Suboptimal initiation predicts short-term prognosis and vulnerability among very elderly patients who start haemodialysis.

AIM: A recent, growing concern regarding haemodialysis in Japan is a sustained increase in the elderly population. Among very elderly people who start haemodialysis, the prognosis is considered to be poor; however, this has not been fully elucidated. This study aimed to discover the short-term prognosis and related factors in very elderly patients who commence haemodialysis. METHODS: Between January 2008 and December 2013, 122 patients aged ≥85 years at haemodialysis initiation were documented in our hospital. Predictors of 90-day and 1-year mortality after haemodialysis initiation were assessed with Cox proportional hazards regression analysis. Selection of covariates for the multivariate model was based on forward stepwise selection using the probability of a likelihood ratio statistics. RESULTS: The subjects' mean age was 87.4 ± 2.5 years, and 48% were female. The most common cause of death was infection (38% of patients) and the leading cause of infectious death was pneumonia. The 90-day and 1-year survival rates were 81% and 62%, respectively. Suboptimal initiation was a significant prognostic factor for 90-day [hazard ratio (HR) 3.98, 95% confidence interval (CI) 1.18-13.43] and 1-year [HR 3.19, 95% CI 1.51-6.76] mortality after adjusting for confounders in multivariate analysis. CONCLUSION: Very elderly patients who started haemodialysis had a poor prognosis, and suboptimal initiation significantly predicted outcome. Shared decision-making with patients and their families is needed for initiating haemodialysis on the conditions that appropriate information on the expected prognosis is provided.

Tonicity-Responsive Enhancer-Binding Protein Mediates Hyperglycemia-Induced Inflammation and Vascular and Renal Injury.

Diabetic nephropathy (DN) has become the single leading cause of ESRD in developed nations. Bearing in mind the paucity of effective treatment for DN and progressive CKD, novel targets for treatment are sorely needed. We previously reported that increased activity of tonicity-responsive enhancer-binding protein (TonEBP) in monocytes was associated with early DN in humans. We now extend these findings by testing the hypotheses that TonEBP in macrophages promotes hyperglycemia-mediated proinflammatory activation and chronic renal inflammation leading to DN and CKD, and TonEBP genetic variability in humans is associated with inflammatory, renal, and vascular function-related phenotypes. In a mouse model of DN, compared with the wild-type phenotype, TonEBP haplodeficiency associated with reduced activation of macrophages by hyperglycemia, fewer macrophages in the kidney, lower renal expression of proinflammatory genes, and attenuated DN. Furthermore, in a cohort of healthy humans, genetic variants within TonEBP associated with renal function, BP, and systemic inflammation. One of the genetic variants associated with renal function was replicated in a large population-based cohort. These findings suggest that TonEBP is a promising target for minimizing diabetes- and stress-induced inflammation and renovascular injury.

Gross Hematuria after the COVID-19 mRNA Vaccination: Nationwide Multicenter Prospective Cohort Study in Japan.

BACKGROUND: In the past three years, cases of gross hematuria (GH) after the vaccination for coronavirus disease 2019 (COVID-19) in IgA nephropathy (IgAN) patients have been frequently reported worldwide. However, the post-event renal prognosis of these patients, their clinical backgrounds, and underlying mechanisms remain unknown. Therefore, we conducted a nationwide multicenter prospective cohort study in Japan. METHODS: We analyzed laboratory findings at the time of the first presentation to the hospital, and 3 and 6 months after in patients with GH after the vaccination, and histopathological findings in their kidney biopsy specimens. Moreover, changes in pathological biomarkers of IgAN such as galactose-deficient IgA1 (Gd-IgA1) and its immune complexes (ICs) were also evaluated. RESULTS: During the study period, 127 newly presenting with GH after the vaccination were enrolled, with a clear female bias (73.2%). GH was observed after the second or subsequent vaccinations in most patients (92.9%). Of the 37 patients undergoing kidney biopsy prior to the vaccination, 36 patients had been diagnosed with IgAN/IgA vasculitis (IgAV). In remaining 90 patients, 69 of the 70 who newly underwent kidney biopsy were diagnosed with IgAN (N=67)/IgAV (N=2). Their histopathology did not show a high incidence of acute lesions such as endocapillary hypercellularity and crescentic lesions. Most cases showed a temporary increase in proteinuria, but no sustained worsening in renal function. Among the biomarkers measured, serum Gd-IgA1 and ICs were comparable throughout the observation period, however, only urinary Gd-IgA1 was increased at the time of GH. CONCLUSIONS: We found that GH after the vaccination is more likely to occur in IgAN/IgAV patients, with a female bias, but without progressive exacerbation of renal function. Although further investigation is needed regarding causal relationship between vaccination and GH, this study provides many insights into the molecular mechanisms of GH.

