Lipopolysaccharide directly inhibits bicarbonate absorption by the renal outer medullary collecting duct

Acidosis is associated with E. coli induced pyelonephritis but whether bacterial cell wall constituents inhibit HCO3 transport in the outer medullary collecting duct from the inner stripe (OMCDi) is not known. We examined the effect of lipopolysaccharide (LPS), on HCO3 absorption in isolated perfused rabbit OMCDi. LPS caused a ~ 40% decrease in HCO3 absorption, providing a mechanism for E. coli pyelonephritis-induced acidosis. Monophosphoryl lipid A (MPLA), a detoxified TLR4 agonist, and Wortmannin, a phosphoinositide 3-kinase inhibitor, prevented the LPS-mediated decrease, demonstrating the role of TLR4-PI3-kinase signaling and providing proof-of-concept for therapeutic interventions aimed at ameliorating OMCDi dysfunction and pyelonephritis-induced acidosis.


Bicarbonate transport and transepithelial voltage. Triplicate collections of 15-20 nl of tubular fluid
were made under water saturated mineral oil and analyzed for [HCO 3 ] using a Nanoflo microfluorometer (World Precision Instruments, Sarasota, FL) [15][16][17] . Net [HCO 3 ] transport was calculated as J HCO3 = (C o -C L ) × V L /L), where C o and C L are the HCO 3 concentrations of perfused and collected fluid, respectively, V L is the rate of collected fluid, and L is the length of the tubule (mm), and no water is net absorbed. When J HCO3 > zero, there is net HCO 3 absorption. Transepithelial voltage (mV) was measured using the luminal perfusion pipette as an electrode.

Basolateral LPS inhibits HCO 3 absorption in OMCDi.
Basolateral LPS exposure is a model for investigating the impact of sepsis on renal tubular transport mechanisms 6 . Baseline HCO 3 transport by the OMCDi averaged 13.65 ± 0.33 pmol/min per mm length and the transepithelial voltage was + 3.7 ± 0.2 mV. LPS applied to the bath (500 ng/ml) resulted in a 40% decrease in HCO 3 transport to 8.22 ± 0.42 pmol/min per mm (p < 0.001) ( Fig. 1, L panel), and there was a corresponding fall in transepithelial voltage to 3.1 ± 0.2 mV (p < 0.001). Removal of LPS resulted in a complete return of transport to 13.79 ± 0.61 pmol/min per mm and voltage (+ 3.6 ± 0.2 mV), neither significantly different from control values.

Luminal LPS inhibits HCO 3 absorption in OMCDi. Luminal exposure to LPS models E. coli-induced
pyelonephritis' impact on HCO 3 transport by OMCDi. Baseline HCO 3 absorption was decreased by 37% by luminal LPS at 500 ng/ml (absorption was 13.28 ± 0.32 pmol/min per mm and decreased to 8.37 ± 0.32, p < 0.001) ( Fig. 1, R panel). Transepithelial voltage was + 2.7 ± 0.2 mV and decreased to + 2.2 ± 0.2, p < 0.001 (Table 1). The inhibition by LPS was reversible by removing it from the luminal fluid. Recovery HCO 3 absorption was 13.14 ± 0.29 pmol/min per mm, 99% of control rate and voltage returned to + 2.6 ± 0.2 mV. The recovery transport rate was not significantly different from control (p > 0.5), and the voltage recovered to 97% of control, slightly but significantly less than control (p = 0.02).

Basolateral MPLA blocks the luminal LPS effect.
To confirm and extend results of Watts et al. 12,13 the effect of MPLA on bicarbonate transport and the LPS response was examined. In two independent experiments, MPLA at 1 μg/ml in the bath had no major effect on HCO 3 transport (11.74-11.52 and 12.79-13.09 pmol/min  Basolateral wortmannin blocks the luminal LPS effect. In the mTAL luminal exposure to LPS inhibits basolateral sodium hydrogen exchange via a phosphoinositide 3-kinase (PI3-K)-dependent pathway 8 .
In the next eight experiments we examined the effect of PI3-K inhibitor Wortmannin 18 on LPS-mediated inhibition of bicarbonate absorption in the OMCDi. Similar to the design above for the MPLA studies, wortmannin (100-200 nM) was added to bath during the control period, and then LPS added to lumen (500 ng/ml) for an experimental period, after which LPS was removed and recovery bicarbonate flux was measured in the presence of wortmannin. In eight independent experiments (Fig. 3, R panel) pretreatment with Wortmannin blocked the effect of LPS; HCO 3 transport in OMCDi treated with LPS + wortmannin was 93 ± 1% of control and recovery values (this change was still significantly different from zero, mean of control and recovery was 12.5 ± 0.2 pmol/min per mm compared to LPS of 11.6 ± 0.2 pmol/min per mm, p < 0.001). In addition, there was only a small but significant decrease in transepithelial voltage such that the voltage during LPS + Wortmannin was 95 ± 1% of control and recovery values (mean of control and recovery was 2.6 mV compared to LPS voltage of 2.4 mV, p < 0.001, Table 1). In the absence of wortmannin, luminal LPS inhibited the HCO 3 absorptive flux by 34 ± 3% (Fig. 3, L panel, n = 3, mean of control and recovery was 14.2 ± 0.6 pmol/min per mm compared to LPS at 9.4 ± 0.4 pmol/min per mm, p < 0.01). These results demonstrate that similar to signaling in mTAL 8 , luminal exposure to LPS inhibits bicarbonate absorption in the OMCDi via a TLR4-PI3-kinase dependent pathway.

