Advanced glycation endproducts (AGEs) contribute to cellular damage of various pathologies, including kidney diseases. Acute kidney injury (AKI) represents a syndrome seldom characterized by a single, distinct pathophysiological cause. Rhabdomyolysis-induced acute kidney injury (RIAKI) constitutes roughly 15% of AKI cases, yet its underlying pathophysiology remains poorly understood. Using a murine model of RIAKI induced by muscular glycerol injection, we observed elevated levels of AGEs and the AGE receptor galectin-3 (LGALS3) in the kidney. Immunofluorescence localized LGALS3 to distal nephron segments. According to transcriptomic profiling via next-generation sequencing, RIAKI led to profound changes in kidney metabolism, oxidative stress, and inflammation. Cellular stress was evident in both proximal and distal tubules, as shown by kidney injury markers KIM-1 and NGAL. However, only proximal tubules exhibited overt damage and apoptosis, as detected by routine morphology, active Caspase-3, and TUNEL assay, respectively. In vitro, distal convoluted tubule (DCT) cells challenged with AGEs underwent apoptosis, which was markedly enhanced by Lgals3 siRNA treatment. Thus, in RIAKI, the upregulation of LGALS3 may protect the distal nephron from AGE-mediated damage, while proximal tubules lacking LGALS3 stay at risk. Thus, stimulating LGALS3 in the proximal nephron, if achievable, may attenuate RIAKI.

AIM:2,3-bisphosphoglycerate mutase (BPGM) is traditionally recognized for its role in modulating oxygen affinity to hemoglobin in erythrocytes. Recent transcriptomic analyses, however, have indicated a significant upregulation of BPGM in acutely injured murine and human kidneys, suggesting a potential renal function for this enzyme. Here we aim to explore the physiological role of BPGM in the kidney. METHODS:A tubular-specific, doxycycline-inducible Bpgm-knockout mouse model was generated. Histological, immunofluorescence, and proteomic analyses were conducted to examine the localization of BPGM expression and the impact of its knockout on kidney structure and function. In vitro studies were performed to investigate the metabolic consequences of Bpgm knockdown under osmotic stress. RESULTS:BPGM expression was localized to the distal nephron and was absent in proximal tubules. Inducible knockout of Bpgm resulted in rapid kidney injury within 4 days, characterized by proximal tubular damage and tubulointerstitial fibrosis. Proteomic analyses revealed involvement of BPGM in key metabolic pathways, including glycolysis, oxidative stress response, and inflammation. In vitro, Bpgm knockdown led to enhanced glycolysis, decreased reactive oxygen species elimination capacity under osmotic stress, and increased apoptosis. Furthermore, interactions between nephron segments and immune cells in the kidney suggested a mechanism for propagating stress signals from distal to proximal tubules. CONCLUSION:BPGM fulfills critical functions beyond the erythrocyte in maintaining glucose metabolism in the distal nephron. Its absence leads to metabolic imbalances, increased oxidative stress, inflammation, and ultimately kidney injury.

Introduction: Mechanic power output (MPO) and oxygen consumption (VO2) reflect endurance capacity and are often stated relative to body mass (BM) but less often per skeletal muscle mass (SMM). Rating of perceived exertion (RPE) has previously shown conflicting results between sexes at submaximal intensities. Individual body composition, however, largely differs due to sex and training status. It was the aim of this study to evaluate RPE of untrained and trained individuals of both sexes considering body composition and to estimate whether RPE could be improved as a tool to determine endurance capacity. Methods: The study included 34 untrained adults (age 26.18 ± 6.34 years, 18 women) and 29 endurance trained (age 27.86 ± 5.19, 14 women) who were measured for body composition (InBody 770, InBody Europe B.V., Germany) and tested on a treadmill (Pulsar, H/P/Cosmos, Germany) for aerobic capacity (Metalyzer 3B, Cortex Biophysik GmbH, Germany) in an all-out exercise test applying the Bruce-protocol. VO2, MPO, heart rate (HR), and RPE were obtained at each exercise stage. VO2 and MPO were calculated per BM and SMM. RPE values were correlated with absolute VO2 and MPO, as well as relative to BM, and SMM. HR values and the parameters' standardized values served for comparison to standard procedures. Results: VO2 and MPO were higher in men compared to women and in trained compared to untrained participants. No differences between groups and sexes exist when VO2 and MPO were calculated per BM. When calculated per SMM, VO2 and MPO indicate opposite results already at low intensity stages of exercise test. RPE values had highest correlation with MPO per SMM (R2 = 0.8345) compared to absolute MPO (R2 = 0.7609), or MPO per BM (R2 = 0.8176). Agreement between RPE and MPO per SMM was greater than between RPE and HR (p = 0.008). Conclusion: Although RPE represents a subjective value at first glance, it was shown that RPE constitutes a valuable tool to estimate endurance capacity, which can be further enhanced if individual body composition is considered. Furthermore, MPO and VO2 should be considered relative to SMM. These findings might help to avoid over-exertion, especially among untrained people, by adjusting the training intensity for each subject according to the individual strain evaluated in an exercise test based on individual body composition.

