Visualization of intracellular ATP dynamics in different nephron segments under pathophysiological conditions using the kidney slice culture system.

ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here, we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated the ATP synthesis pathway, and analyzed intracellular ATP dynamics using an ex vivo IR-mimicking model and a cisplatin nephropathy model. Proximal tubules (PTs) were found to be strongly dependent on oxidative phosphorylation (OXPHOS) using the inhibitor oligomycin A, whereas podocytes relied on both OXPHOS and glycolysis using phloretin an active transport inhibitor of glucose. We also confirmed that an ex vivo IR-mimicking model could recapitulate ATP dynamics in vivo; ATP recovery in PTs after reoxygenation varied depending on anoxic time length, whereas ATP in distal tubules (DTs) recovered well even after long-term anoxia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor, cimetidine, suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent (Mitochonic Acid 5) delayed the cisplatin-induced ATP decrease in PTs. Thus, our novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.

Expression pattern of Runt-related transcription factor (RUNX) family members and the role of RUNX1 during kidney development.

Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1f/f:HoxB7-Cre and Runx1f/f:R26-CreERT2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development.

Amino acid catabolite markers for early prognostication of pneumonia in patients with COVID-19.

Effective early-stage markers for predicting which patients are at risk of developing SARS-CoV-2 infection have not been fully investigated. Here, we performed comprehensive serum metabolome analysis of a total of 83 patients from two cohorts to determine that the acceleration of amino acid catabolism within 5 days from disease onset correlated with future disease severity. Increased levels of de-aminated amino acid catabolites involved in the de novo nucleotide synthesis pathway were identified as early prognostic markers that correlated with the initial viral load. We further employed mice models of SARS-CoV2-MA10 and influenza infection to demonstrate that such de-amination of amino acids and de novo synthesis of nucleotides were associated with the abnormal proliferation of airway and vascular tissue cells in the lungs during the early stages of infection. Consequently, it can be concluded that lung parenchymal tissue remodeling in the early stages of respiratory viral infections induces systemic metabolic remodeling and that the associated key amino acid catabolites are valid predictors for excessive inflammatory response in later disease stages.

Tertiary Lymphoid Tissues Are Microenvironments with Intensive Interactions between Immune Cells and Proinflammatory Parenchymal Cells in Aged Kidneys.

SIGNIFICANCE STATEMENT: Ectopic lymphoid structures called tertiary lymphoid tissues (TLTs) develop in several kidney diseases and are associated with poor renal prognosis. However, the mechanisms underlying TLT expansion and their effect on renal regeneration remain unclear. The authors report that single-nucleus RNA sequencing and validation experiments demonstrate that TLTs potentially amplify inflammation in aged injured kidneys. Lymphocytes within TLTs promote proinflammatory phenotypes of the surrounding proximal tubules and fibroblasts within the TLTs via proinflammatory cytokine production. These proinflammatory parenchymal cells then interact with immune cells by chemokine or cytokine production. Such cell-cell interactions potentially increase inflammation, expand TLTs, and exacerbate kidney injury. These findings help illuminate renal TLT pathology and suggest potential therapeutic targets. BACKGROUND: Ectopic lymphoid structures called tertiary lymphoid tissues (TLTs) develop in several kidney diseases and are associated with poor renal prognosis. However, the mechanisms that expand TLTs and underlie exacerbation of kidney injury remain unclear. METHODS: We performed single-nucleus RNA sequencing (snRNA-seq) on aged mouse kidneys with TLTs after ischemia-reperfusion injury. The results were validated using immunostaining, in situ hybridization of murine and human kidneys, and in vitro experiments. RESULTS: Using snRNA-seq, we identified proinflammatory and profibrotic Vcam1+ injured proximal tubules (PTs) with NF κ B and IFN-inducible transcription factor activation. VCAM1 + PTs were preferentially localized around TLTs and drove inflammation and fibrosis via the production of multiple chemokines or cytokines. Lymphocytes within TLTs expressed Tnf and Ifng at high levels, which synergistically upregulated VCAM1 and chemokine expression in cultured PT cells. In addition, snRNA-seq also identified proinflammatory and profibrotic fibroblasts, which resided within and outside TLTs, respectively. Proinflammatory fibroblasts exhibited STAT1 activation and various chemokine or cytokine production, including CXCL9/CXCL10 and B cell-activating factor, contributing to lymphocyte recruitment and survival. IFN γ upregulated the expression of these molecules in cultured fibroblasts in a STAT1-dependent manner, indicating potential bidirectional interactions between IFN γ -producing CXCR3 + T cells and proinflammatory fibroblasts within TLTs. The cellular and molecular components described in this study were confirmed in human kidneys with TLTs. CONCLUSIONS: These findings suggest that TLTs potentially amplify inflammation by providing a microenvironment that allows intense interactions between renal parenchymal and immune cells. These interactions may serve as novel therapeutic targets in kidney diseases involving TLT formation.

