not reflected by standard measures of kidney function. Hence, our data might aid in designing kidney recovery studies and suggest the need for biomarkers specifically monitoring intra-kidney tissue injury.

SIRT2 Affects Cell Proliferation and Apoptosis by Suppressing the Level of Autophagy in Renal Podocytes

Purpose Despite the discovery of many important molecules in diabetic nephropathy, there has been very limited progress in the management of diabetic kidney diseases and the design of new drugs. To fill this gap, the present study explored the expression of SIRT2 in high-glucose murine kidney foot cells and its impact on cell biological functions. Methods Expression levels of SIRT2 in the MPC-5 of murine kidney foot cells after high and normal glucose treatment or in cells targeted with siRNA were detected using qRT-PCR. Cellular proliferation and programmed cell death were analyzed via the CCK8 assay and flow cell technique, separately. Levels of autophagy markers were measured by western blotting, and chloroquine treatment was applied to the cells to observe the effect of SIRT2 on cell proliferation and apoptosis after treatment. Results The expression level of SIRT2 was remarkably upregulated in the high-GLU group in contrast to the low-GLU group. The cell proliferation and autophagy levels were significantly reduced, and apoptosis was remarkably reinforced in the high-GLU group in contrast to the normal GLU group. However, knocking down the expression level of SIRT2 caused an increase in cell proliferation and cell autophagy levels and significantly weakened apoptosis. Chloroquine influenced cell proliferation and apoptosis in cells targeted with SIRT2 siRNA. Conclusion SIRT2 expression was upregulated in hyperglycaemic murine kidney foot cells, and knocking down the expression level of SIRT2 affected the biological function of the cells. We found that SIRT2 may modulate cell proliferation and apoptosis by regulating cell autophagy.

Sulfatide with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs in renal intercalated cells

Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H+-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H+-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH3 and protons into the urine.

Acute adaptation of renal phosphate transporters in the murine kidney to oral phosphate intake requires multiple signals.

Dietary inorganic phosphate (Pi) modulates renal Pi reabsorption by regulating the expression of the NaPi-IIa and NaPi-IIc Pi transporters. Here, we aimed to clarify the role of several Pi-regulatory mechanisms including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) and inositol hexakisphosphate kinases (IP6-kinases) in the acute regulation of NaPi-IIa and NaPi-IIc. Wildtype (WT) and PTH-deficient mice (PTH-KO) with/without inhibition of FGF23 signalling were gavaged with Pi/saline and examined at 1, 4 and 12h. Pi-gavage elevated plasma Pi and decreased plasma Ca2+ in both genotypes after 1h Within 1h, Pi-gavage decreased NaPi-IIa abundance in WT and PTH-KO mice. NaPi-IIc was downregulated 1h post-administration in WT and after 4h in PTH-KO. PTH increased after 1h in WT animals. After 4h Pi-gavage, FGF23 increased in both genotypes being higher in the KO group. PTHrp and dopamine were not altered by Pi-gavage. Blocking FGF23 signalling blunted PTH upregulation in WT mice and reduced NaPi-IIa downregulation in PTH-KO mice 4h after Pi-gavage. Inhibition of IP6-kinases had no effect. (1) Acute downregulation of renal Pi transporters in response to Pi intake occurs also in the absence of PTH and FGF23 signalling, (2) when FGF23 signalling is blocked, a partial contribution of PTH is revealed, (3) IP6 kinases, intracellular Pi-sensors in yeast and bacteria, are not involved, and (4) Acute Pi does not alter PTHrp and dopamine. Thus, signals other than PTH, PTHrp, FGF23 and dopamine contribute to renal adaption.

Conventional NK Cells and Type 1 Innate Lymphoid Cells Do Not Influence Pathogenesis of Experimental Glomerulonephritis.