Multiple cell death pathways are independently activated by lethal hypertonicity in renal epithelial cells.

When hypertonicity is imposed with sufficient intensity and acuteness, cells die. Here we investigated the cellular pathways involved in death using a cell line derived from renal epithelium. We found that hypertonicity rapidly induced activation of an intrinsic cell death pathway-release of cytochrome c and activation of caspase-3 and caspase-9-and an extrinsic pathway-activation of caspase-8. Likewise, a lysosomal pathway of cell death characterized by partial lysosomal rupture and release of cathepsin B from lysosomes to the cytosol was also activated. Relationships among the pathways were examined using specific inhibitors. Caspase inhibitors did not affect cathepsin B release into the cytosol by hypertonicity. In addition, cathepsin B inhibitors and caspase inhibitors did not affect hypertonicity-induced cytochrome c release, suggesting that the three pathways were independently activated. Combined inhibition of caspases and cathepsin B conferred significantly more protection from hypertonicity-induced cell death than inhibition of caspase or cathepsin B alone, indicating that all the three pathways contributed to the hypertonicity-induced cell death. Similar pattern of sensitivity to the inhibitors was observed in two other cell lines derived from renal epithelia. We conclude that multiple cell death pathways are independently activated early in response to lethal hypertonic stress in renal epithelial cells.

Renal recovery following daratumumab, lenalidomide, and dexamethasone therapy in a patient with newly diagnosed dialysis-dependent multiple myeloma.

An 81-year-old Japanese woman was diagnosed with Bence Jones protein κ-type multiple myeloma with acute kidney injury and severe anemia, complicated by congestive heart failure with triple vessel coronary artery disease. Her serum κ-free light-chain (FLC) level was 49,400 mg/L and κ/λ ratio was extremely high at 2373. Her kidney function deteriorated rapidly and required hemodialysis before initiating chemotherapy. A combination therapy of daratumumab (16 mg/kg), lenalidomide, and dexamethasone was initiated as a first-line treatment; the infusion rate of daratumumab was adjusted to reduce the heart load. The level of κ-FLC was rapidly reduced by 75% in only one week and by 99% after three weeks. Furthermore, she was dialysis-independent after the fourth dose of daratumumab. We report the first case of untreated patient with myeloma who had been successfully treated with daratumumab, lenalidomide, and dexamethasone therapy even in dialysis requiring state. Daratumumab may benefit patients with acute kidney injury caused by multiple myeloma, owing to the immediate need of FLC level reduction. Daratumumab and lenalidomide combination therapy could be a valuable treatment option for patients requiring dialysis when bortezomib may be hesitate to use due to severe heart disease.

Bortezomib-induced glomerular microangiopathy complicated with monoclonal immunoglobulin deposition disease.

A 75-year-old man admitted with IgG λ-type myeloma with creatinine level of 2.3 mg/dL. Serum lactate dehydrogenase level and platelet count were normal. Urinalysis demonstrated massive proteinuria dominated by albuminuria. Weekly bortezomib and dexamethasone therapy were started to treat myeloma but failed to be continued because of rapid deterioration of renal function and increase in proteinuria 1 week after the treatment. His renal function exacerbated to require hemodialysis for a month. There was no clinical evidence of tumor lysis syndrome or thrombocytopenia throughout the course of his acute kidney injury (AKI). After he became dialysis independent, a renal biopsy was performed to clarify myeloma-related renal involvement and the cause of AKI. As a result, IgG2-λ monoclonal immunoglobulin deposition disease (MIDD) and severe endothelial injury were revealed. There was no evidence of cast nephropathy. Bortezomib-induced glomerular microangiopathy (GMA) superimposed on MIDD. Bortezomib has a potential risk to cause drug-induced GMA without systemic thrombotic microangiopathy, in which vascular endothelial growth factor-nuclear factor-κ B pathway could be involved. This is the first case of biopsy-proven bortezomib-induced GMA. If proteinuria (mainly albuminuria) increases after using bortezomib, GMA should be suspected as an adverse effect of bortezomib even absent of clinical signs of systemic thrombotic microangiopathy.

