Paracellular transport as a strategy for energy conservation by multicellular organisms?

ABSTRACT

Paracellular transport of solutes and water accompanies transcellular transport across epithelial barriers and together they serve to maintain internal body composition. However, whether paracellular transport is necessary and why it evolved is unknown. In this commentary I discuss our recent studies to address this question in the proximal tubule of the kidney. Paracellular reabsorption of sodium occurs in the proximal tubule and is mediated by claudin-2. However, deletion of claudin-2 in mice does not affect whole kidney sodium excretion because it can be completely compensated by downtream transcellular transport mechanisms. This occurs at the expense of increased oxygen consumption, tissue hypoxia and increased susceptibility to ischemic injury. It is concluded that paracellular transport acts as an energy saving mechanism to increase transport without consuming additional oxygen. It is speculated that this might be why paracellular transport evolved in leaky epithelia with high transport needs.

Introduction

Epithelia are sheets of cells that separate body compartments in multicellular organisms. All eumetazoa, and some sponges,¹ have epithelia.² Polarized epithelia are essential for internal homeostasis. They act as barriers to protect the organism from the external environment, and mediate vectorial transepithelial transport to regulate the complex composition of the internal medium.

Transepithelial transport can occur by the transcellular or paracellular route. Transcellular transport occurs through the epithelial cell, passing sequentially through transmembrane proteins in the apical and basal plasma membranes, and the intervening cytoplasm. It is the predominant route for most transepithelial transport. Moreover, transcellular transport is the only route available for active transport, in other words, transport occurring against an uphill electrochemical gradient. This is because active transport requires energy from the hydrolysis of ATP, which is mediated by a class of transmembrane transport proteins known as pumps or ATPases. Transcellular transport occurs through transmembrane transport proteins that can be highly specific. For example, transmembrane voltage-dependent K⁺ channels are more than 100 times more selective for K⁺ than Na⁺.³ Transcellular transport is also highly regulated by extracellular ligands, proteases and other signals, intracellular signaling molecules, and transmembrane voltage.

Paracellular transport refers to transport that occurs in between cells, passing through an intercellular shunt pathway.⁴ Paracellular transport appears to be exclusively passive and downhill, occurring by diffusion or convection, and driven by existing transepithelial gradients. Paracellular transport pathways tend to be relatively unselective, primarily selecting permeant molecules on the basis of size and charge, and do not appear to be as tightly regulated as transcellular transporters. We now know that the rate-limiting barrier for paracellular transport is the tight junction, which is constituted by a complex containing a large number of proteins. A family of 27 tetraspanning transmembrane proteins, the claudins, form paracellular channels that mediate transport through the tight junction.⁵

Why paracellular transport evolved, and the reason why such a large family of paracellular channel isoforms is needed, is unknown. One could envision a much simpler system in which the intercellular junctions are completely sealed and all transport occurs transcellularly. Our recent studies to investigate the role of paracellular transport in the proximal tubule of the kidney⁶ led us to one plausible answer to this fascinating question.

The renal proximal tubule as a representative leaky transporting epithelium

The kidney consists of a mechanical filter of the blood, the glomerulus, together with a tubule lined with epithelium down which the filtrate flows. During this transit, the tubule fluid composition is continuously modified by transepithelial transport so as to excrete precisely the right amount of each of the hundreds of different solutes and water in the urine and thereby maintain internal homeostasis. The early part of the tubule, referred to as the proximal tubule, does the heavy lifting, performing bulk transport (largely reabsorption) of solutes and water (Fig. 1). For example, 60% of filtered Na⁺ or ∼300 g daily is reabsorbed in the proximal tubule (compare this to an average dietary Na⁺ intake of 3–4 g per day). The late part, or distal tubule, is involved in fine-tuning of the final urine composition.

Figure 1.