Innate lymphoid cells (ILCs) that express NK cell receptors (NCRs) and the transcription factor T-bet populate nonlymphoid tissues and are crucial in immune responses against viral infections and malignancies. Recent studies highlighted the heterogeneity of this ILC population and extended their functional spectrum to include important roles in tissue homeostasis and autoimmunity. In this article, we provide detailed profiling of NCR+T-bet+ ILC populations in the murine kidney, identifying conventional NK (cNK) cells and type 1 ILCs (ILC1s) as the two major subsets. Induction of renal inflammation in a mouse model of glomerulonephritis did not substantially influence abundance or phenotype of cNK cells or ILC1s in the kidney. For functional analyses in this model, widely used depletion strategies for total NCR+ ILCs (anti-NK1.1 Ab application) and cNK cells (anti-asialoGM1 serum application) were unreliable tools, because they were accompanied by significant off-target depletion of kidney NKT cells and CD8+ T cells, respectively. However, neither depletion of cNK cells and ILC1s in NKT cell-deficient mice nor specific genetic deletion of cNK cells in Ncr1 Cre/wt × Eomes fl/fl mice altered the clinical course of experimental glomerulonephritis. In summary, we show in this article that cNK cells and ILC1s are dispensable for initiation and progression of immune-mediated glomerular disease and advise caution in the use of standard Ab depletion methods to study NCR+ ILC function in mouse models.

Selective kidney targeting increases the efficacy of mesenchymal stromal/stem cells for alleviation of murine stenotic-kidney senescence and damage.

Chronic ischemia triggers senescence in renal tubules and at least partly mediates kidney dysfunction and damage through a p16Ink4a -related mechanism. We previously showed that mesenchymal stromal/stem cells (MSCs) delivered systemically do not effectively decrease cellular senescence in stenotic murine kidneys. We hypothesized that selective MSC targeting to injured kidneys using an anti-KIM1 antibody (KIM-MSC) coating would enhance their ability to abrogate cellular senescence in murine renal artery stenosis (RAS). KIM-MSC were injected into transgenic INK-ATTAC mice, which are amenable for selective eradication of p16Ink4a+ cells, 4weeks after induction of unilateral RAS. To determine whether KIM-MSC abolish p16Ink4a -dependent cellular senescence, selective clearance of p16Ink4a+ cells was induced in a subgroup of RAS mice using AP20187 over 3weeks prior to KIM-MSC injection. Two weeks after KIM-MSC aortic injection, renal senescence, function, and tissue damage were assessed. KIM-MSC delivery decreased gene expression of senescence and senescence-associated secretory phenotype factors, and improved micro-MRI-derived stenotic-kidney glomerular filtration rate and perfusion. Renal fibrosis and tubular injury also improved after KIM-MSC treatment. Yet, their efficacy was slightly augmented by prior elimination of p16Ink4a+ senescent cells. Therefore, selective targeting of MSC to the injured kidney markedly improves their senolytic potency in murine RAS, despite incomplete eradication of p16+ cells. KIM-MSC may constitute a useful therapeutic strategy in chronic renal ischemic injury.

Blockade of STAT3 signaling alleviates the progression of acute kidney injury to chronic kidney disease through antiapoptosis

Signal transducer and activator of transcription 3 (STAT3) is a pivotal mediator of IL-6-type cytokine signaling. However, the roles of its full-length and truncated isoforms in acute kidney injury (AKI) and its transition to chronic kidney disease (CKD) remain elusive. Herein, the role of STAT3 isoforms in the AKI-to-CKD transition was characterized using an ischemia-reperfusion injury (IRI) mouse model. The STAT3 inhibitor Stattic was administered to C57BL/6 mice 3 h before IRI. Intrarenal cytokine expression was quantified using real-time PCR and FACS. The effect of Stattic on human tubular epithelial cells cultured under hypoxic conditions was also evaluated. Phosphorylated (p)STAT3 isoforms were detected by Western blot analysis. Stattic treatment attenuated IRI-induced tubular damage and inflammatory cytokine/chemokine expression while decreasing macrophage infiltration and fibrosis in mouse unilateral IRI and unilateral ureteral obstruction models. Similarly, in vitro STAT3 inhibition downregulated fibrosis and apoptosis in 72-h hypoxia-induced human tubular epithelial cells and reduced pSTAT3α-mediated inflammation. Moreover, pSTAT3 expression was increased in human acute tubular necrosis and CKD tissues. STAT3 activation is associated with IRI progression, and STAT3α may be a significant contributor. Hence, STAT3 may affect the AKI-to-CKD transition, suggesting a novel strategy for AKI management with STAT3 inhibitors.NEW & NOTEWORTHY We found that IRI increased expression of STAT3 in murine kidneys, along with inflammation markers. Through the investigation of the role of STAT3 in the AKI-to-CKD transition mechanism using mouse unilateral IRI and unilateral ureteral obstruction models and 24- or 72-h hypoxic induction of primary cultured human tubular epithelial cells, we found that STAT3 could affect the AKI-to-CKD transition. We also observed different degrees of expression in STAT3 isoforms in these processes.

