Mitochondria In Proximal Tubules Research Articles

Presence of mitochondrial dysfunction in a case of Fanconi syndrome with normoglycemic MODY1.

Maturity-onset diabetes of the young 1 (MODY1) is characterized by macrosomia and transient hypoglycemia in neonates, in addition to diabetes mellitus (DM). Only patients with MODY1 harboring a pathogenic variant (c.187C > T; p.R63W) in HNF4A are sure to develop Fanconi syndrome (FS). Here we report the successful diagnosis of MODY1 in a patient harboring p.R63W before confirmation of DM-related hyperglycemia after being alerted to the presence of abnormal mitochondria in a kidney-biopsy specimen revealed by electron microscopy. The patient was born at 39weeks of gestation with macrosomia, elevated levels of liver enzymes, and transient hypoglycemia. At three years of age, proteinuria was found by chance, and further laboratory investigations revealed metabolic acidosis, mild renal dysfunction, hypouricemia, proteinuria, aminoaciduria, and glycosuria. On this basis, we diagnosed the patient as having FS and performed percutaneous renal biopsy. Light microscopy revealed no evidence of proximal tubule disorder, but electron microscopy demonstrated mitochondria with disordered cristae in glomerular podocytes and giant mitochondria in proximal tubules. Mitochondrial nephropathy was suspected, and skin fibroblasts from the patient grown on galactose medium showed decreased oxygen consumption suggestive of mitochondrial dysfunction. Therefore, genetic testing was performed and a pathogenic variant (c.187C > T; p.R63W) in HNF4A was detected. Mitochondrial dysfunction in a Drosophila and murine model of patients with both MODY1 and FS has already been reported, and we detected it in this human MODY1/FS patient on the basis of functional tests and imaging. We believe mitochondrial dysfunction may be involved in the pathogenesis of MODY1/FS.

A comparative modeling study of the mitochondrial function of the proximal tubule and thick ascending limb cells in the rat kidney.

To reabsorb >99% of the glomerular filtrate, the metabolic demand of the kidney is high. Interestingly, renal blood flow distribution exhibits marked inhomogeneity, with typical tissue oxygen tension (Po2) of 50-60 mmHg in the well-perfused cortex and 10-20 mmHg in the inner medulla. Cellular fluid composition and acidity also varies substantially. To understand how different renal epithelial cells adapt to their local environment, we have developed and applied computational models of mitochondrial function of proximal convoluted tubule cell (baseline Po2 = 50 mmHg, cytoplasmic pH = 7.20) and medullary thick ascending limb (mTAL) cell (baseline Po2 = 10 mmHg, cytoplasmic pH = 6.85). The models predict key cellular quantities, including ATP generation, P/O (phosphate/oxygen) ratio, proton motive force, electrical potential gradient, oxygen consumption, the redox state of key electron carriers, and ATP consumption. Model simulations predict that close to their respective baseline conditions, the proximal tubule and mTAL mitochondria exhibit qualitatively similar behaviors. Nonetheless, because the mTAL mitochondrion has adapted to a much lower Po2, it can sustain a sufficiently high ATP production at Po2 as low as 4-5 mmHg, whereas the proximal tubule mitochondria would not. Also, because the mTAL cytosol is already acidic under baseline conditions, the proton motive force (pmf) exhibits higher sensitivity to further acidification. Among the different pathways that lead to oxidative phosphorylation impairment, the models predict that both the proximal tubule and mTAL mitochondria are most sensitive to reductions in Complex III activity.NEW & NOTEWORTHY Tissue fluid composition varies substantially within the mammalian kidney. The renal cortex is well perfused and pH neutral, whereas some medullary regions are hypoxic and acidic. How do these environments affect the mitochondrial function of proximal convoluted tubule and medullary thick ascending limb cells, which reside in the cortex and medulla, respectively? This computational modeling study demonstrates that these mitochondria can adapt to their contrasting environments and exhibit different sensitivities to perturbations to local environments.

