A Mathematical Model of Mitochondria in Proximal Tubule and Thick Ascending Limb Cells

Mitochondria are a key player in several kinds of tissue injury, and are even the ultimate cause of certain diseases. In this work we introduce new models of mitochondrial ATP generation in multiple tissues, including liver hepatocytes and the medullary thick ascending limb in the kidney. Using this model, we predict these tissues' responses to hypoxia, uncoupling, ischemia-reperfusion, and oxidative phosphorylation dysfunction. Our results suggest mechanisms explaining differences in robustness of mitochondrial function across tissues. The medullary thick ascending limb and proximal tubule in the kidney both experience a high metabolic demand, while having lower baseline activity of oxidative phosphorylation relative to the liver. These factors make these tissues susceptible to dysfunction of Complex III. A lower baseline oxygen tension observed in the thick ascending limb makes it susceptible to Complex IV. On the other hand, since the liver lacks these risk factors, and has higher baseline rates of glycolysis, it is less susceptible to all kinds of oxidative phosphorylation dysfunction.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through induction of Tollip and negative regulation of IRAK-1

LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-extracellular signal-regulated kinase (ERK) pathway that is upregulated by sepsis. Pretreatment with the nontoxic immunomodulator monophosphoryl lipid A (MPLA) prevents inhibition by LPS through activation of a TLR4-TIR-domain-containing adaptor-inducing interferon-β (TRIF)-phosphatidylinositol 3-kinase (PI3K) pathway that prevents LPS-induced ERK activation. Here, we identified the molecular mechanisms that underlie the protective inhibitory interaction between the MPLA-PI3K and LPS-ERK pathways. Treatment of mouse MTALs with LPS in vitro increased phosphorylation of IL-1 receptor-associated kinase (IRAK)-1, a critical mediator of LPS signaling downstream of TLR4-MyD88. Activation of ERK by LPS was eliminated by a selective IRAK-1 inhibitor, establishing IRAK-1 as the upstream mediator of ERK activation. Pretreatment of MTALs with MPLA in vitro prevented LPS-induced IRAK-1 activation; this effect was dependent on PI3K. Treatment of MTALs with MPLA increased expression of Toll-interacting protein (Tollip), an inducible protein that negatively regulates LPS signaling by inhibiting IRAK-1. The MPLA-induced increase in Tollip protein level was prevented by PI3K inhibitors. In coimmunoprecipitation experiments, MPLA increased the amount of Tollip stably bound to IRAK-1, an interaction that inhibits IRAK-1 activation. These results support a mechanism whereby MPLA increases Tollip expression in the MTAL through a PI3K-dependent pathway. Tollip, in turn, inhibits LPS-induced TLR4 signaling by suppressing activation of IRAK-1, thereby preventing activation of ERK that inhibits [Formula: see text] absorption. These studies show that MPLA induces reprogramming of MTAL cells that protects against LPS stimulation and identify IRAK-1 and Tollip as new therapeutic targets to prevent renal tubule dysfunction in response to infectious and inflammatory stimuli.

Kidney outer medulla mitochondria are more efficient compared with cortex mitochondria as a strategy to sustain ATP production in a suboptimal environment

The kidneys receive ~25% of cardiac output, which is a prerequisite to maintain sufficient glomerular filtration rate. However, both intrarenal regional renal blood flow and tissue oxygen levels are heterogeneous with decreasing levels in the inner part of the medulla. These differences, in combination with the heterogeneous metabolic activity of the different nephron segment located in the different parts of the kidney, may constitute a functional problem when challenged. The proximal tubule and the medullary thick ascending limb of Henle are considered to have the highest metabolic rate, which is related to the high mitochondria content needed to sustain sufficient ATP production from oxidative phosphorylation to support high electrolyte transport activity in these nephron segments. Interestingly, the cells located in kidney medulla function at the verge of hypoxia, and the mitochondria may have adapted to the surrounding environment. However, little is known about intrarenal differences in mitochondria function. We therefore investigated functional differences between mitochondria isolated from kidney cortex and medulla of healthy normoglycemic rats by using high-resolution respirometry. The results demonstrate that medullary mitochondria had a higher degree of coupling, are more efficient, and have higher oxygen affinity, which would make them more suitable to function in an environment with limited oxygen supply. Furthermore, these results support the hypothesis that mitochondria of medullary cells have adapted to the normal hypoxic in vivo situation as a strategy of sustaining ATP production in a suboptimal environment.

