Characterization of gene expression in the kidney of renal tubular cell-specific NFAT5 knockout mice.

Nuclear factor of activated T cells 5 (NFAT5; also called TonEBP/OREBP) is a transcription factor that is activated by hypertonicity and induces osmoprotective genes to protect cells against hypertonic conditions. In the kidney, renal tubular NFAT5 is known to be involved in the urine concentration mechanism. Previous studies have suggested that NFAT5 modulates the immune system and exerts various effects on organ damage, depending on organ and disease states. Pathophysiological roles of NFAT5 in renal tubular cells, however, still remain obscure. We conducted comprehensive analysis by performing transcription start site (TSS) sequencing on the kidney of inducible and renal tubular cell-specific NFAT5 knockout (KO) mice. Mice were subjected to unilateral ureteral obstruction to examine the relevance of renal tubular NFAT5 in renal fibrosis. TSS sequencing analysis identified 722 downregulated TSSs and 1,360 upregulated TSSs, which were differentially regulated ≤-1.0 and ≥1.0 in log2 fold, respectively. Those TSSs were annotated to 532 downregulated genes and 944 upregulated genes, respectively. Motif analysis showed that sequences that possibly bind to NFAT5 were enriched in TSSs of downregulated genes. Gene Ontology analysis with the upregulated genes suggested disorder of innate and adaptive immune systems in the kidney. Unilateral ureteral obstruction significantly exacerbated renal fibrosis in the renal medulla in KO mice compared with wild-type mice, accompanied by enhanced activation of immune responses. In conclusion, NFAT5 in renal tubules could have pathophysiological roles in renal fibrosis through modulating innate and adaptive immune systems in the kidney.NEW & NOTEWORTHY TSS-Seq analysis of the kidney from renal tubular cell-specific NFAT5 KO mice uncovered novel genes that are possibly regulated by NFAT5 in the kidney under physiological conditions. The study further implied disorders of innate and adaptive immune systems in NFAT5 KO mice, thereby exacerbating renal fibrosis at pathological states. Our results may implicate the involvement of renal tubular NFAT5 in the progression of renal fibrosis. Further studies would be worthwhile for the development of novel therapy to treat chronic kidney disease.

A Serine Protease Inhibitor, Camostat Mesilate, Suppresses Urinary Plasmin Activity and Alleviates Hypertension and Podocyte Injury in Dahl Salt-Sensitive Rats.

In proteinuric renal diseases, the serine protease (SP) plasmin activates the epithelial sodium channel (ENaC) by cleaving its γ subunit. We previously demonstrated that a high-salt (HS) diet provoked hypertension and proteinuria in Dahl salt-sensitive (DS) rats, accompanied by γENaC activation, which were attenuated by camostat mesilate (CM), an SP inhibitor. However, the effects of CM on plasmin activity in DS rats remain unclear. In this study, we investigated the effects of CM on plasmin activity, ENaC activation, and podocyte injury in DS rats. The DS rats were divided into the control diet, HS diet (8.0% NaCl), and HS+CM diet (0.1% CM) groups. After weekly blood pressure measurement and 24-h urine collection, the rats were sacrificed at 5 weeks. The HS group exhibited hypertension, massive proteinuria, increased urinary plasmin, and γENaC activation; CM treatment suppressed these changes. CM prevented plasmin(ogen) attachment to podocytes and mitigated podocyte injury by reducing the number of apoptotic glomerular cells, inhibiting protease-activated receptor-1 activation, and suppressing inflammatory and fibrotic cytokine expression. Our findings highlight the detrimental role of urinary plasmin in the pathogenesis of salt-sensitive hypertension and glomerular injury. Targeting plasmin with SP inhibitors, such as CM, may be a promising therapeutic approach for these conditions.

Chapter 3: Management of kidney injury caused by cancer drug therapy, from clinical practice guidelines for the management of kidney injury during anticancer drug therapy 2022.