10-OR: The Triple Roles of Tubular Mitochondria AKT in the Regulation of Renal Function, Insulin Resistance, and Body Composition during Development of Diabetic Nephropathy

Diabetic nephropathy is a major cause of end-stage renal disease. We have recently shown translocation of AKT to mitochondria in proximal tubules played a renoprotective role in murine ischemia/reperfusion injury. In this study, we tested the hypothesis that activation of mitochondrial AKT (mito-AKT) signaling in renal proximal tubules improves kidney function in diabetic nephropathy and modulates overall glucose homeostasis. We used renal tubule-specific bigenic mice that harbor tamoxifen-inducible mitochondria-targeting constitutively active AKT1 (KMCAKT), which were fed with either normal chow diet or high fat and fructose diet (HFFD). Wild type mice fed with HFFD developed nephropathy after 24 wks. Activation of renal tubular mito-AKT significantly attenuated urinary albumin, creatinine, and glucose excretion in the KMCAKT mice on HFFD. Interestingly, activation of tubular mito-AKT also decreased fasting hyperglycemia, and improved glucose tolerance of HFFD-fed mice. Insulin resistance (HOMA-IR) was significantly reduced when tubular mito-AKT was activated. In addition, mito-AKT activation reduced plasma cholesterol. Furthermore, mito-AKT activation increased total body weight and fat mass of HFFD-fed mice, but had no effect on their water and food intake. To dissect the underlying mechanism, we used 18F-fluoro-4-thia-oleate PET scan to define organ specific fatty acid uptake, and revealed a significant increase of uptake in white and brown adipose tissues in KMCAKT mice on HFFD. In conclusion, this is the first report to demonstrate critical roles of mitochondrial AKT in renal tubules in the regulation of whole-body metabolism, insulin sensitivity, and body composition beyond its local effect on kidney function in diabetic nephropathy, which suggests kidney function and nephropathy associated metabolic dysfunction could be mediated through a common pathway in tubular mitochondria. Disclosure E.Salem: None. A.Ta: None. H.Y.H.Lin: None. J.Li: None. P.H.Wang: Research Support; ViaCyte, Inc. Funding Shannon & Amy Ko Foundation

Intrinsic TGF-β signaling attenuates proximal tubule mitochondrial injury and inflammation in chronic kidney disease

Excessive TGF-β signaling and mitochondrial dysfunction fuel chronic kidney disease (CKD) progression. However, inhibiting TGF-β failed to impede CKD in humans. The proximal tubule (PT), the most vulnerable renal segment, is packed with giant mitochondria and injured PT is pivotal in CKD progression. How TGF-β signaling affects PT mitochondria in CKD remained unknown. Here, we combine spatial transcriptomics and bulk RNAseq with biochemical analyses to depict the role of TGF-β signaling on PT mitochondrial homeostasis and tubulo-interstitial interactions in CKD. Male mice carrying specific deletion of Tgfbr2 in the PT have increased mitochondrial injury and exacerbated Th1 immune response in the aristolochic acid model of CKD, partly, through impaired complex I expression and mitochondrial quality control associated with a metabolic rewiring toward aerobic glycolysis in the PT cells. Injured S3T2 PT cells are identified as the main mediators of the maladaptive macrophage/dendritic cell activation in the absence of Tgfbr2. snRNAseq database analyses confirm decreased TGF-β receptors and a metabolic deregulation in the PT of CKD patients. This study describes the role of TGF-β signaling in PT mitochondrial homeostasis and inflammation in CKD, suggesting potential therapeutic targets that might be used to mitigate CKD progression.

Study of the Involvement of Mitochondria in Dent’s Disease Using a Mice Model

Dent's disease is a rare renal tubular disorder characterized by low‐molecular‐weight‐proteinuria, hypercalciuria with nephrocalcinosis or nephrolithiasis, and progressive renal failure. It is frequently associated with inactivating mutations of the CLCN5gene coding for the 2Cl‐/H+ exchanger ClC‐5. In the kidney, ClC‐5 is mainly expressed in early endosomes of proximal tubules (PT) cells where it optimizes the function of the vacuolar H+‐ATPase to ensure proper endocytosis of low‐molecular‐weight‐proteins.To better understand the cellular mechanisms involved in Dent’s disease, we have generated a novel Knock‐In mouse model carrying the ClC‐5 N340K pathogenic mutation. We have previously demonstrated that these mice display similar clinical features to those observed in patients with Dent’s disease. Interestingly, we observed an increase of oxidative stress in their kidney, as evidenced by higher expression of the NADPH oxidase 4, increased H2O2production, and ROS‐related total protein damage in PT cells.Such cellular stress may arise from dysfunctional mitochondria in PT. Here, we therefore investigated the aspect of these organelles in PT cells of WT and N340K 3 month old mice using transmission electron microscopy. Compared to WT mice, mitochondria from mice carrying the N340K mutation exhibited morphologic alterations such as increased roundness, indicating that their function may be altered. Furthermore, to identify potential markers whose urinary excretion could be associated with the progression of Dent’s disease, using mass spectrometry, we performed metabolomic analyses of urine form WT and transgenic mice aged of 4, 7 and 10 months. We demonstrated changes in the excretion of metabolites that are crucial of mitochondrial metabolism (tryptophan, quinolinic acid, malate, oxaloadipate, linolenic acid and butyric acid), suggesting that the NAD+ synthesis pathway, the citric acid cycle, and beta oxidation are affected.The present study shed more light on the pathophysiology of Dent’s disease by suggesting that a mitochondrial dysfunction is involved in this disorder.