Lineage tracing analysis defines erythropoietin-producing cells as a distinct subpopulation of resident fibroblasts with unique behaviors.

Erythropoietin (Epo) is produced by a subpopulation of resident fibroblasts in the healthy kidney. We have previously demonstrated that, during kidney fibrosis, kidney fibroblasts including Epo-producing cells transdifferentiate into myofibroblasts and lose their Epo-producing ability. However, it remains unclear whether Epo-producing cells survive and transform into myofibroblasts during fibrosis because previous studies did not specifically label Epo-producing cells in pathophysiological conditions. Here, we generated EpoCreERT2/+ mice, a novel mouse strain that enables labeling of Epo-producing cells at desired time points and examined the behaviors of Epo-producing cells under pathophysiological conditions. Lineage-labeled cells that were producing Epo when labeled were found to be a small subpopulation of fibroblasts located in the interstitium of the kidney, and their number increased during phlebotomy-induced anemia. Around half of lineage-labeled cells expressed Epo mRNA, and this percentage was maintained even 16 weeks after recombination, supporting the idea that a distinct subpopulation of cells with Epo-producing ability makes Epo repeatedly. During fibrosis caused by ureteral obstruction, EpoCreERT2/+-labeled cells were found to transdifferentiate into myofibroblasts with concomitant loss of Epo-producing ability, and their numbers and the proportion among resident fibroblasts increased during fibrosis, indicating their high proliferative capacity. Finally, we confirmed that EpoCreERT2/+-labeled cells that lost their Epo-producing ability during fibrosis regained their ability after kidney repair due to relief of the ureteral obstruction. Thus, our analyses have revealed previously unappreciated characteristic behaviors of Epo-producing cells, which had not been clearly distinguished from those of resident fibroblasts.

CD153/CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury.

Tertiary lymphoid tissues (TLTs) facilitate local T and B cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here, we identified TNF superfamily CD153/CD30 signaling between 2 unique age-dependent lymphocyte subpopulations, CD153+PD-1+CD4+ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL-21 and IFN-γ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis, and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153/CD30 signaling in TLT formation and propose targeting the CD153/CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.

Rapidly progressive glomerulonephritis caused by tegafur/gimeracil/oteracil resulted in diabetes nephropathy, in a patient with minor risk of diabetes nephropathy: a case report.

A 79-year-old Japanese male with a history of type 2 diabetes mellitus (T2DM) for 16 years was admitted to evaluate possible renal disease. The T2DM was well controlled in this patient using nutrition therapy without the need for any diabetes medication, and both diabetes retinopathy and proteinuria were negative. At the age of 78 advanced colorectal cancer (stage IIIa) was diagnosed and laparoscopic-assisted colectomy was performed. Following this procedure, the patient began treatment with tegafur/gimeracil/oteracil (S-1), 80 mg twice daily for 28 days of 42-day cycle. The patient received S-1 for 6 months, during which time, serum albumin decreased from 3.0 g/dL to 1.1 g/dL, urinary protein increased from negative to 3.0 g/day, and serum creatinine increased from 0.9 mg/dL to 2.1 mg/dL. Treatment with S-1 was discontinued, and furosemide 180 mg and prednisolone 30 mg treatment was initiated; however, serum creatinine levels continued to increase to 7.2 mg/dL and proteinuria continued to increase reaching a nephrotic range. A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity was decreased to 27.0%. Renal biopsy showed Kimmelstiel-Wilson nodules, while immunofluorescence intensity of IgG subclass was IgG1 dominant, which was not compatible with diabetic nephropathy (DN). Plasma exchange was not affected. However, hemodialysis was initiated.The results of this investigation suggest that when S-1 monotherapy is performed in the case with DN, rapidly progressive glomerulonephritis (RPGN) may develop due to a condition similar to thrombotic microangiopathy, even in patients with a minor risk factor of DN.