Cisplatin should be administered with diuretics and Magnesium supplementation under adequate hydration to avoid renal impairment. Patients should be evaluated for eGFR (estimated glomerular filtration rate) during the treatment with pemetrexed, as kidney injury has been reported. Pemetrexed should be administered with caution in patients with a CCr (creatinine clearance) < 45 mL/min. Mesna is used to prevent hemorrhagic cystitis in patients receiving ifosfamide. Febuxostat is effective in avoiding hyperuricemia induced by TLS (tumor lysis syndrome). Preventative rasburicase is recommended in high-risk cases of TLS. Thrombotic microangiopathy could be triggered by anticancer drugs and there is no evidence of efficacy of plasma exchange therapy. When proteinuria occurs during treatment with anti-angiogenic agents or multi-kinase inhibitors, dose reductions or interruptions based on grading should be considered. Grade 3 proteinuria and renal dysfunction require urgent intervention, including drug interruption or withdrawal, and referral to a nephrologist should be considered. The first-line drugs used for blood pressure elevation due to anti-angiogenic agents are ACE (angiotensin-converting enzyme) inhibitors and ARBs (angiotensin receptor blockers). The protein binding of drugs and their pharmacokinetics are considerably altered in patients with hypoalbuminemia. The clearance of rituximab is increased in patients with proteinuria, and the correlation with urinary IgG suggests similar pharmacokinetic changes when using other antibody drugs. AIN (acute interstitial nephritis) is the most common cause of ICI (immune checkpoint inhibitor)-related kidney injury that is often treated with steroids. The need for renal biopsy in patients with kidney injury that occurs during treatment with ICI remains controversial.

Suppression of Indoxyl Sulfate Accumulation Reduces Renal Fibrosis in Sulfotransferase 1a1-Deficient Mice.

Renal fibrosis is the final manifestation of chronic kidney disease (CKD); its prevention is vital for controlling CKD progression. Indoxyl sulfate (IS), a typical sulfate-conjugated uremic solute, is produced in the liver via the enzyme sulfotransferase (SULT) 1A1 and accumulates significantly during CKD. We investigated the toxicopathological role of IS in renal fibrosis using Sult1a1-KO mice and the underlying mechanisms. The unilateral ureteral obstruction (UUO) model was created; kidney IS concentrations, inflammation, and renal fibrosis were assessed on day 14. After UUO treatment, inflammation and renal fibrosis were exacerbated in WT mice, with an accumulation of IS in the kidney. However, they were significantly suppressed in Sult1a1-KO mice. CD206+ expression was upregulated, and ß-catenin expression was downregulated in Sult1a1-KO mice. To confirm the impact of erythropoietin (EPO) on renal fibrosis, we evaluated the time-dependent expression of EPO. In Sult1a1-KO mice, EPO mRNA expression was improved considerably; UUO-induced renal fibrosis was further attenuated by recombinant human erythropoietin (rhEPO). Thus, UUO-induced renal fibrosis was alleviated in Sult1a1-KO mice with a decreased accumulation of IS. Our findings confirmed the pathological role of IS in renal fibrosis and identified SULT1A1 as a new therapeutic target enzyme for preventing and attenuating renal fibrosis.

Chapter 1: Evaluation of kidney function in patients undergoing anticancer drug therapy, from clinical practice guidelines for the management of kidney injury during anticancer drug therapy 2022.

The prevalence of CKD may be higher in patients with cancer than in those without due to the addition of cancer-specific risk factors to those already present for CKD. In this review, we describe the evaluation of kidney function in patients undergoing anticancer drug therapy. When anticancer drug therapy is administered, kidney function is evaluated to (1) set the dose of renally excretable drugs, (2) detect kidney disease associated with the cancer and its treatment, and (3) obtain baseline values for long-term monitoring. Owing to some requirements for use in clinical practice, a GFR estimation method such as the Cockcroft-Gault, MDRD, CKD-EPI, and the Japanese Society of Nephrology's GFR estimation formula has been developed that is simple, inexpensive, and provides rapid results. However, an important clinical question is whether they can be used as a method of GFR evaluation in patients with cancer. When designing a drug dosing regimen in consideration of kidney function, it is important to make a comprehensive judgment, recognizing that there are limitations regardless of which estimation formula is used or if GFR is directly measured. Although CTCAEs are commonly used as criteria for evaluating kidney disease-related adverse events that occur during anticancer drug therapy, a specialized approach using KDIGO criteria or other criteria is required when nephrologists intervene in treatment. Each drug is associated with the different disorders related to the kidney. And various risk factors for kidney disease associated with each anticancer drug therapy.