In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms

Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection indicating this bacterium overcomes host nutritional immunity. Despite this, there exists a significant knowledge gap regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis can usurp hydroxamate siderophores and staphyloferrin A and B from Staphylococcus aureus. These transport activities all required a functional FhuC ATPase. Moreover, we show that the acquisition of catechol siderophores and catecholamine stress hormones by S. lugdunensis required the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitated the ferrous iron transport system encoded by feoAB. Heme iron was acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice, we demonstrated that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci (isd, fhuC, sst-1, and feo), we demonstrate that only a strain deficient for all of these systems was attenuated in its ability to proliferate to high numbers in the murine kidney. We propose the concerted action of heme and non-heme iron acquisition systems also enable S. lugdunensis to cause human infection.

Analysis of Mononuclear Phagocytes Disclosed the Establishment Processes of Two Macrophage Subsets in the Adult Murine Kidney.

The interstitium of kidney involves a variety of components including resident immune cells, in particular mononuclear phagocytes. However, many proposed markers for distinguishing macrophages or dendritic cells are, in fact, shared by the majority of renal mononuclear phagocytes, which impedes the research of kidney diseases. Here, by employing a flow cytometry strategy and techniques of fate mapping, imaging and lineage depletion, we were able to demarcate renal monocytes, macrophages and dendritic cells and their subsets in mice. In particular, using this strategy, we found that even in steady state, the renal macrophage pool was continuously replenished by bone marrow-derived monocytes in a stepwise process, i.e., from infiltration of classical monocyte, to development of nonclassical monocyte and eventually to differentiation to macrophages. In mechanism, we demonstrated that the ligation of tissue-anchored CX3CL1 and monocytic CX3CR1 was required for promoting monocyte differentiation to macrophages in the kidney, but CX3CL1-CX3CR1 signaling was dispensable in monocyte infiltrating into the kidney. In addition to the bone marrow-derived macrophages, fate mapping in adult mice revealed another population of renal resident macrophages which were embryo-derived and self-maintaining. Thus, the dissecting strategies developed by us would assist in exploration of the biology of renal mononuclear phagocytes.

Effects of obesity on reparative function of human adipose tissue-derived mesenchymal stem cells on ischemic murine kidneys.

Introduction:Obesity is a health burden that impairs cellular processes. Mesenchymal stem/stromal cells (MSCs) are endowed with reparative properties and can ameliorate renal injury. Obesity impairs human MSC function in-vitro, but its effect on their in-vivo reparative potency remains unknown.Subjects and Methods:Abdominal adipose tissue-derived MSC were harvested from patients without (‘lean’) or with obesity (‘obese’) (body mass index<30 or ≥30kg/m2, respectively) during kidney donation or bariatric surgery, respectively. MSC (5x105/200μL) or vehicle were then injected into 129S1 mice 2 weeks after renal artery stenosis (RAS) or sham surgery (n=8/group). Two weeks later, mice underwent magnetic resonance imaging to assess renal perfusion and oxygenation in-vivo, and kidneys then harvested for ex-vivo studies.Results:Similar numbers of lean and obese-MSCs engrafted in stenotic mouse kidneys. Vehicle-treated RAS mice had reduced stenotic-kidney cortical and medullary perfusion and oxygenation. Lean (but not obese) MSC normalized ischemic kidney cortical perfusion, whereas both effectively mitigated renal hypoxia. Serum creatinine and blood pressure were elevated in RAS mice and lowered only by lean-MSC. Both types of MSCs alleviated stenotic-kidney fibrosis, but lean-MSC more effectively than obese-MSC. MSC senescence-associated beta-gal activity, and gene expression of p16, p21, and vascular endothelial growth factor correlated with recipient kidney perfusion and tissue injury, linking MSC characteristics with their in-vivo reparative capacity.Discussion:Human obesity impairs the reparative properties of adipose-tissue-derived MSCs, possibly by inducing cellular senescence. Dysfunction and senescence of the endogenous MSC repair system in patients with obesity may warrant targeting interventions to restore MSC vitality.