Sex-specific effects of metformin and liraglutide on renal pathology and expression of connexin 45 and pannexin 1 following long-term high-fat high-sugar diet

The comparative effects of the two commonly used antidiabetic drugs metformin and liraglutide on renal pathology and expression of connexin 45 (Cx45) and pannexin 1 (Panx1) in adult obese rats fed high-fat high-sugar diet (HFHSD) were studied. Considering recent data on the profound influence of sex on metformin and liraglutide effects, we compared the effects of both drugs between male and female animals. 44-week-old Sprague-Dawley rats were separated into 4 groups that were fed: standard diet, HFHSD, HFHSD treated with metformin (s.c., 50 mg/kg/day) and HFHSD treated with liraglutide (s.c., 0.3 mg/kg/day). Treatment with metformin or liraglutide lasted for 14 weeks. Histology and immunohistochemistry were performed to quantify renal pathological changes and Cx45 and Panx1 expression. HFHSD caused thickening of the Bowman’s capsule (BC). Both metformin and liraglutide failed to ameliorate the BC thickening; metformin even worsened it. Effects on the tubulointerstitial fibrosis score, BC thickness and Cx45 and Panx1 expression were sex-dependent. We found a 50% increase in mitochondria in proximal tubules of metformin- and liraglutide-treated HFHSD-fed rats, but these effects were not dependent on the sex. This is a first study showing that the effects of metformin and liraglutide on kidney pathology in rats fed HFHSD are mostly sex-dependent and that these effects are not necessarily beneficial. Both drugs changed the Cx45 and Panx 1 expression; hence their effects could be related to amelioration of disruptions in intercellular communication.

Changes in NAD and Lipid Metabolism Drive Acidosis-Induced Acute Kidney Injury.

The kidney plays an important role in maintaining normal blood pH. Metabolic acidosis (MA) upregulates the pathway that mitochondria in the proximal tubule (PT) use to produce ammonia and bicarbonate from glutamine, and is associated with AKI. However, the extent to which MA causes AKI, and thus whether treating MA would be beneficial, is unclear. Gavage with ammonium chloride induced acute MA. Multiphoton imaging of mitochondria (NADH/membrane potential) and transport function (dextran/albumin uptake), oxygen consumption rate (OCR) measurements in isolated tubules, histologic analysis, and electron microscopy in fixed tissue, and urinary biomarkers (KIM-1/clara cell 16) assessed tubular cell structure and function in mouse kidney cortex. MA induces an acute change in NAD redox state (toward oxidation) in PT mitochondria, without changing the mitochondrial energization state. This change is associated with a switch toward complex I activity and decreased maximal OCR, and a major alteration in normal lipid metabolism, resulting in marked lipid accumulation in PTs and the formation of large multilamellar bodies. These changes, in turn, lead to acute tubular damage and a severe defect in solute uptake. Increasing blood pH with intravenous bicarbonate substantially improves tubular function, whereas preinjection with the NAD precursor nicotinamide (NAM) is highly protective. MA induces AKI via changes in PT NAD and lipid metabolism, which can be reversed or prevented by treatment strategies that are viable in humans. These findings might also help to explain why MA accelerates decline in function in CKD.

Angiotensinogen import in isolated proximal tubules: evidence for mitochondrial trafficking and uptake.

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled 125I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; n = 4). Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min-1·mg protein-1; n = 3). Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; P < 0.05, n = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.

Ultrastructure of mitochondria and the endoplasmic reticulum in renal tubules of Dahl salt-sensitive rats

Metabolic and functional abnormalities in the kidney precede or coincide with the initiation of overt hypertension in the Dahl salt-sensitive (SS) rat. However, renal histological injury in SS rats is mild before the development of overt hypertension. We performed electron microscopy analysis in 7-wk-old SS rats and salt-insensitive consomic SS.13(BN) rats and Sprague-Dawley (SD) rats fed a 4% NaCl diet for 7 days. Long mitochondria (>2 μm) accounted for a significantly smaller fraction of mitochondria in medullary thick ascending limbs in SS rats (4% ± 1%) than in SS.13(BN) rats (8% ± 1%, P < 0.05 vs. SS rats) and SD rats (9% ± 1%, P < 0.01 vs. SS rats), consistent with previous findings of mitochondrial functional insufficiency in the medulla of SS rats. Long mitochondria in proximal tubules, however, were more abundant in SS rats than in SS.13(BN) and SD rats. The width of the endoplasmic reticulum, an index of endoplasmic reticulum stress, was significantly greater in medullary thick ascending limbs of SS rats (107 ± 1 nm) than in SS.13(BN) rats (95 ± 2 nm, P < 0.001 vs. SS rats) and SD rats (74 ± 3 nm, P < 0.01 vs. SS or SS.13(BN) rats). The tubules examined were indistinguishable between rat strains under light microscopy. These data indicate that ultrastructural abnormalities occur in the medullary thick ascending limbs of SS rats before the development of histological injury in renal tubules, providing a potential structural basis contributing to the subsequent development of overt hypertension.