Development of treatment strategies for kidney diseases largely relies on experimental models of ureteral obstruction/tubularinterstitial fibrosis,1,2 acute kidney injury/ ischemia/reperfusion,3,4 cyclosporine toxicity5,6 or diabetic nephropathy.7 However, experimental models only partly replicate the respective human disease. Moreover, despite an impressive leap in mechanistic knowledge, the therapeutic yield for human has remained rather poor. One notable exception is novel, orally available, highly specific, small molecule activators of hypoxiainducible factor (HIF). Within two decades of basic and clinical research, these compounds made their way from bench to bedside, crossing European Medicines Agency's goal line for approval, under the cheers of the Nobel Prize Committee. Is such fanfare justified? It's up to you to decide: Gregg Semenza, Sir Peter Ratcliffe, and William Kaelin have identified the cellular oxygen sensor, an enzyme named HIF prolyl hydroxylase (PHD), and HIF as the ubiquitous cellular conductor to orchestrate adaptation to hypoxia. This brings us back to the kidney, a very sophisticated organ, often hardly accessible to clinical research. Imagine a Swiss watch's clockwork with hundreds of delicate cogwheels perfectly fitting together. You may admire its thrilling design and impeccable function, yet, you would probably not dare to look inside if it were at the risk of damaging it. Accordingly, kidney biopsies are often performed for diagnostic purposes nonetheless, they are rarely available for scientific research. Hence, we seldom get a look inside the kidney's sophisticated clockwork. Thus, noninvasive methods are under development to gain insight into the pathophysiology of various kidney disorders.8 For example, blood oxygenation leveldependent (BOLD) MRI to assess kidney hypoxia is of great interest.6,9 Is there a link between kidney disease and hypoxia? Certainly yes, both, acute and chronic kidney disease are often accompanied by kidney hypoxia. What if a small pill could help the kidney cope with hypoxia? This exactly is the promise of PHD inhibitors, beyond the treatment of renal anemia, the label obtained by regulatory authorities. Roxadustat is the first PHD inhibitor that was authorized for use in the EU in 2021. What is more, further HIF activators, with daprodustat being an example, are undergoing clinical investigations.10 To gain a better understanding of their medical indication, let us catch a glimpse of their specific mechanism of action. As mentioned above, PHD inhibitors activate the HIF pathway. Under normoxic conditions, HIFα is ubiquitinylated by the von Hippel– Lindau tumor supressor (VHL) and subsequently degraded through the proteasomal pathway.11 The binding ability of VHL is dependent on the hydroxylation of HIFα by PHDs which need oxygen as a substrate. When oxygen is missing or PHDs are blocked, HIFα accumulates, and translocates to the nucleus. There, it forms dimers with HIFβ and binds to hypoxia response elements of target genes, thus activating transcription.12 In a nutshell, PHD inhibitors prevent the degradation of HIFα, which results in stimulation of erythropoietin (Epo). Epo transcription and release is the consequence of low blood oxygen level, which is sensed by fibroblastlike cells in the kidney.13 This leads to an increased number of red blood cells and hemoglobin levels. Therefore, the therapeutic area of PHD inhibitors is the treatment of symptomatic anemia that may occur in patients with chronic kidney disease (CKD). A major risk factor for the development of CKD is acute kidney injury, depending on its frequency, severity, and duration.14 Further, CKD may progress to endstage renal disease (ESRD), a condition, where the only treatment options are dialysis or kidney transplantation. To prevent allograft rejection, immunosuppressive therapy is essential. A widely used class of immunosuppressant drugs are the socalled calcineurin inhibitors, such as CyclosporineA (CsA).15 Let us take another peek at the mechanism of action of these class of therapeutics. Under normal conditions, calmodulin binds— together with calcium ions— the phosphatase calcineurin, thus, increasing its activity. The calcineurin inhibitors CsA and tacrolimus form a complex with