Using Polyethylene Glycol 3350 Plus Electrolytes in Constipated Hemodialysis Patients: A Case Series.

Appropriate management of constipation in hemodialysis patients has not been established, although constipation is the most frequent gastrointestinal complication in dialysis patients. We herein report the efficacy and safety of polyethylene glycol in constipated hemodialysis patients assessed prospectively. Seven patients using stimulant laxatives participated in this study. Polyethylene glycol was administered to reduce stimulant laxatives during the six-week intervention period. The amount of stimulant laxatives decreased and spontaneous bowel movements with ideal stool consistency increased significantly after the intervention. No serious adverse effects were observed throughout this study. In conclusion, polyethylene glycol can be a useful tool for managing constipated hemodialysis patients.

Successful management of hemodialysis-dependent refractory myeloma with modified daratumumab, bortezomib and dexamethasone regimen.

A 71-year-old petite Japanese woman was diagnosed with IgG λ-type multiple myeloma with acute kidney injury, severe anemia, and a pathological rib fracture. Emergent hemodialysis was initiated combined with chemotherapy including bortezomib, lenalidomide, and pomalidomide, but myeloma had become refractory due to the treatments. Therefore, a combination therapy with weekly daratumumab (16 mg/kg), bortezomib (0.7 mg/m2), and dexamethasone was started. Daratumumab was administered on a non-dialysis day with a reduced infusion speed to avoid acute water load. No infusion-related adverse events were observed throughout the treatment. Daratumumab and bortezomib were administrated weekly for three times in the first cycle and a hematological very good partial response was achieved. Then, the treatment schedule was reduced to once every three weeks from the 2nd cycle, the very good partial response had been maintained. Fourteen months after the initiation of maintenance hemodialysis, the patient was able to reduce dialysis frequency due to improvement of renal function. A modified daratumumab, bortezomib and dexamethasone regimen could be a valuable treatment option for dialysis-dependent myeloma patients.

Potential for erythropoietin synthesis in kidney of uraemic rat alters depending on severity of renal failure.

AIM: Renal anaemia is a common early complication of chronic renal failure (CRF) that is characterized by relative erythropoietin (EPO) deficiency. Although a lowered renal function is considered to induce limited EPO production, potential EPO production capacity in CRF remains unclear. The aim of this study was to determine the mechanisms underlying this relative deficiency. METHODS: Male Sprague-Dawley rats were underwent 5/6 nephrectomy with different severities of CRF. These rats were assigned to two groups - mild CRF or advanced CRF - and subjected to haemodilution by exchange of blood with Ringer's solution or haemoconcentration by blood transfusion. Serum EPO and EPO transcript levels in remnant kidney were examined. Expression levels of hypoxia-related genes, including heme oxygenase-1 (HO-1) and glucose transporter-1 (Glut-1), were also examined. RESULTS: Haemodilution increased both serum EPO and EPO transcript levels in mild CRF, as observed in sham-operated controls, whereas the extents of such increases were significantly smaller in advanced CRF. HO-1 and Glut-1 transcript levels also increased by haemodilution in mild CRF, but not in advanced CRF. Haemoconcentration markedly decreased serum EPO and EPO transcript levels in mild CRF as in controls. Rats with advanced CRF did not survive after blood transfusion. CONCLUSION: Potential EPO regulation capacity in mild CRF is as conserved as that in normal control, whereas that in advanced CRF is impaired, suggesting that underlying mechanisms of low EPO production alters according to the stage of CRF.

Chemokine Receptor 8 Can Distinguish Antineutrophil Cytoplasmic Antibody-Associated Vasculitis From Infectious Complications.