Abstract 479: Shortening of Mitochondria and Dilation of Endoplasmic Reticulum in the Medullary Thick Ascending Limb of Dahl Salt-Sensitive Rats

Functional abnormalities in the kidney appear to precede or coincide with the initiation of salt-induced hypertension in the Dahl salt-sensitive SS rat. However, the structural basis or mechanism of early renal abnormalities, especially in the renal tubules, is poorly understood. We performed electron microscopy analysis in 7 week old SS rats and salt-insensitive consomic SS.13 BN rats and Sprague-Dawley (SD) rats fed a 4% NaCl diet for 7 days. Long mitochondria (&gt;2 μm), an indication of healthy mitochondria, accounted for a significantly smaller fraction of all mitochondria in medullary thick ascending limbs in SS rats (4% ± 1%) than in SS.13 BN rats (8% ± 1%, P&lt;0.05 vs. SS) and SD rats (9% ± 1%, P&lt;0.01 vs. SS). This is consistent with our previous findings of mitochondrial insufficiency in the medulla of SS rats. Long mitochondria in proximal tubules, however, were more abundant in SS rats than in SS.13 BN and SD rats. The width of endoplasmic reticulum, an index of endoplasmic reticulum stress, was significantly greater in medullary thick ascending limbs of SS rats (107 ± 1 nm) than SS.13 BN rats (95 ± 2 nm, P&lt;0.001 vs. SS) and SD rats (74 ± 3 nm, P&lt;0.01 vs. SS or SS.13 BN ). The rat strain differences in ultrastructure were observed even though the tubules were indistinguishable by light microscopy and less than 5% of the kidney area was affected by inflammation, interstitial fibrosis, or tubular casts. These data indicate that ultrastructural abnormalities in the mitochondria and endoplasmic reticulum in the medullary thick ascending limbs occur in the early stage of, and could contribute to, the development of salt-induced hypertension.

Hexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3β (GSK3β) and Bax caused mitochondrial membrane injury and apoptosis. ATP depletion led to a progressive HK II dissociation from mitochondria, released mitochondrial apoptosis inducing factor and cytochrome c into the cytosol, activated caspase-3, and reduced cell survival. Compared with control, adenoviral-mediated HK I or II overexpression improved cell survival following stress, but did not prevent GSK3β or Bax activation, improve ATP content, or reduce mitochondrial fragmentation. HK I or HK II overexpression increased mitochondria-associated isoform-specific HK content, and decreased mitochondrial membrane injury and apoptosis after stress. In vivo, HK II localized exclusively to the proximal tubule. Ischemia reduced total renal HK II content and dissociated HK II from proximal tubule mitochondria. In cells overexpressing HK II, Bax and HK II did not interact before or after stress. While the mechanism by which HK antagonizes Bax-mediated apoptosis is unresolved by these studies, one possible scenario is that the two proteins compete for a common binding site on the outer mitochondrial membrane.

Testosterone-dependent sexual dimorphism of the mouse kidney is mediated by polyamines

Male mice exhibit higher levels of kidney ornithine decarboxylase and polyamines and excrete larger amounts of polyamines into the urine than female mice. Orchiectomy elicits the female pattern and testosterone induces the male pattern of polyamine synthesis and excretion. α-Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, suppressed the testosterone-induced increase in kidney ornithine decarboxylase and polyamines and urinary excretion of polyamines. It also suppressed the testosterone-induced increment in kidney cytochrome c oxidase, lysosomal hydrolases and lysosomal enzymuria, and attenuated the renal hypertrophy. Administration of exogenous putrescine increased kidney polyamine levels and abolished the inhibitory effect of α-difluoromethylornithine. These data indicate that enhanced ornithine decarboxylase activity and polyamine synthesis are essential in mediating androgenic effects on proximal tubule mitochondria and lysosomes. Testosterone-induced kidney growth is at least partially mediated by ornithine decarboxylase and the polyamines.