A case of chyluria with nephrotic-range proteinuria caused by lymphatic malformation, leading to a diagnosis of Klippel-Trenaunay syndrome.

Klippel-Trenaunay syndrome (KTS) is a rare syndrome, which is clinically diagnosed by the presence of unilateral limb hypertrophy with vascular malformation including cutaneous capillaries, veins and lymphatic vessels. Most cases typically exhibit cutaneous manifestations such as port-wine stains and limb hypertrophy from infancy, but cases with mild manifestations may remain undiagnosed. We here report a case of KTS who was diagnosed by chance chyluria. A 15-year-old girl who exhibited hematochyluria with nephrotic-range proteinuria was referred to our hospital. She had been diagnosed as idiopathic scoliosis accompanied by left lower limb hypertrophy in the past. She noticed her milky urine for the first time two months before. Immediately thereafter, she noticed edema of her left leg. Hematochyluria with nephrotic-range proteinuria was found by our initial urine examination. Magnetic resonance imaging suggested venous or lymphatic malformation along the left common iliac vein at the retroperitoneal side. Lymphoscintigraphy showed congestion of radioisotope around backside of the pancreas to the left renal hilus, suggesting an existence of lymphostasis. Based on the findings, we diagnosed the patient as KTS. After admission, hematochyluria and proteinuria were decreased and became insignificant by three days with bed rest. Her left leg edema was reduced. After taking a guidance to avoid intensive exercise, she was discharged in two weeks. Because the present case exhibited mild manifestations, diagnosis was made by urine abnormalities for the first time. The case suggests that we should be aware of the presence of undiagnosed patients of KTS due to relatively mild manifestations.

The serine protease plasmin plays detrimental roles in epithelial sodium channel activation and podocyte injury in Dahl salt-sensitive rats.

Salt-sensitive hypertension is associated with poor clinical outcomes. The epithelial sodium channel (ENaC) in the kidney plays pivotal roles in sodium reabsorption and blood pressure regulation, in which its γ subunit is activated by extracellular serine proteases. In proteinuric nephropathies, plasmin filtered through injured glomeruli reportedly activates γENaC in the distal nephron and causes podocyte injury. We previously reported that Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet developed hypertension and proteinuria along with γENaC activation and that a synthetic serine protease inhibitor, camostat mesilate, mitigated these changes. However, the role of plasmin in DS rats remained unclear. In this study, we evaluated the relationship between plasmin and hypertension as well as podocyte injury and the effects of plasmin inhibitors in DS rats. Five-week-old DS rats were divided into normal-salt diet, HS diet, and HS+plasmin inhibitor (either tranexamic acid [TA] or synthetic plasmin inhibitor YO-2) groups. After blood pressure measurement and 24 h urine collection over 5 weeks, rats were sacrificed for biochemical analyses. The HS group displayed severe hypertension and proteinuria together with activation of plasmin in urine and γENaC in the kidney, which was significantly attenuated by YO-2 but not TA. YO-2 inhibited the attachment of plasmin(ogen) to podocytes and alleviated podocyte injury by inhibiting apoptosis and inflammatory/profibrotic cytokines. YO-2 also suppressed upregulation of protease-activated receptor-1 and phosphorylated ERK1/2. These results indicate an important role of plasmin in the development of salt-sensitive hypertension and related podocyte injury, suggesting plasmin inhibition as a potential therapeutic strategy.

Increased numbers of pre-operative circulating monocytes predict risk of developing cardiac surgery-associated acute kidney injury in conditions requiring cardio pulmonary bypass.

BACKGROUND: Evaluating patients' risk for acute kidney injury (AKI) is crucial for positive outcomes following cardiac surgery. Our aims were first to select candidate risk factors from pre- or intra-operative real-world parameters collected from routine medical care and then evaluate potential associations between those parameters and risk of onset of post-operative cardiac surgery-associated AKI (CSA-AKI). METHOD: We conducted two cohort studies in Japan. The first was a single-center prospective cohort study (n = 145) to assess potential association between 115 clinical parameters collected from routine medical care and CSA-AKI (≥ Stage1) risk in the population of patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB). To select candidate risk factors, we employed random forest analysis and applied survival analyses to evaluate association strength. In a second retrospective cohort study, we targeted patients undergoing cardiac surgery with CPB (n = 619) and evaluated potential positive associations between CSA-AKI incidence and risk factors suggested by the first cohort study. RESULTS: Variable selection analysis revealed that parameters in clinical categories such as circulating inflammatory cells, CPB-related parameters, ventilation, or aging were potential CSA-AKI risk factors. Survival analyses revealed that increased counts of pre-operative circulating monocytes and neutrophils were associated with CSA-AKI incidence. Finally, in the second cohort study, we found that increased pre-operative circulating monocyte counts were associated with increased CSA-AKI incidence. CONCLUSIONS: Circulating monocyte counts in the pre-operative state are associated with increased risk of CSA-AKI development. This finding may be useful in stratifying patients for risk of developing CSA-AKI in routine clinical practice.