Therapeutic efficacy of heterogeneously distributed radiolabelled peptides: Influence of radionuclide choice

PurposeTo model dose-response relationships for in vivo experiments with radiolabelled peptides enabling maximum therapeutic efficacy while limiting toxicity to kidney and bone marrow. MethodsA multiregional murine kidney phantom, with a kinetic model for cortex and outer medulla distribution, were used to predict renal toxicity.Maximum tolerated activities to avoid nephrotoxicity (at 40 Gy Biological Effective Dose BED) and hematologic toxicity (at 2 Gy) were compared.The therapeutic efficacy of 90Y, 161Tb, 177Lu and 213Bi was assessed at their respective maximum tolerated activities based on cellular-level dosimetry accounting for activity and tumor heterogeneity. These results were compared with average tumor-dosimetry-based predictions. ResultsThe kidney was found to be the dose-limiting organ for all radionuclides, limiting the administered activity to 44 MBq 177Lu, 34 MBq 161Tb, 19 MBq 90Y and 13 MBq 213Bi , respectively.The average S-values for the initial heterogeneous activity distribution in the tumor volume are not significantly different from the homogeneous ones. The in vivo tumor cell survivals predicted by assuming uniform dose rate-distributions are not significantly different from those for heterogeneous dose rate-based predictions.The lowest in vivo survival was found for 213Bi (2%) followed by 161Tb (30%), 177Lu (37%) and 90Y (60%). The minimal effective dose rate for cell kill is 13–14 mGy/h for β-emitters and 2.2 mGy/h for the α-particle emitter 213Bi, below these values proliferation takes over. ConclusionsRadionuclides emitting α-particles have the highest potential for improving therapeutic efficacy in tumors and metastases with uniform receptor expression, after careful evaluation of their burden to the healthy organs.

The ILEI/LIFR complex induces EMT via the Akt and ERK pathways in renal interstitial fibrosis

BackgroundChronic kidney disease (CKD) is characterized by high morbidity and mortality and is difficult to cure. Renal interstitial fibrosis (RIF) is a major determinant of, and commonly occurs within, CKD progression. Epithelial mesenchymal transition (EMT) has been identified as a crucial process in triggering renal interstitial fibrosis (RIF). Interleukin‐like EMT inducer (ILEI) is an important promotor of EMT; this study aims to elucidate the mechanisms involved.MethodsMale C57BL6/J mouse were randomly divided into 6 groups: sham (n = 10), sham with negative control (NC) shRNA (sham + NC, n = 10), sham with ILEI shRNA (sham + shILEI, n = 10), unilateral ureteral obstruction (UUO, n = 10), UUO with NC (UUO + NC, n = 10) and UUO with ILEI shRNA (UUO + shILEI, n = 10). Hematoxylin and eosin (H&E), Masson, and immunohistochemical (IHC) staining and western blotting (WB) were performed on murine kidney tissue to identify the function and mechanism of ILEI in RIF. In vitro, ILEI was overexpressed to induce EMT in HK2 cells and analyzed via transwell, WB, real-time PCR, and co-immunoprecipitation. Finally, tissue from 12 pediatric CKD patients (seven with RIF and five without RIF) were studied with H&E, Masson, and IHC staining.ResultsOur in vitro model revealed that ILEI facilitates RIF in the UUO model via the Akt and ERK pathways. Further experiments in vivo and in vitro revealed that ILEI promotes renal tubular EMT by binding and activating leukemia inhibitory factor receptor (LIFR), in which phosphorylation of Akt and ERK is involved. We further find markedly increased expression levels of ILEI and LIFR in kidneys from pediatric CKD patients with RIF.ConclusionOur results indicate that ILEI may be a useful biomarker for renal fibrosis and a potential therapeutic target for modulating RIF.