INTRODUCTION: Diagnosing vasculitis is frequently difficult because its clinical symptoms are similar to those of common infectious diseases and other inflammatory disorders. This study focused on chemokine receptor 8 (CCR8) in peripheral blood mononuclear cells to find a new biomarker that distinguishes vasculitis from infectious complications. METHODS: A cross-sectional study was conducted among 113 patients with systemic vasculitis who were referred to Japan Health Care Organization Sendai Hospital from 2014 to 2016, including those with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, anti-glomerular basement membrane disease, lupus nephritis, and Henoch-Schonlein purpura. Peripheral blood mononuclear cells were extracted from blood, and CCR8 expression was examined by real time polymerase chain reaction and flow cytometry. RESULTS: CCR8 gene expression was significantly higher in patients with ANCA-associated vasculitis, which was confirmed by upregulated CCR8 protein expression in flow cytometry (P < 0.001 and P = 0.01, respectively). Neither lupus nephritis nor Henoch-Schonlein purpura showed upregulated CCR8. Elevated CCR8 in the active phase decreased significantly in remission (P = 0.002), which was correlated with decreased serum inflammatory markers. Despite elevated serological inflammatory markers, the CCR8 levels at the time of infection, including bacterial, viral, and fungal, did not increase, indicating that infectious complications did not affect CCR8 expression (P = 0.02). CONCLUSION: CCR8 in peripheral blood mononuclear cells may be a useful diagnostic marker for ANCA-associated vasculitis to differentiate between active vasculitis and infectious inflammation.

Transcriptional Regulator TonEBP Mediates Oxidative Damages in Ischemic Kidney Injury.

TonEBP (tonicity-responsive enhancer binding protein) is a transcriptional regulator whose expression is elevated in response to various forms of stress including hyperglycemia, inflammation, and hypoxia. Here we investigated the role of TonEBP in acute kidney injury (AKI) using a line of TonEBP haplo-deficient mice subjected to bilateral renal ischemia followed by reperfusion (I/R). In the TonEBP haplo-deficient animals, induction of TonEBP, oxidative stress, inflammation, cell death, and functional injury in the kidney in response to I/R were all reduced. Analyses of renal transcriptome revealed that genes in several cellular pathways including peroxisome and mitochondrial inner membrane were suppressed in response to I/R, and the suppression was relieved in the TonEBP deficiency. Production of reactive oxygen species (ROS) and the cellular injury was reproduced in a renal epithelial cell line in response to hypoxia, ATP depletion, or hydrogen peroxide. The knockdown of TonEBP reduced ROS production and cellular injury in correlation with increased expression of the suppressed genes. The cellular injury was also blocked by inhibitors of necrosis. These results demonstrate that ischemic insult suppresses many genes involved in cellular metabolism leading to local oxidative stress by way of TonEBP induction. Thus, TonEBP is a promising target to prevent AKI.

Characteristics of young-onset white coat hypertension identified by targeted screening for hypertension at a university health check-up.

Previously we estimated the prevalence of essential hypertension (EH) as around 0.1% and suggested that male gender, obesity, and strong genetic background (hypertension in parents) were risk factors for EH in a young population aged less than 30 based on targeted screening for hypertension at a university health check-up. This study also revealed a high incidence of white coat hypertension (WCH) in university students, and thus, we continued this screening for four consecutive years, and examined the prognosis and clinical characteristics of young-onset WCH. Three occasions of casual blood pressure (BP) measurement and additional home BP measurement revealed 72 WCH and 15 EH students (all males) during the 4-year study period. None of the WCH students had elevated home BP to the level of hypertension during their stay at university, and 26 out of 38 WCH students participating screening in the following years showed normal casual BP. Although WCH students showed a significantly higher pulse rate than controls, WCH could not be fully differentiated from EH either by pulse rate or by correlation between casual BP value and pulse rate. These findings indicate the requirement of longer follow-up after graduation to determine the prognosis of young-onset WCH, though EH and WCH in the young population share the same risk factors and, possibly, autonomic nervous system dysfunction. Since diagnosis of WCH has limited importance for university students, screening of EH following a general health check-up would elevate the clinical validity of casual BP measurement at the university.

Efficacy of statin on vascular access patency in diabetic hemodialysis patients.

INTRODUCTION: An effective approach to prevent hemodialysis vascular access dysfunction is still unclear despite previous studies, which have shown conflicting results of several drugs on vascular access outcomes. In this study, we focused on diabetic hemodialysis patients with native arteriovenous fistula and evaluated the impact of statin treatment on vascular access patency. METHODS: A retrospective cohort study of 268 consecutive patients who newly started hemodialysis due to diabetic nephropathy between January 2011 and December 2013 at Japan Community Health Care Organization Sendai Hospital was performed and the patients were followed for two years. The primary outcome was vascular access dysfunction. Effect of statin treatment was examined using Kaplan Meier analysis and Cox proportional hazard, after adjusting for covariates. RESULTS: The mean follow-up period was 426.7 days, and 117 (52.2%) patients developed vascular access dysfunction. The two-year patency rate was 55.0% among statin users and 36.1% in non-users. Vascular access survival period was significantly longer among statin users (log-rank test, p = 0.004). In multivariable analysis, statin treatment is significantly associated with better vascular access outcomes, in which the hazard ratio was 0.71 (95% CI, 0.52 to 0.97; p = 0.028) in the unadjusted model and 0.63 (95% CI, 0.45 to 0.88; p = 0.007) after adjustment for covariates. CONCLUSIONS: Statin treatment could be associated with improved vascular access dysfunction among diabetic hemodialysis patients.