Non-resolving inflammation plays a critical role during the transition from renal injury towards end-stage renal disease. The glucocorticoid-inducible protein annexin A1 has been shown to function as key regulator in the resolution phase of inflammation, but its role in immune-mediated crescentic glomerulonephritis has not been studied so far. Methods: Acute crescentic glomerulonephritis was induced in annexin A1-deficient and wildtype mice using a sheep serum against rat glomerular basement membrane constituents. Animals were sacrificed at d5 and d10 after nephritis induction. Renal leukocyte abundance was studied by immunofluorescence and flow cytometry. Alterations in gene expression were determined by RNA-Seq and gene ontology analysis. Renal levels of eicosanoids and related lipid products were measured using lipid mass spectrometry. Results: Histological analysis revealed an increased number of sclerotic glomeruli and aggravated tubulointerstitial damage in the kidneys of annexin A1-deficient mice compared to the wildtype controls. Flow cytometry analysis confirmed an increased number of CD45+ leukocytes and neutrophil granulocytes in the absence of annexin A1. Lipid mass spectrometry showed elevated levels of prostaglandins PGE2 and PGD2 and reduced levels of antiinflammatory epoxydocosapentaenoic acid regioisomers. RNA-Seq with subsequent gene ontology analysis revealed induction of gene products related to leukocyte activation and chemotaxis as well as regulation of cytokine production and secretion. Conclusion: Intrinsic annexin A1 reduces proinflammatory signals and infiltration of neutrophil granulocytes and thereby protects the kidney during crescentic glomerulonephritis. The annexin A1 signaling cascade may therefore provide novel targets for the treatment of inflammatory kidney disease.

Chronic Cyclosporine-A treatment is associated with serious side effects, including kidney toxicity and anemia. Although pathophysiology of Cyclosporine-A-induced kidney injury remains incompletely understood, hypoxia is likely involved. Here, we investigated the effect of the hypoxia inducible factor activator daprodustat on Cyclosporine-A -induced kidney toxicity. As Cyclosporine-A profoundly alters protein phosphorylation by inhibiting the phosphatase calcineurin, special attention was directed towards the kidney phospho-proteome. Mice received Cyclosporine-A with or without daprodustat for up to eight weeks. In kidney homogenates, 1360 selected proteins were analyzed at expression and phosphorylation levels. Of these, Cyclosporine-A changed the expression of 79 and the phosphorylation of 86 proteins. However, when Cyclosporine-A treatment was combined with daprodustat, the expression of 95 proteins and phosphorylation of only six proteins was altered suggesting that daprodustat prevented most protein phosphorylation brought about by Cyclosporine-A. Although daprodustat showed only marginal effect on its own, angiogenesis-related pathways were among the most profoundly impacted by daprodustat when given on top of Cyclosporine-A. Additionally, Cyclosporine-A lowered the blood hemoglobin concentration and caused kidney capillary rarefaction, which daprodustat prevented. Thus, combined daprodustat/Cyclosporine-A treatment prevented deleterious Cyclosporine-A effects on microcirculation and hemoglobin, and the protective action of daprodustat involves suppression of broad protein phosphorylation changes caused by Cyclosporine-A.

Acute kidney injury (AKI) causes multiple organ dysfunction. Here, we identify a possible mechanism that can drive brain vessel injury after AKI. We induced 30-minute bilateral renal ischemia-reperfusion injury in C57Bl/6 mice and isolated brain microvessels and macrovessels 24 hours or 1 week later to test their responses to vasoconstrictors and found that after AKI brain vessels were sensitized to Ang II (angiotensin II). Upregulation of FGF2 (fibroblast growth factor 2) and FGFBP1 (FGF binding protein 1) expression in both serum and kidney tissue after AKI suggested a potential contribution to the vascular sensitization. Administration of FGF2 and FGFBP1 proteins to isolated healthy brain vessels mimicked the sensitization to Ang II after AKI. Brain vessels in Fgfbp1(-/-) AKI mice failed to induce Ang II sensitization. Complementary to this, systemic treatment with the clinically used FGF receptor kinase inhibitor BGJ398 (Infigratinib) reversed the AKI-induced brain vascular sensitization to Ang II. All these findings lead to the conclusion that FGFBP1 is especially necessary for AKI-mediated brain vascular sensitization to Ang II and inhibitors of FGFR pathway may be beneficial in preventing AKI-induced brain vessel injury.