Regression of renal amyloid deposits by VAD therapy plus autologous stem cell transplantation in a patient with primary AL amyloidosis.

We report a 58-year-old Japanese woman who presented with nephrotic syndrome. Steroid therapy and cyclosporine A administration were initiated, but hematological remission and renal response were not achieved. Renal biopsy revealed amyloid deposits in the mesangial region and the small arteries. Proteomic analysis based on laser microdissection and mass spectrometry showed that the amyloid deposits were composed of the constant region of the lambda light chain. She received vincristine, adriamycin, and dexamethasone therapy followed by high-dose melphalan and autologous stem cell transplantation, resulting in hematological complete remission and renal response with negative urinary Bence-Jones protein and proteinuria. Renal biopsy was performed four times during follow-up, demonstrating that amyloid deposits decreased gradually, while glomeruli showing global sclerosis increased from 3 to 62%. This case suggests that glomerular amyloid deposits can be cleared via tissue remodeling, if stem cells producing amyloid precursors are completely replaced by unrelated cells after stem cell transplantation.

Increase of 1,25 dihydroxyvitamin D in sarcoidosis patients with renal dysfunction.

INTRODUCTION: In sarcoidosis, renal involvement includes hypercalcemia-related nephrocalcinosis and granulomatous tubulointerstitial nephritis. Hypercalcemia is thought to be due to increased production of 1,25 dihydroxyvitamin D (1-25D), but 1-25D levels have not been evaluated in sarcoidosis patients with renal dysfunction. MATERIALS AND METHODS: We enrolled 9 sarcoidosis patients who underwent renal biopsy, and compared the serum 1-25D concentration and eGFR with those in 428 non-sarcoidosis patients who had renal dysfunction (stage 2 or higher CKD with an estimated glomerular filtration rate < 90). RESULTS: Serum calcium and 1-25D levels were significantly higher in the sarcoidosis patients than in the non-sarcoidosis patients (p < 0.01 and p = 0.01, respectively). There was a positive correlation between 1-25D and eGFR in the patients without sarcoidosis (r = 0.693; p < 0.01). As the renal function of sarcoidosis patients was improved by steroid therapy, the serum 1-25D and adjusted serum calcium levels decreased to near the median values in non-sarcoidosis patients. On renal biopsy, CD68 staining was positive for tissue macrophages in all 8 patients who had tubulointerstitial nephritis (with or without typical granulomas), while Von Kossa staining showed calcification of tubules near or inside granulomas in 6 of these 8 patients. CONCLUSION: While tissue macrophages promote development of tubulointerstitial nephritis and 1-25D overproduction in renal sarcoidosis, hypercalcemia secondary to elevation of 1-25D may be related to renal calcification and granuloma formation.

Long-term outcome of biopsy-proven cholesterol crystal embolism.