Camostat mesilate, a serine protease inhibitor, exerts aquaretic effects and decreases urinary exosomal AQP2 levels.

Serine proteases (SPs) play physiological roles in the kidney. We previously reported that a synthetic SP inhibitor, camostat mesilate (CM), suppressed sodium reabsorption in the renal tubule and showed natriuretic effects in aldosterone-infused rats. Here, we aimed to explore novel physiological roles of SPs in the renal tubule and understand the mechanism of actions of SP inhibitors, by administering CM to healthy rats. Sprague-Dawley rats were classified into control and CM (subcutaneous sustained-release pellet) groups and sacrificed on day 7. CM significantly increased urine volumes by approximately two-fold in a urinary sodium- and osmolyte excretion-independent manner, indicating the occurrence of free water excretion. Serum vasopressin, potassium, and calcium levels and the osmolality in the renal medulla, which all affect free water reabsorption in the renal tubule, remained unchanged after CM administration. CM decreased urinary exosomal AQP2 excretion, suggesting suppression of AQP2 activity in the collecting duct. These changes were reversed by desmopressin infusion. Water diuresis caused by CM was independent of its action on prostasin or TMPRSS4. Our results revealed the association of SP inhibition with free water handling and demonstrated that CM administration exerted diuretic effects with AQP2 downregulation, suggesting SP inhibitors as a new class of aquaretic drugs.

A novel VCP modulator KUS121 exerts renoprotective effects in ischemia-reperfusion injury with retaining ATP and restoring ERAD-processing capacity.

Acute kidney injury (AKI) is a life-threatening condition and often progresses to chronic kidney disease or the development of other organ dysfunction even after recovery. Despite the increased recognition and high prevalence of AKI worldwide, there has been no established treatment so far. The aim of this study was to investigate the renoprotective effect of Kyoto University substance 121 (KUS121), a novel valosin-containing protein modulator, on AKI. In in vitro experiments, we evaluated cell viability and ATP levels of proximal tubular cells with or without KUS121 under endoplasmic reticulum (ER) stress conditions. In in vivo experiments, the effects of KUS121 were examined in mice with AKI caused by ischemia-reperfusion injury. ER-associated degradation (ERAD)-processing capacity was evaluated by quantification of the ERAD substrate CD3delta-YFP. KUS121 protected proximal tubular cells from cell death under ER stress. The apoptotic response was mitigated as indicated by the suppression of C/EBP homologous protein expression and caspase-3 cleavage, with maintained intracellular ATP levels by KUS121 administration. KUS121 treatment suppressed the elevation of serum creatinine and neutrophil gelatinase-associated lipocalin levels and attenuated renal tubular damage after ischemia-reperfusion. The expression of inflammatory cytokines in the kidney was also suppressed in the KUS121-treated group. Valosin-containing protein expression levels were not altered by KUS121 both in vitro and in vivo. KUS121 treatment restored ERAD-processing capacity associated with potentiation of its upstream pathway, phosphorylated inositol-requiring enzyme-1α, and spliced X box-binding protein-1. In conclusion, these findings indicate that KUS121 can protect renal tubular cells from ER stress-induced injury, suggesting that KUS121 could be a novel and promising therapeutic compound for ischemia-associated AKI.NEW & NOTEWORTHY Novel findings of this study are as follows: 1) Kyoto University substance 121 (KUS121), a novel valosin-containing protein (VCP) modulator, can reduce ATP consumption of VCP; 2) KUS121 reduced endoplasmic reticulum (ER) stress and improved cell viability in proximal tubular cells; 3) KUS121 exerted renoprotective effects against ischemia-reperfusion injury; and 4) KUS121 may prevent ischemic acute kidney injury with ATP retention and restoring ER-associated degradation capacity.