Oxygen affinity to haemoglobin is indicated by the p50 value (pO2 at 50% O2Hb) and critically determines cellular oxygen availability. Although high Hb-O2 affinity can cause tissue hypoxia under conditions of well O2 saturated blood, individual differences in p50 are commonly not considered in clinical routine. Here, we investigated the diversity in Hb-O2 affinity in the context of physiological relevance. Oxyhaemoglobin dissociation curves (ODCs) of 60 volunteers (18–40 years, both sexes, either endurance trained or untrained) were measured at rest and after maximum exercise (VO2max) test. At rest, p50 values of all participants ranged over 7 mmHg. For comparison, right shift of ODC after VO2max test, representing the maximal physiological range to release oxygen to the tissue, indicated a p50 difference of up to 10 mmHg. P50 at rest differs significantly between women and men, with women showing lower Hb-O2 affinity that is determined by higher 2,3-BPG and BPGM levels. Regular endurance exercise did not alter baseline Hb-O2 affinity. Thus, p50 diversity is already high at baseline level and needs to be considered under conditions of impaired tissue oxygenation. For fast prediction of Hb-O2 affinity by blood gas analysis, only venous but not capillary blood samples can be recommended.

Christin Boldt,* Tom Röschel,* Nina Himmerkus, Allein Plain, Markus Bleich, Robert Labes, Maximilian Blum, Hans Krause, Ahmed Magheli, Torsten Giesecke, Kerim Mutig, Michael Rothe, Steven M. Weldon, Duska Dragun, Wolf-Hagen Schunck, Sebastian Bachmann, and Alexander Paliege Department of Anatomy, Charité-Universitätsmedizin Berlin, Berlin, Germany; Department of Physiology, Christian-Albrechts-University, Kiel, Germany; Max Delbrueck Center for Molecular Medicine, Berlin, Germany; Department of Urology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Lipidomix, Berlin, Germany; Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut; Department of Nephrology, Charité-Universitätsmedizin Berlin, Berlin, Germany; and Berlin Institute of Health, Berlin, Germany

Calcineurin dephosphorylates nuclear factor of activated T cells transcription factors, thereby facilitating T cell-mediated immune responses. Calcineurin inhibitors are instrumental for immunosuppression after organ transplantation but may cause side effects, including hypertension and electrolyte disorders. Kidneys were recently shown to display activation of the furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb and the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule upon calcineurin inhibition using cyclosporin A (CsA). An involvement of major hormones like angiotensin II or arginine vasopressin (AVP) has been proposed. To resolve this issue, the effects of CsA treatment in normal Wistar rats, AVP-deficient Brattleboro rats, and cultured renal epithelial cells endogenously expressing either NKCC2 or NCC were studied. Acute administration of CsA to Wistar rats rapidly augmented phosphorylation levels of NKCC2, NCC, and their activating kinases suggesting intraepithelial activating effects. Chronic CsA administration caused salt retention and hypertension, along with stimulation of renin and suppression of renal cyclooxygenase 2, pointing to a contribution of endocrine and paracrine mechanisms at long term. In Brattleboro rats, CsA induced activation of NCC, but not NKCC2, and parallel effects were obtained in cultured cells in the absence of AVP. Stimulation of cultured thick ascending limb cells with AVP agonist restored their responsiveness to CsA. Our results suggest that the direct epithelial action of calcineurin inhibition is sufficient for the activation of NCC, whereas its effect on NKCC2 is more complex and requires concomitant stimulation by AVP.