BACKGROUND: Cholesterol crystal embolism (CCE) causes renal damage, and there is an extremely high risk of end-stage renal disease. However, the time course of CCE-related renal deterioration varies and little is known about the subsequent risk of dialysis among patients with biopsy-proven CCE. METHODS: We performed a retrospective cohort study of 38 Japanese patients in whom a histological diagnosis of CCE was made from September 1992 to July 2005. Competing risk regression analysis was used to investigate the association between declining renal function ( ≥ 1.5 elevation of serum creatinine within 26 weeks after CCE) or its subtypes (acute [ < 1 week after CCE], subacute [1 to < 6 weeks], and chronic [6 to < 26 weeks]) and the risk of dialysis, with adjustment for age, baseline serum creatinine, and the precipitating event (iatrogenic or spontaneous). RESULTS: During a median follow-up period of 25.9 weeks, 14 patients (35.9%) started dialysis. Multivariable analysis showed that patients with declining renal function had a higher risk of commencing dialysis than those without declining function (subdistribution hazard ratio [SHR] 9.47; 95% confidence interval [CI] 1.34-66.8). Patients with different renal presentations had a similarly increased risk of commencing dialysis, with the risk being significantly higher for the subacute and chronic patterns of declining renal function (adjusted SHR [95% CI] for acute, subacute, and chronic declining renal function[vs. no decline]: 7.36 [0.85-63.6], 11.9 [1.36-101], and 10.7 [1.49-77.0], respectively). CONCLUSION: Declining renal function after CCE, even later than 6 weeks, was significantly associated with the subsequent risk of dialysis.

Renal-Limited Thrombotic Microangiopathy due to Bevacizumab Therapy for Metastatic Colorectal Cancer: A Case Report.

An 88-year-old Japanese man received bevacizumab for colorectal cancer with liver and peritoneal metastasis, during which nephrotic range proteinuria occurred (7.66 g/day). Renal biopsy showed endothelial damage with subendothelial swelling and a double contour of the glomerular basement membrane, which indicated a diagnosis of thrombotic microangiopathy (TMA). After bevacizumab was stopped, proteinuria decreased to 1 g/day. During the clinical course, this patient had no extrarenal manifestations. This case suggests that renal injury induced by bevacizumab is characterized by nephrotic range proteinuria and histological TMA, and is a renal-limited condition that differs from systemic TMA related to thrombotic thrombocytopenic purpura.

Brown tumor diagnosed three years after parathyroidectomy in a patient with nail-patella syndrome: A case report.

We report a 48-year-old Japanese man with a brown tumor of the right distal tibia. At the age of 25 years, hemodialysis was initiated due to nail-patella syndrome. Severe secondary hyperparathyroidism and osteoporosis progressed over time, so parathyroidectomy was performed at age 45. Spontaneous fracture of the right distal tibia occurred suddenly at age 48. Imaging studies revealed a bone tumor-like lesion and surgery was performed. The resected specimen was a brown mass consisting of multinucleated giant cells on a fibrous tissue background, and these findings were consistent with a diagnosis of brown tumor. Immunohistochemistry revealed that multinucleated giant cells near areas of bone matrix were positive for tartrate-resistant acid phosphatase and cathepsin K, but the majority of the giant cells in the lesion were negative for these markers. Even after parathyroidectomy, brown tumor should be considered in the differential diagnosis of bone tumor-like lesions in patients on long-term dialysis. This case suggests that osteoclast activation may not contribute to development of brown tumors, although these lesions are generally considered to arise from subperiosteal bone resorption related to osteoclast overactivity in patients with hyperparathyroidism.

A case of nephrotic syndrome showing contemporary presence of apolipoprotein E2 homozygote glomerulopathy and membranous nephropathy-like findings modified by apolipoprotein E Toyonaka.

A 79-year-old man was admitted to our hospital for proteinuria due to nephrotic syndrome. Renal biopsy revealed focal sclerosis and foam cell infiltration in the glomerulus. In addition, electron microscopic findings (EM) revealed peculiar electron-dense deposits (EDDs) in both sides of the glomerular basement membrane. Although subepithelial deposits had spike formation highly resembling those seen in membranous nephropathy (MN), immunoglobulins and complements were not identified by immunofluorescence study, and microbubbles appeared in high magnification of EM different from the immune disease. The analysis of apolipoprotein (Apo) E showed an elevated concentration of plasma ApoE. The phenotype, genotype, and DNA sequence studies revealed homozygous ApoE2/2 and a novel missense mutation called ApoE Toyonaka (Ser197Cys). This case may confirm the independent responsibility of ApoE2/2 and ApoE Toyonaka for ApoE2 homozygote glomerulopathy and MN-like EDD findings, respectively.