Microtubule-associated protein tau in murine kidney: role in podocyte architecture

Tau is a cytoskeletal protein that is expressed mainly in neurons and is involved in several cellular processes, such as microtubule stabilization, axonal maintenance, and transport. Altered tau metabolism is related to different tauopathies being the most important Alzheimer’s disease where aberrant hyperphosphorylated and aggregated tau is found in the central nervous system. Here, we have analyzed that function in kidney by using tau knockout mice generated by integrating GFP-encoding cDNA into exon 1 of MAPT (here referred to as TauGFP/GFP). IVIS Lumina from PerkinElmer demonstrated GFP expression in the kidney. We then demonstrated by qPCR that the main tau isoform in the kidney is Tau4R. The GFP reporter allowed us to demonstrate that tau is found in the glomeruli of the renal cortex, and specifically in podocytes. This was further confirmed by immunohistochemistry. TauGFP/GFP mice present a podocyte cytoskeleton more dynamic as they contain higher levels of detyrosinated tubulin than wild-type mice. In addition, transmission electron microscopy studies demonstrated glomerular damage with a decrease in urinary creatinine. Our results prove that tau has an important role in kidney metabolism under normal physiological conditions.

Stx2 Induces Differential Gene Expression and Disturbs Circadian Rhythm Genes in the Proximal Tubule.

Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na+-glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.

Disruption of the TCA cycle reveals an ATF4-dependent integration of redox and amino acid metabolism

The Tricarboxylic Acid (TCA) Cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), and combined metabolomics, transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in murine kidney epithelial cells. Our comparative approach shows that TCAi elicits a convergent rewiring of redox and amino acid metabolism dependent on the activation of ATF4 and the integrated stress response (ISR). Furthermore, we also uncover a divergent metabolic response, whereby acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis. Our work highlights an important interplay between the TCA cycle, redox biology, and amino acid homeostasis.

Human MSC-Derived Exosomes Reduce Cellular Senescence in Renal Epithelial Cells.

Cellular senescence of renal tubular cells is associated with chronic diseases and age-related kidney disorders. Therapies to antagonize senescence are, therefore, explored as novel approaches in nephropathy. Exosomes derived from human mesenchymal stroma-/stem-like cells (MSC) entail the transfer of multiple bioactive molecules, exhibiting profound regenerative potential in various tissues, including therapeutic effects in kidney diseases. Here, we first demonstrate that exosomes promote proliferation and reduce senescence in aged MSC cultures. For potential therapeutic perspectives in organ rejuvenation, we used MSC-derived exosomes to antagonize senescence in murine kidney primary tubular epithelial cells (PTEC). Exosome treatment efficiently reduced senescence while diminishing the transcription of senescence markers and senescence-associated secretory phenotype (SASP) factors. Concomitantly, we observed less DNA damage foci and more proliferating cells. These data provide new information regarding the therapeutic property of MSC exosomes in the development of renal senescence, suggesting a contribution to a new chapter of regenerative vehicles in senotherapy.

Dimethyl fumarate attenuates LPS induced septic acute kidney injury by suppression of NFκB p65 phosphorylation and macrophage activation