Leukocyte infiltration presents a hallmark feature of immune‐mediated acute kidney injury and defective resolution of inflammation may promote progression towards chronic kidney failure. Leukocytes exert distinctive effects during this process and may either aggravate kidney damage or foster renal repair. The glucocorticoid‐inducible protein Annexin A1 has been shown to induce resolution of inflammation and to shift macrophage polarization towards the anti‐inflammatory and repair‐promoting M2 phenotype. The cellular source of the putative Annexin A1 signal and its regulation during the course of renal inflammation remains to be elucidated. Adult Wistar rats were injected with an antibody directed against the Thy‐1.1 protein located on mesangial cells to induce mesangioproliferative glomerulonephritis. Animals were examined after 24h (initiation phase), 5d (proliferation phase), and 15d (resolution phase). Regulation of Annexin A1 was studied by qPCR and immunohistochemistry. Annexin A1 + cells were characterized by triple labelling immunofluorescence using antibodies for CD68 (monocytes and macrophages), CD206 (macrophages with M2 polarization), CD3, CD4, and CD8 (T‐cell subpopulations), CD20 (B‐lymphocytes), and myeloperoxidase (neutrophil granulocytes). Quantification of immunoreactive cells was performed by cell counting on confocal micrographs. Renal Annexin A1 mRNA levels increased rapidly after induction of anti‐Thy‐1.1 nephritis (24h: +92±16%; 5d: +128±19%; 15d: +78±33% relative to controls; p<.05). Immunofluorescence labelling showed an interstitial accumulation of total, and CD206+ M2 macrophages during the proliferation and resolution phase (5d: +150±40% and +260±29%; 15d: +378±43% and +600±100% relative to controls; p<.05). Elevated numbers of CD68+/CD206+/Annexin A1 + interstitial M2 macrophages were revealed by triple labelling studies at d5 and d15 (+240±27% and +450±80% relative to controls; p<.05). Abundant expression of Annexin A1 was further found in CD4+ and CD8+ T‐lymphocytes but their number remained unaffected by renal inflammation. CD20+ B‐lymphocytes were negative for Annexin A1. Granulocytes were found in subset of glomeruli at 24h and d5. All granulocytes stained positive for Annexin A1. In conclusion, we have shown a significant increase of Annexin A1 gene expression and protein abundance during the course of anti‐Thy‐1.1 nephritis. The increased amount of Annexin A1 + leukocytes in animals with anti‐Thy‐1.1 nephritis suggest that these cells present a major source for anti‐inflammatory and pro‐resolving signals during renal inflammation. Support or Funding Information Deutsche Forschungsgemeinschaft; FOR1368

Activation of the thick ascending limb (TAL) Na+-K+-2Cl- cotransporter (NKCC2) by the antidiuretic hormone arginine vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here, we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5 ng/h, 3 days) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 μM 14,15-EET for 30 min reduced phosphorylation of NKCC2 at the AVP-sensitive threonine residues T96 and T101 (-66 ± 5%; P < 0.05), while 14,15-DHET had no effect. Concomitantly, isolated perfused cortical thick ascending limb pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symmetric chloride concentrations. In summary, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis.

The anti‐inflammatory protein annexin A1 (AnxA1) and its formyl peptide receptor 2 (FPR2) have protective effects in organ fibrosis. Their role in chronic kidney disease (CKD) has not yet been elucidated. Our aim was to characterize the AnxA1/FPR2 system in models of renal fibrosis.

The G‐protein coupled receptor formyl peptide receptor 2 (FPR2) integrates signals of multiple anti‐inflammatory and anti‐fibrotic mediators including lipoxin A4, resolvin D1, and annexin A1. Previous studies have demonstrated protective effects of FPR2 in animal models of renal disease but the localization of FPR2 in the healthy kidney and its regulation in chronic kidney disease (CKD) have not been elucidated. Aim of this study was to determine the renal localization of FPR2 under control conditions and in a rat model of CKD. Newborn rats were treated with the AT1R antagonist Candesartan from postnatal d1‐14 to impair nephrogenesis and examined at 11 moth of age (CKD rats). Expression of FPR2 was studied using immunohistochemistry (IHC) and double labeling immunofluorescence (IF) for FPR2 and markers for fibroblasts, myofibroblasts, macrophages, and endothelial cells. CKD rats showed signs of renal inflammation and fibrosis with a focal accumulation of macrophages, myofibroblasts and extracellular matrix components. IHC revealed expression of FPR2 in the tubulointerstitium of controls and a strong accumulation of FPR2 expressing cells in fibrotic areas of CKD animals. IF identified fibroblasts and endothelial cells as the predominant FPR2 expressing cell types in both groups. Abundant signal was further detected in macrophages and myofibroblasts in the fibrotic areas of CKD animals. Our results show that FPR2 is abundantly expressed in the kidneys of control and CKD animals. It may therefore play an important role in the regulation of normal renal function and exert anti‐inflammatory and anti‐fibrotic effects during CKD. FPR2 may thus provide a promising target for pharmacological intervention in the treatment of CKD.