Osteocrin, a bone-derived humoral factor, exerts a renoprotective role in ischemia-reperfusion injury in mice.

BACKGROUND: Osteocrin (OSTN), a bone-derived humoral factor, was reported to act on heart and bone by potentiating the natriuretic peptide (NP) system. Ostn gene polymorphisms have been associated with renal function decline, but its pathophysiological role in the kidney remains unclear. METHODS: The role of endogenous OSTN was investigated using systemic Ostn-knockout (KO) mice. As a model for OSTN administration, liver-specific Ostn-overexpressing mice crossed with KO (KO-Tg) were generated. These mice were subjected to unilateral ischemia-reperfusion injury (IRI) and renal lesions after 21 days of insult were evaluated. A comprehensive analysis of the Wnt/ß-catenin pathway was performed using a polymerase chain reaction (PCR) array. Reporter plasmid-transfected proximal tubular cells (NRK52E) were used to investigate the mechanism by which OSTN affects the pathway. RESULTS: After injury, KO mice showed marginal worsening of renal fibrosis compared with wild-type mice, with comparable renal atrophy. KO-Tg mice showed significantly ameliorated renal atrophy, fibrosis and tubular injury, together with reduced expressions of fibrosis- and inflammation-related genes. The PCR array showed that the activation of the Wnt/ß-catenin pathway was attenuated in KO-Tg mice. The downstream targets Mmp7, Myc and Axin2 showed similar results. MMP7 and Wnt2 were induced in corticomedullary proximal tubules after injury, but not in KO-Tg. In NRK52E, OSTN significantly potentiated the inhibitory effects of NP on transforming growth factor ß1-induced activation of the Wnt/ß-catenin pathway, which was reproduced by a cyclic guanosine monophosphate analog. CONCLUSIONS: Ectopic Ostn overexpression ameliorated subsequent renal injury following ischemia-reperfusion. OSTN could represent possible renoprotection in acute to chronic kidney disease transition, thus serving as a potential therapeutic strategy.

Hyperglycemia and Thrombocytopenia　- Combinatorially Increase the Risk of Mortality in Patients With Acute Myocardial Infarction Undergoing Veno-Arterial Extracorporeal Membrane Oxygenation.

Background: Patients with cardiogenic shock due to acute myocardial infarction (AMI) can rapidly undergo veno-arterial extracorporeal membrane oxygenation (VA-ECMO) therapy to recover cardiac output and decrease mortality. However, the clinical indicators predictive of mortality in these patients remain unknown. Methods and Results: We conducted a single-center retrospective cohort study targeting AMI patients undergoing VA-ECMO. All 63 patients undergoing VA-ECMO for AMI at the Japanese Red Cross Kumamoto Hospital between January 1, 2010 and June 30, 2020 were enrolled. An exploratory analysis was conducted using a survival tree model and variables selected in a univariate Cox proportional hazard model. The median survival time from the start of VA-ECMO was 6.3 days, and 77.8% (n=49) of patients died. Survival analysis divided patients into 3 groups based on 2 parameters at the initial medical examination: Group 1, patients with neither hyperglycemia (blood glucose ≥213 mg/dL) nor thrombocytopenia (platelets ≤145,100/µL); Group 2, patients with hyperglycemia; and Group 3, patients with hyperglycemia plus thrombocytopenia. Relative to Group 1, the risk of in-hospital mortality was significantly increased in Group 2 (hazard ratio [HR] 2.25; 95% confidence interval [CI] 1.13-4.46), and that risk further increased in Group 3 (HR 7.60; 95% CI 3.21-17.95). Conclusions: Hyperglycemia plus thrombocytopenia on initial medical examination combinatorially increase the risk of mortality in patients with cardiogenic shock due to AMI undergoing VA-ECMO.

Vaccine targeting ANGPTL3 ameliorates dyslipidemia and associated diseases in mouse models of obese dyslipidemia and familial hypercholesterolemia.