Marked recovery of severe renal lesions in POEMS syndrome with high-dose melphalan therapy supported by autologous blood stem cell transplantation.

POEMS syndrome is a rare plasma cell disorder, characterized by polyneuropathy, organomegaly, endocrinopathy, serum monoclonal protein, and skin lesions. Although not included in the acronym, renal lesions also are characteristic of this disease and sometimes require dialysis therapy. We treated a 61-year-old woman with POEMS syndrome with high-dose melphalan therapy (HDT) supported by autologous blood stem cell transplantation (SCT), and clinical remission was achieved. A repeated renal biopsy showed the striking effectiveness of this therapy on renal lesions. Pathological features of the renal lesions, such as membranoproliferative glomerulonephritis-like lesions, microangiopathic glomerulopathy, and mesangiolytic lesions with microcapillaries, almost completely disappeared. This treatment also markedly decreased serum levels of vascular endothelial growth factor (VEGF). These findings indicate that HDT with SCT is effective, even on renal lesions in patients with POEMS syndrome, and suggest that high serum VEGF concentrations are associated closely with the development of renal lesions in patients with this type of plasma cell disorder.

Characteristics of young-onset hypertension identified by targeted screening performed at a university health check-up.

Since the prevalence and clinical characteristics of young-onset hypertension are still to be elucidated, we performed targeted-screening at an annual university health check-up for two consecutive years. Out of 16,464 subjects in 2003 and 17,032 in 2004 that were aged less than 30 years, 22 and 26 students (all males) exhibited high blood pressure (BP), respectively, on three occasions during casual BP measurements at the Tohoku University Health Center (systolic and diastolic BP of 140 and/or 90 mmHg or greater, respectively). These students were asked to measure their BP at home, and 9 subjects in total were diagnosed as having essential hypertension (EH). The remaining students were diagnosed as having white coat hypertension (WCH). In 8 out of 9 EH students, their father and/or mother had also been treated with antihypertensive medication. Adjustment by attendance ratio for each BP measurement suggested that the incidence of EH was around 0.1% and that of hypertension (EH and WCH) was around 0.5% in university students aged less than 25 years, since most of the subjects and hypertensive students were between 18 and 24 years old. Body mass index of the EH, which was more than 25 kg/m2 (overweight), was significantly higher than that with WCH. In conclusion, the combination of repeated casual BP measurements and home BP effectively identified young-onset EH. The clinical parameters indicated that male gender, genetic background, and excessive weight were risk factors for young-onset hypertension.

Transient body fluid accumulation and enhanced NKCC2 expression in gerbils with brain infarction.

BACKGROUND: Enhanced expression of a kidney-specific sodium co-transporter (NKCC2: Na-K-2Cl co-transporter) in the thick ascending limb of Henle has been identified in rat models of congestive heart failure and liver cirrhosis, suggesting that high NKCC2 expression underlies edema formation. An increased abundance of NKCC2, however, has also been noted in rats with the syndrome of inappropriate secretion of antidiuretic hormone; hyponatremia without edema. In the present study, we examined NKCC2 expression in non-edematous disease, such as a brain infarction, and investigated the physiological and/or pathological characterization of NKCC2 expression. METHODS: We initially examined NKCC2 expression in an animal model of brain infarction. Mongolian gerbils (around 60 g body weight) underwent bilateral clamping of the common carotid arteries for 5 min for the induction of brain infarction. NKCC2 and apical water channel (AQP2) protein levels in the collecting duct were examined by Western blotting in kidney tissues 2, 7, and 14 days after the brain infarction. Gerbils with brain infarction were then fed either a normal low-sodium diet (0.3 g/kg/day) or a high-sodium diet (3.0 g/kg/day), and body weight, urine volume and urinary osmolality were examined daily. Blood parameters were measured on day 14 after the brain infarction. RESULTS: Histochemical examination of the brain confirmed the presence of brain infarction, as manifested by altered cresyl violet staining in the hippocampus. Protein levels of NKCC2 were significantly increased in gerbils with brain infarction on days 2 and 7 after brain infarction, whereas AQP2 protein signals remained unaltered. However, the increased NKCC2 intensity disappeared on day 14. Body weight gain was slightly, but significantly greater in gerbils with brain infarction than in sham-operated gerbils up to 7 days after the brain infarction. The high-sodium diet resulted in significant urinary concentration and enhanced weight gain in infarcted gerbils. CONCLUSION: We noted increased NKCC2 abundance in non-edematous disease, which enhanced body fluid accumulation, likely via the sodium loading-dependent concentration of the urine. These results suggest that the physiological process of edema formation is based on specific NKCC2 expression. The transient duration of these findings in the present animal model suggests two different characteristics of specific NKCC2 expression, an immediate, transient appearance as a common response in serious conditions and more chronic expression that leads to edema formation.