Septic acute kidney injury (AKI) always accounts for high mortality of septic patients in ICU. Due to its not well understood mechanism for infection and immune-regulation in kidney dysfunction, there is a lack of effective therapy without side effects. Dimethyl fumarate (DMF) as an immunomodulatory molecule has been approved for treatment to multiple sclerosis. However, the therapeutic effect and immunomodulatory role underlying DMF action in septic AKI is unclear. This study aimed to elucidate the role of DMF in lipopolysaccharide (LPS)-induced septic AKI involving macrophage regulation. In current study, we administered DMF by oral gavage to mice with LPS-induced AKI, then harvested serum and kidney at three different time points. We further isolated Bone marrow-derived macrophages (BMDMs) from mice and stimulated them with LPS followed by DMF treatment. To explore immunomodulatory role of DMF in macrophages, we depleted macrophages in mice using liposomal clodronate after DMF treatment upon LPS-induced septic AKI. Then we observed that DMF attenuated renal dysfunction and murine pathological kidney injury after LPS injection. DMF could inhibit translocation of phosphorylated NF-κB p65 and suppress macrophage activation in LPS-induced AKI. DMF reduced the secretion of TNF-α and IL-6 whereas increased the secretion of IL-10 and Arg-1 in BMDMs after LPS stimulation. DMF also inhibited NF-κB p65 phosphorylation in BMDMs after LPS stimulation. Importantly, the effect of DMF against LPS-induced AKI, macrophage activation, and translocation of phosphorylated NF-κB p65 was impaired upon macrophage depletion. Thus, DMF could attenuate LPS-induced septic AKI by suppression of NF-κB p65 phosphorylation and macrophage activation. This work suggested the potential therapeutic role of DMF for patients in ICU threatened by septic AKI.

Subchronic oral exposure of tungsten induces myofibroblast transformation and various markers of kidney fibrosis.

Tungsten is a naturally occurring transition element used in a broad range of applications. As a result of its extensive use, we are increasingly exposed to tungsten from our environment, including potable water, since tungsten can become bioaccessible in ground sources. The kidneys are particularly susceptible to tungsten exposure as this is the main site for tungsten excretion. In this study, we investigated the prolonged effects of tungsten on the kidneys and how this may impact injury and function. When mice were exposed to tungsten in their drinking water for 1 mo, kidney function had not significantly changed. Following 3-mo exposure, mice were presented with deterioration in kidney function as determined by serum and urine creatinine levels. During 3 mo of tungsten exposure, murine kidneys demonstrated significant increases in the myofibroblast marker α-smooth muscle actin (αSMA) and extracellular matrix products: fibronectin, collagen, and matricellular proteins. In addition, Masson's trichrome and hematoxylin-eosin (H&E) staining revealed an increase in fibrotic tissue and vacuolization of tubular epithelial cells, respectively, from kidneys of tungsten-treated mice, indicative of renal injury. In vitro treatment of kidney fibroblasts with tungsten led to increased proliferation and upregulation of transforming growth factor β1 (TGFβ1), which was consistent with the appearance of fibroblast-to-myofibroblast transition (FMT) markers. Our data suggest that continuous exposure to tungsten impairs kidney function that may lead to the development of chronic kidney disease (CKD).

Ultrasound and Microbubbles Enhance Uptake of Doxorubicin in Murine Kidneys.

The use of ultrasound and microbubble-enhanced drug delivery, commonly referred to as sonoporation, has reached numerous clinical trials and has shown favourable results. Nevertheless, the microbubbles and acoustic path also pass through healthy tissues. To date, the majority of studies have focused on the impact to diseased tissues and rarely evaluated the impact on healthy and collateral tissue. The aim of this study was to test the effect and feasibility of low-intensity sonoporation on healthy kidneys in a mouse model. In our work here, we used a clinical diagnostic ultrasound system (GE Vivid E9) with a C1-5 ultrasound transducer combined with a software modification for 20-µs-long pulses to induce the ultrasound-guided drug delivery of doxorubicin (DOX) in mice kidneys in combination with SonoVue® and Sonazoid™ microbubbles. The acoustic output settings were within the commonly used diagnostic ranges. Sonoporation with SonoVue® resulted in a significant decrease in weight vs. DOX alone (p = 0.0004) in the first nine days, whilst all other comparisons were not significant. Ultrasound alone resulted in a 381% increase in DOX uptake vs. DOX alone (p = 0.0004), whilst SonoVue® (p = 0.0001) and Sonazoid™ (p < 0.0001) further increased the uptake nine days after treatment (419% and 493%, respectively). No long-standing damage was observed in the kidneys via histology. In future sonoporation and drug uptake studies, we therefore suggest including an “ultrasound alone” group to verify the actual contribution of the individual components of the procedure on the drug uptake and to perform collateral damage studies to ensure there is no negative impact of low-intensity sonoporation on healthy tissues.