Dyslipidemia is a risk factor for cardiovascular disease (CVD), a major cause of death worldwide. Angiopoietin-like protein 3 (ANGPTL3), recognized as a new therapeutic target for dyslipidemia, regulates the metabolism of low-density lipoprotein-cholesterol (LDL-C) and triglycerides. Here, we design 3 epitopes (E1-E3) for use in development of a peptide vaccine targeting ANGPTL3 and estimate effects of each on obesity-associated dyslipidemia in B6.Cg-Lepob /J (ob/ob) mice. Vaccination with the E3 (32EPKSRFAMLD41) peptide significantly reduces circulating levels of triglycerides, LDL-C, and small dense (sd)-LDL-C in ob/ob mice and decreases obese-induced fatty liver. Moreover, E3 vaccination does not induce cytotoxicity in ob/ob mice. Interestingly, the effect of E3 vaccination on dyslipidemia attenuates development of atherosclerosis in B6.KOR/StmSlc-Apoeshl mice fed a high-cholesterol diet, which represent a model of severe familial hypercholesterolemia (FH) caused by ApoE loss of function. Taken together, ANGPTL3 vaccination could be an effective therapeutic strategy against dyslipidemia and associated diseases.

Osteocrin ameliorates adriamycin nephropathy via p38 mitogen-activated protein kinase inhibition.

Natriuretic peptides exert multiple effects by binding to natriuretic peptide receptors (NPRs). Osteocrin (OSTN) binds with high affinity to NPR-C, a clearance receptor for natriuretic peptides, and inhibits degradation of natriuretic peptides and consequently enhances guanylyl cyclase-A (GC-A/NPR1) signaling. However, the roles of OSTN in the kidney have not been well clarified. Adriamycin (ADR) nephropathy in wild-type mice showed albuminuria, glomerular basement membrane changes, increased podocyte injuries, infiltration of macrophages, and p38 mitogen-activated protein kinase (MAPK) activation. All these phenotypes were improved in OSTN- transgenic (Tg) mice and NPR3 knockout (KO) mice, with no further improvement in OSTN-Tg/NPR3 KO double mutant mice, indicating that OSTN works through NPR3. On the contrary, OSTN KO mice increased urinary albumin levels, and pharmacological blockade of p38 MAPK in OSTN KO mice ameliorated ADR nephropathy. In vitro, combination treatment with ANP and OSTN, or FR167653, p38 MAPK inhibitor, reduced Ccl2 and Des mRNA expression in murine podocytes (MPC5). OSTN increased intracellular cyclic guanosine monophosphate (cGMP) in MPC5 through GC-A. We have elucidated that circulating OSTN improves ADR nephropathy by enhancing GC-A signaling and consequently suppressing p38 MAPK activation. These results suggest that OSTN could be a promising therapeutic agent for podocyte injury.

Salt-Sensitive Hypertension of the Renal Tubular Cell-Specific NFAT5 (Nuclear Factor of Activated T-Cells 5) Knockout Mice.

[Figure: see text].

A serine protease inhibitor camostat mesilate prevents podocyte apoptosis and attenuates podocyte injury in metabolic syndrome model rats.

Metabolic syndrome (MetS) is associated with chronic kidney disease and proteinuria. Previously, we reported that a synthetic serine protease inhibitor, camostat mesilate (CM), mitigated hypertension and proteinuria in rodent disease models. The present study evaluated the anti-hypertensive and anti-proteinuric effects of CM in MetS model rats (SHR/ND mcr-cp). Rats were divided into normal salt-fed (NS), high salt-fed (HS), HS and CM-treated (CM), and HS and hydralazine-treated (Hyd) groups. Rats were sacrificed after four weeks of treatment. Severe hypertension and proteinuria were observed in the HS group. Although CM and Hyd equally alleviated hypertension, CM suppressed proteinuria and glomerular sclerosis more efficiently than Hyd. The HS group revealed a decrease in podocyte number and podocyte-specific molecules, together with an increase in glomerular apoptotic cells and apoptosis-related proteins in the kidney. These changes were significantly attenuated by CM, but not by Hyd. Furthermore, CM ameliorated the apoptotic signals in murine cultured podocytes stimulated with the high glucose and aldosterone medium. In conclusion, CM could exert renoprotective effects in MetS model rats, together with the inhibition of podocyte apoptosis. Our study suggests that serine protease inhibition may become a new therapeutic strategy against MetS-related hypertension and renal injuries.