LPS-induced NFκB enhanceosome requires TonEBP/NFAT5 without DNA binding.

NFκB is a central mediator of inflammation. Present inhibitors of NFκB are mostly based on inhibition of essential machinery such as proteasome and protein kinases, or activation of nuclear receptors; as such, they are of limited therapeutic use due to severe toxicity. Here we report an LPS-induced NFκB enhanceosome in which TonEBP is required for the recruitment of p300. Increased expression of TonEBP enhances the NFκB activity and reduced TonEBP expression lowers it. Recombinant TonEBP molecules incapable of recruiting p300 do not stimulate NFκB. Myeloid-specific deletion of TonEBP results in milder inflammation and sepsis. We discover that a natural small molecule cerulenin specifically disrupts the enhanceosome without affecting the activation of NFκB itself. Cerulenin suppresses the pro-inflammatory activation of macrophages and sepsis without detectable toxicity. Thus, the NFκB enhanceosome offers a promising target for useful anti-inflammatory agents.

A case report of the effect of plasma exchange on reactive hemophagocytic syndrome associated with toxic shock syndrome.

We report here the case of a patient suffering from hemophagocytic syndrome (HPS) associated with toxic shock syndrome (TSS). A 50-year-old man was admitted because of fever, watery diarrhea and shortness of breath. Clinical analysis revealed systemic cyanosis, sunburn-like erythema and septic shock. Staphylococcus aureus was identified from both blood and sputum culture and the serum enterotoxin A antibody test was positive, suggesting that this was a case of TSS. Though the respiratory and hemodynamic status improved by the mechanical ventilation, fluid resuscitation with catecholamine and antibiotic therapy, the platelet count decreased rapidly. Bone marrow aspiration revealed a large quantity of hemophagocytosis by macrophages. This reactive HPS was treated not with immunosuppressive drugs but with therapeutic plasma exchange in order to prevent worsening of S. aureus infection. After plasma exchange, the circulating macrophage colony-stimulating factor (M-CSF) level was reduced and the platelet count increased rapidly. Bacteria associated HPS remains a difficult diagnosis with high mortality and there is a crucial question of whether this should be treated with immunosuppressive drugs. The patient's clinical course would suggest that the therapeutic plasma exchange should be considered as a therapeutic tool for the bacteria associated HPS instead of immunosuppressive drugs.

Modulation of TonEBP activity by SUMO modification in response to hypertonicity.

TonEBP is a DNA binding transcriptional enhancer that enables cellular adaptation to hypertonic stress by promoting expression of specific genes. TonEBP expression is very high in the renal medulla because local hypertonicity stimulates its expression. Given the high level of expression, it is not well understood how TonEBP activity is modulated. Here we report that TonEBP is post-translationally modified by SUMO, i.e., sumoylated, in the renal medulla but not in other isotonic organs. The sumoylation is reproduced in cultured cells when switched to hypertonicity. Analyses of site-directed TonEBP mutants reveal that K556 and K603 are independently sumoylated in response to hypertonicity. DNA binding is required for the sumoylation. Functional analyses of non-sumoylated mutants and SUMO-conjugated constructs show that sumoylation inhibits TonEBP in a dose-dependent manner but independent of the site of SUMO conjugation. Sumoylation inhibits transactivation without affecting nuclear translocation or DNA binding. These data suggest that sumoylation modulates the activity of TonEBP in the hypertonic renal medulla to prevent excessive action of TonEBP.