Function of the renal tubule. (A) Filtration of blood through the glomerulus. Filtrate composition is identical to plasma. (B) Bulk transport in the proximal tubule. Transepithelial flux of solutes and water is high. Tubule fluid composition is moderately different from plasma and transepithelial gradients are just beginning to be established. (C) Fine-tuning in the distal tubule. Transepithelial transport capacity is low but largely driven uphill against steep and thermodynamically unfavorable transepithelial gradients, thereby requiring considerable energy consumption for active transport.

The proximal tubule is a leaky epithelium, meaning that paracellular permeability is high, which suggests that the paracellular pathway might play an important role in bulk transepithelial transport in this segment. By contrast, the distal tubule is a tight epithelium and the paracellular pathway functions primarily as a barrier, protecting transepithelial gradients for solutes like Na⁺, K⁺, H⁺ and preventing them from being dissipated by backflux.⁷

Elucidating the functional role of paracellular transport in the proximal tubule

Claudin-2 is highly expressed in the proximal tubule with the highest levels in the late or S3 segment.^8-10 When expressed in vitro, claudin-2 functions as a high-conductance paracellular Na⁺ channel.^11-13 Consistent with this, claudin-2 null mice exhibit 37% lower Na⁺ reabsorption in their proximal tubules, compared with control mice, demonstrating that claudin-2 plays a major role in proximal tubule paracellular Na⁺ transport.¹⁴ However, these mice have normal urinary Na⁺ excretion and are able to conserve Na⁺ even when placed on a very low salt diet. In our studies we found that this was because the defect in proximal paracellular Na⁺ reabsorption is fully compensated by increased Na⁺ reabsorption further downstream in a tubule segment called the thick ascending limb of Henle's loop.⁶ This raised the question of what is the true role of paracellular transport in the proximal tubule. We propose that paracellular transport exists in the proximal tubule because it allows transepithelial transport to be accomplished in a more energy-efficient manner. To understand this, a brief review of energy use in the kidney is needed.¹⁵

Energy and oxygen utilization by the kidney

The kidney consumes more oxygen per gram of tissue than any other organ in the body except the brain. Although the kidneys receive 25% of cardiac output, the kidney tissue oxygenation is low, ranging from 10 mmHg in the inner medulla to 40 mmHg in the cortex.¹⁶ So the efficiency with which oxygen is used by the kidney becomes a critical determinant of kidney oxygen tension.

Renal oxygen consumption is determined primarily by the energy required to drive tubular Na⁺ reabsorption.¹⁷ Since all transcellular transport of Na⁺ is ultimately driven by the Na, K-ATPase either directly or indirectly by the potential energy from the Na⁺ gradients it generates, the theoretical maximum efficiency of oxygen usage can be calculated. The Na-K-ATPase uses 1 molcule of ATP to directly drive the transport of 3 Na⁺ ions. Ninety-five percent of ATP in the kidney is generated by aerobic respiration,¹⁸ which produces 6 molcules of ATP for each molecule of O₂ consumed. Thus, the ratio of suprabasal renal oxygen consumption (QO₂) to Na⁺ reabsorption (T_Na) is predicted to be 1:18 (mol:mol). However, experimentally measured values of QO₂/T_Na are in the range of 1:25 to 1:29,^19-22 indicating that the kidney uses oxygen much more efficiently than expected.

We suggest that paracellular transport optimizes the efficiency of oxygen usage by leveraging the excess free energy in solute gradients established by active transcellular transport to drive additional, paracellular reabsorption of Na⁺, Cl⁻ and other solutes in a purely passive manner (i.e. not requiring additional energy expenditure). In the case of the proximal tubule, it is transcellular reabsorption of Na⁺ in the early proximal tubule coupled either to HCO₃⁻ or to neutral organic solutes, in conjunction with isosmotic water reabsorption, that generates a transepithelial Cl⁻ concentration gradient and a lumen-negative electrical gradient.^23,24 This drives passive reabsorption of Cl⁻ via paracellular diffusion, leading to the development of a lumen-positive electrical potential²⁵ that ultimately provides the driving force for paracellular reabsorption of the counter-ion, Na⁺.

