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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Multiparametric imaging reveals that mitochondria-rich intercalated cells in the kidney collecting duct have a very high glycolytic capacity.

Alpha intercalated cells (αICs) in the kidney collecting duct (CD) belong to a family of mitochondria rich cells (MRCs) and have a crucial role in acidifying the urine via apical V-ATPase pumps. The nature of metabolism in αICs and its relationship to transport was not well-understood. Here, using multiphoton live cell imaging in mouse kidney tissue, FIB-SEM, and other complementary techniques, we provide new insights into mitochondrial structure and function in αICs. We show that αIC mitochondria have a rounded structure and are not located in close proximity to V-ATPase containing vesicles. They display a bright NAD(P)H fluorescence signal and low uptake of voltage-dependent dyes, but are energized by a pH gradient. However, expression of complex V (ATP synthase) is relatively low in αICs, even when stimulated by metabolic acidosis. In contrast, anaerobic glycolytic capacity is surprisingly high, and sufficient to maintain intracellular calcium homeostasis in the presence of complete aerobic inhibition. Moreover, glycolysis is essential for V-ATPase-mediated proton pumping. Key findings were replicated in narrow/clear cells in the epididymis, also part of the MRC family. In summary, using a range of cutting-edge techniques to investigate αIC metabolism in situ, we have discovered that these mitochondria dense cells have a high glycolytic capacity.

Targeting glycolysis in proliferative kidney diseases.

Glycolytic activity is increased in proliferating cells, leading to the concept that glycolysis could be a therapeutic target in cystic diseases and kidney cancer. Preclinical studies using the glucose analog 2-deoxy-d-glucose have shown promise; however, inhibiting glycolysis in humans is unlikely to be without risks. While proximal tubules are predominantly aerobic, later segments are more glycolytic. Understanding exactly where and why glycolysis is important in the physiology of the distal nephron is thus crucial in predicting potential adverse effects of glycolysis inhibitors. Live imaging techniques could play an important role in the process of characterizing cellular metabolism in the functioning kidney. The goal of this review is to briefly summarize recent findings on targeting glycolysis in proliferative kidney diseases and to highlight the necessity for future research focusing on glycolysis in the healthy kidney.

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis.

BACKGROUND: The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown. METHODS: To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach. RESULTS: Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology. CONCLUSIONS: Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

Live Imaging of Mitochondria in Kidney Tissue.

Kidneys are highly aerobic organs and their function is tightly coupled to mitochondrial energy production. Renal tubular cells, particularly the proximal tubule (PT), require an abundance of mitochondria to provide sufficient energy for regulating fluid and electrolyte balance. Meanwhile, mitochondrial defects are implicated in a range of different kidney diseases. Multiphoton microscopy (MP) is a powerful tool that allows detailed study of mitochondrial morphology, dynamics, and function in kidney tissue. Here, we describe how MP can be used to image mitochondria in kidney tubular cells, either ex vivo in tissue slices or in vivo in living rodents, using both endogenous and exogenous fluorescent molecules. Moreover, changes in mitochondrial signals can be followed in real time in response to different insults or stimuli, in parallel with other important readouts of kidney tubular function, such as solute uptake and trafficking.

The iron chelator Deferasirox causes severe mitochondrial swelling without depolarization due to a specific effect on inner membrane permeability.

The iron chelator Deferasirox (DFX) causes severe toxicity in patients for reasons that were previously unexplained. Here, using the kidney as a clinically relevant in vivo model for toxicity together with a broad range of experimental techniques, including live cell imaging and in vitro biophysical models, we show that DFX causes partial uncoupling and dramatic swelling of mitochondria, but without depolarization or opening of the mitochondrial permeability transition pore. This effect is explained by an increase in inner mitochondrial membrane (IMM) permeability to protons, but not small molecules. The movement of water into mitochondria is prevented by altering intracellular osmotic gradients. Other clinically used iron chelators do not produce mitochondrial swelling. Thus, DFX causes organ toxicity due to an off-target effect on the IMM, which has major adverse consequences for mitochondrial volume regulation.