Renal-limited Cryoglobulinemic Vasculitis: Two Case Reports.

Cryoglobulinemic vasculitis (CV) presents with systemic manifestations, including renal disease, arthritis, peripheral neuropathy, and muscle weakness. We encountered two patients who developed severe nephrotic range proteinuria; however, extrarenal manifestations were not noted during the clinical course. A renal biopsy revealed typical membranoproliferative glomerulonephritis (MPGN) with huge thrombus-like endothelial deposits and predominant IgM positivity, but electron microscopy did not reveal any definite microtubules. Immunosuppressive therapy and plasmapheresis were only partially effective, and the improvement was not durable. Biological therapy with rituximab (RTX) had no effect. Renal-limited CV should be recognized as a subset of essential CV.

Everolimus Reduces the Size of Tuberous Sclerosis Complex-Related Huge Renal Angiomyolipomas Exceeding 20 cm in the Longest Diameter.

We evaluated the efficacy of everolimus in 3 patients who had huge renal angiomyolipomas associated with tuberous sclerosis complex. Two patients with large lipid-rich angiomyolipomas had a history of renal transarterial embolization for renal bleeding, but the effect had only been temporary and the embolized kidneys had continued to enlarge. In case 1, case 2, and case 3, total renal volume was respectively 3,891, 4,035, and 1,179 cm3 before administration of everolimus, decreasing to 3,016 (77%), 3,043 (75%), and 1,051 (89%) cm3 after 1 year of everolimus therapy and to 2,832 (73%), 3,209 (80%), and 1,102 (93%) cm3 after 3 years. New renal bleeding did not occur, but elevation of serum creatinine and urinary protein were noted in 2 patients. While previous reports have largely assessed the effect of everolimus for angiomyolipomas of < 10 cm in the longest diameter, our findings suggest that this drug might also be effective for huge lesions of > 20 cm in diameter. However, total renal volume still exceeds 2,000 cm3 in 2 of our patients, suggesting limited size reduction of lipid-rich angiomyolipomas. In addition, occurrence of everolimus-related nephropathy needs to be monitored carefully.

Transcatheter Arterial Embolization Therapy for Huge Renal Cysts: Two Case Reports.

We encountered 2 patients with symptomatic huge simple renal cysts. In case 1, 4,000 mL of cyst fluid was drained via a catheter, but intracystic bleeding occurred immediately afterwards. Transcatheter arterial embolization (TAE) was performed, after which the bleeding stopped, and cyst drainage was repeated successfully. After 2 years, the total cyst volume was reduced from 11,775 mL to 75.4 mL. In case 2, TAE was performed prophylactically before drainage. Subsequently, 9,400 mL of fluid was removed from multiple cysts. After 1 year, the total cyst volume was reduced from 9,215 mL to 633 mL without bleeding. Based on these 2 cases, prophylactic TAE before drainage may be useful in patients with huge renal cysts.

Preservation of renal function by intensive glycemic control.

We report the case of a 67-year-old Japanese woman with type 1 diabetes mellitus. At 47 years of age, her hemoglobin A1c (HbA1c) was 10.0%, and she had overt nephropathy. The first renal biopsy yielded a diagnosis of diabetic nephropathy. Intensive glycemic control was initiated and her HbA1c improved to 6.0%. Renal dysfunction showed no progression for 15 years. At 62 years of age, a second renal biopsy was performed. Glomerular lesions did not show progression but tubulointerstitial fibrosis and vascular lesions showed progression compared with the first biopsy. Intensive glycemic control can prevent the progression of glomerular lesions, but might not be effective for interstitial and vascular lesions. LEARNING POINTS: Intensive control of blood glucose can prevent the progression of glomerular lesions.Intensive control of blood glucose may not be able to prevent progression of interstitial and vascular lesions.CSII reduces HbA1c without increasing the risk of hypoglycemia.