Role of claudin-2 in energy saving by the kidney

Since claudin-2 plays a major role in proximal tubule paracellular Na⁺ reabsorption, we tested the hypothesis that knockout of claudin-2 would impair energy efficiency in the kidney.⁶ We found that claudin-2 null mice have similar rates of renal tubule Na⁺ reabsorption (T_Na) to wild-type mice, but that the rate of renal oxygen consumption (QO₂) is substantially higher (Fig. 2). Consequently T_Na/QO₂ was 40% lower in the claudin-2 null mice. Because of this the renal medulla of these mice was more hypoxic than that of wild-type mice. Moreover, this predisposed the mice to ischemic kidney injury. After bilateral renal ischemia-reperfusion injury, plasma creatinine levels were 4 times higher in claudin-2 null mice and histological scores for acute tubular necrosis were significantly worse. From these results, we concluded that paracellular transport in the proximal tubule is required for efficient utilization of oxygen in the service of Na⁺ transport.

Figure 2.

Role of claudin-2 in renal O₂ utilization. (A) Relationship between T_Na and QO₂ in individual claudin-2 wild-type (WT) and knockout (KO) mice, expressed per gram of kidney weight (kwt). (B) Efficiency of O₂ utilization for renal Na⁺ transport, T_Na/QO₂, calculated from the data in A (n = 7 per group). *P < 0.01, by Student's t test. Reproduced with permission from Pei L. et al., Paracellular epithelial sodium transport maximizes energy efficiency in the kidney. J Clin Invest, 126: 2509–2518, 2016.

Implications for paracellular transport in other tissues

Our findings provide an intellectually satisfying explanation to the question of why paracellular transport exists at all. To wit, if the functions of transepithelial transport and internal homeostasis can be achieved with transcellular transport alone and this can be done with greater accuracy and exquisite regulation, why is paracellular transport necessary and why did the complex barrier-pore structure of the tight junction evolve?

We speculate that paracellular transport is a general energy-saving mechanism that enhances the efficiency of transporting epithelia. A useful analogy is to the hybrid car (Fig. 3). Hybrid cars use energy from gasoline to achieve propulsion. However, some of the kinetic energy from movement of the car is wasted when the car needs to slow down. Braking dissipates that energy as heat. Hybrids make use of that energy to recharge a battery (“regenerative braking”) which can then provide additional propulsion to the car via an electric motor. Paracellular transport works in the same way as the electric engine in a hybrid, making use of energy that would otherwise be wasted (potential energy from electrochemical gradients) to drive additional transport.

Figure 3.

Analogous energy-saving strategies in biology and automotive engineering. (A) Hybrid car. Gasoline provides the fuel for a gasoline engine that provides the primary source of propulsion. Regenerative braking uses kinetic energy to recharge a battery, thereby driving additional propulsion via an electric motor. (B) Transporting epithelium. ATP provides the energy for transcellular transport driven by a membrane ATPase. Electrochemical gradients generated by transcellular transport constitute a store of potential energy available to drive additional passive transport via paracellular diffusion.

It would be interesting to examine the first appearance of paracellular transport in simple metazoa to see if this might have evolved for energy conservation. Unfortunately current knowledge of this is very limited. Septate junctions, which are primordial occluding junctions in invertebrates, have been identified as early in phylogeny as Ctenophores, and claudin homologs are expressed, but their function is unknown. The structural components of septate junctions such as neurexin, contactin and neuroglian, arose later at the time of Placozoa/Cnidaria/Bilateria radiation.²⁶

Could impaired paracellular transport make other epithelial tissues with high rates of transport susceptible to ischemia? It is possible. Admittedly, few other tissues struggle with marginal oxygenation to the same degree as the kidney. But certainly the intestine must perform high rates of solute transport, and intestinal ischemia is a significant clinical problem in patients with atherosclerotic vascular disease and in critically ill patients with impaired organ perfusion. Future studies to investigate the importance of claudins and paracellular transport in the oxygen consumption of other epithelial tissues are needed to shed light on this.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

Studies reported here that were conducted in the author's laboratory were supported by a grant from the NIDDK (R01DK062283).