Monophosphoryl lipid A prevents impairment of medullary thick ascending limb HCO3− absorption and improves plasma HCO3− concentration in septic mice

Metabolic acidosis is the most common acid-base disorder in septic patients and is associated with increased mortality. Previously, we demonstrated that sepsis induced by cecal ligation and puncture (CLP) impairs [Formula: see text] absorption in the medullary thick ascending limb (MTAL) by 1) decreasing the intrinsic [Formula: see text] absorptive capacity and 2) enhancing inhibition of [Formula: see text] absorption by LPS through upregulation of Toll-like receptor (TLR) 4 signaling. Both effects depend on ERK activation. Monophosphoryl lipid A (MPLA) is a detoxified TLR4 agonist that enhances innate antimicrobial immunity and improves survival following sepsis. Pretreatment of MTALs with MPLA in vitro prevents LPS inhibition of [Formula: see text] absorption. Here we examined whether pretreatment with MPLA would protect the MTAL against sepsis. Vehicle or MPLA was administered to mice 48 h before sham or CLP surgery, and MTALs were studied in vitro 18 h postsurgery. Pretreatment with MPLA prevented the effects of sepsis to decrease the basal [Formula: see text] absorption rate and enhance inhibition by LPS. These protective effects were mediated through MPLA stimulation of a Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β-(TRIF)-dependent phosphatidylinositol 3-kinase-Akt pathway that prevents sepsis- and LPS-induced ERK activation. The effects of MPLA to improve MTAL [Formula: see text] absorption were associated with marked improvement in plasma [Formula: see text] concentration, supporting a role for the kidneys in the pathogenesis of sepsis-induced metabolic acidosis. These studies support detoxified TLR4-based immunomodulators, such as MPLA, that enhance antimicrobial responses as a safe and effective approach to prevent or treat sepsis-induced renal tubule dysfunction and identify cell signaling pathways that can be targeted to preserve MTAL [Formula: see text] absorption and attenuate metabolic acidosis during sepsis.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through a TLR4-TRIF-PI3K signaling pathway

Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that induces tolerance to LPS and augments host resistance to bacterial infections. Previously, we demonstrated that LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a basolateral Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-ERK pathway. Here we examined whether pretreatment with MPLA would attenuate LPS inhibition. MTALs from rats were perfused in vitro with MPLA (1 µg/ml) in bath and lumen or bath alone for 2 h, and then LPS was added to (and MPLA removed from) the bath solution. Pretreatment with MPLA eliminated LPS-induced inhibition of [Formula: see text] absorption. In MTALs pretreated with MPLA plus a phosphatidylinositol 3-kinase (PI3K) or Akt inhibitor, LPS decreased [Formula: see text] absorption. MPLA increased Akt phosphorylation in dissected MTALs. The Akt activation was eliminated by a PI3K inhibitor and in MTALs from TLR4−/−or Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF)−/−mice. The effect of MPLA to prevent LPS inhibition of [Formula: see text] absorption also was TRIF dependent. Pretreatment with MPLA prevented LPS-induced ERK activation; this effect was dependent on PI3K. MPLA alone had no effect on [Formula: see text] absorption, and MPLA pretreatment did not prevent ERK-mediated inhibition of [Formula: see text] absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit [Formula: see text] absorption in the MTAL. This protective effect is mediated directly through MPLA stimulation of a TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced ERK activation. These studies identify detoxified TLR4-based immunomodulators as novel potential therapeutic agents to prevent or treat renal tubule dysfunction in response to bacterial infections.