Discussion
The key finding of this study is that either luminal or basolateral exposure of the isolated perfused OMCDi to LPS, a major E. coli cell wall constituent, reversibly inhibits bicarbonate absorption. Results presented herein are the first to show that LPS signaling targets acid-base transport mechanisms in the OMCDi. In the mTAL luminal LPS-induced signaling via TLR4 inhibits basolateral sodium/hydrogen exchange (NHE) activity 8,19 , whereas basolateral TLR4 signaling, which is TLR2 dependent 20 targets apical NHE3 activity 19 . Consistent with the results of Good and colleagues 8 luminal TLR4 signaling in the OMCDi inhibited bicarbonate absorption via a PI3-K dependent pathway; the PI3-K inhibitor wortmannin blocked much of the effect of luminal exposure to LPS (see Fig. 3). However the target of TLR4 signaling in the OMCDi is distinct, as bicarbonate absorption   (Table 1). MPLA is a detoxified derivative of LPS as well as a partial TLR4 agonist that effectively blunts pathophysiological responses to LPS 21 . The systemic toxicity of MPLA compared to native LPS is estimated to be 99% reduced 22 . Because it can enhance the adaptive immune response with a minimum of inflammatory side effects, MPLA has been used as an immunoadjuvant in humans 12,22 . Pretreatment with MPLA induces resistance to endotoxemia in animals and humans 21,[23][24][25] . Good and colleagues reported that MPLA induces TLR4 signaling via a TRIF-PI3K-AKT pathway that prevented LPS induced ERK activation 12 . Basolateral exposure of the OMCDi to MPLA and wortmannin likely blocked the effect of luminal LPS on bicarbonate absorption (Fig. 2) and transepithelial voltage via a similar mechanism (see Table 1). Thus, these agents may prevent OMCDi dysfunction associated with pyelonephritis. The medullary collecting duct is the first nephron segment exposed to E. coli pyelonephritis during an ascending UTI, and so this result provides proof-of-concept for attenuation of pyelonephritis via pharmacologic interventions that target basolateral TLR4 signaling. Indeed, partial TLR4 agonists represent a viable antibiotic-sparing therapy for treatment of acute pyelonephritis [26][27][28][29] .
An association between abnormalities in electrolyte and acid-base balance and acute pyelonephritis is common in young children 9 . Recent studies in our laboratory have focused on the intersection of metabolic acidosis and innate immune defense 30,31 . Intracellular acidification promotes the accumulation of 2-hydroxyglutarate, which in turn triggers HIF-1 stabilization via prolyl hydroxylase inhibition 32 . HIF-1 elicits adaptive responses to both acidosis and microbial invasion via induction of SDF-1 (CXCL12) and antimicrobial peptide expression, respectively 30,33,34 . Despite HIF-dependent upregulation of AMPs, metabolic acidosis markedly impairs clearance of urinary tract infection with uropathogenic E. coli (UPEC-UTI) and thus exacerbates pyelonephritis in innate immune competent C3H strains mice that are prone to vesicoureteral reflux (VUR) 35 . Although acidification of culture media or urine (pH ≤ 6) limits bacterial growth in vitro 36 , urine acidification per se was not a major contributor to clearance of UPEC-UTI in this study as neutralization of urine in the setting of metabolic acidosis via concurrent administration acetazolamide did not affect UPEC burden 35 . UPEC burden in TLR4-deficient mice (C3H-HeJ) mice was unaffected by acidosis suggesting that metabolic acidosis impairs some aspect of the TLR-4-dependent innate immune response. Collectively, these studies suggest that other aspects of pathophysiology associated with metabolic acidosis impair clearance of UPEC UTI and thus supersede any benefit of AMP production and urine acidification by α-ICs 36 .
Thus, acidosis represents a key comorbidity with E. coli pyelonephritis and this association may be explained, at least in part, by LPS-induced TLR4 signaling that inhibits bicarbonate absorption by the OMCDi. MPLA and PI3-K inhibition may mitigate acidosis as well as OMCDi injury induced by LPS. Finally, formal correction of acidosis may speed recovery from urinary tract infections and thus represent a key antibiotic-sparing therapy adjunct for treatment of acute pyelonephritis.