LABORATORIO DE URGENCIAS

Embarazo y Función Renal

Adaptive changes in single-nephron GFR, tubular morphology, and transport in a pregnant rat nephron.

Melissa M. Stadt and Anita T. Layton
        Melisa M. Stad: University of Waterloo Ph.D. in Applied Mathematics.Canada
        Anita Layton: Prof.titular Cátedra de Investigación en Biología Matemática y Medicina.University of Wateroo.Canadá

American J.. Physiology, Jan 2022 / https://doi.org/10.1152/ajprenal.00264.2021

During pregnancy, the female body must undergo major adaptations to support the solute and volume demands of a developing fetus and placenta. A large plasma volume expansion is required for a healthy pregnancy for both the mother and fetus (1–4). Remarkably, blood pressure typically decreases during pregnancy despite this large volume change. Pregnancy disorders, such as gestational hypertension and preeclampsia, as well as fetal growth restriction are associated with a failure to expand plasma volume (1, 5–10).
In a virgin or nonpregnant (NP) rat, almost all Na⁺ intake is excreted through urine. In contrast, net Na⁺ retention begins from early pregnancy. Na⁺ retention largely drives the plasma volume expansion. Similarly, in a NP rat, ∼90% of K⁺ intake is excreted through urine, whereas the remaining 10% is excreted through feces (16). However, during late pregnancy, there is net K⁺ retention (8, 12, 13, 15, 17).
The majority of retained electrolytes are taken up by the fetoplacental unit (12, 14, ).
The kidneys regulate volume and solute homeostasis (18). Hence, to support the electrolyte and volume retention required in pregnancy, the kidneys undergo major adaptations in morphology (13, 19, 20), hemodynamics (4, 8–10, 13–15, 19, 21–25), and nephron transport (2, 3, 11, 13, 17, 21, 26–34). One of the first gestational renal adaptations observed is the large increase in the glomerular filtration rate (GFR). GFR increases by 40–50% during human pregnancy (8, 10, 25) and about ∼30% in rats (9, 21, 25). This phenomenon is remarkable as there are no other nonpathological states that sustain such a high increase in GFR. That marked increase in filtration must be matched by appropriate changes in nephron transport. Otherwise, the excessive water and electrolyte loss will likely prove fatal.
Further studies found that the kidney increases in size during pregnancy (13, 19, 20, 23). In particular, the proximal tubule lengthens (19, 23). When taken in isolation, this should increase renal transport capacity. Along the nephron, the activity of several key transporters has been found to change during pregnancy; such adaptations are complex and sometimes counterintuitive when considered in isolation (2). For example, Mahaney et al. found that the activity of the major Na⁺ transporter Na⁺-K⁺-ATPase is downregulated during pregnancy despite the large increase in Na⁺ reabsorption. Other Na⁺ transporters are also down regulated (29, 34), whereas others are up-regulated (11, 30, 33, 35). Up-regulation of key water channels has also been found during mid pregnancy (MP) and late pregnancy (LP), likely facilitating increased water reabsorption (27, 28).
Renal adaptations are different during MP and LP. Specifically, during LP, net K⁺  retention occurs in addition to Na⁺  retention, which starts from early pregnancy. West et al. (17) found significant changes in K⁺ transport in distal nephron segments during LP. Na⁺ and K⁺ transporter adaptations during pregnancy in the rat have been discussed in a recent review by de Souza and West (2).
In recent years, substantial progress has been made in identifying gestational renal adaptations. Studies have generally focused on one segment or a few transporters (understandable due to the difficulty and cost of conducting these complicated experiments). Therefore, how these complex adaptations come together to support the altered solute and volume demands during pregnancy is not fully understood. A comprehensive understanding of gestational kidney function is important for understanding how the female body undergoes massive adaptations required to sustain the growing fetus and placenta. Furthermore, altered renal function from normal pregnancy values has been found in gestational diseases such as preeclampsia (36), gestational hypertension (32), and gestational diabetes (37). Hence, understanding normal renal function during pregnancy is a step toward ultimately understanding not only female physiology but these complicated gestational diseases as well.
Computational models have been used to investigate the complex processes involved in renal function (38–47). Recently, sex-specific epithelial transport models have been built for a rat proximal tubule (48), rat superficial nephron (49), rat kidney (50), and human kidney (51) based on experimental results that found sexual dimorphisms in morphology and transporter activities described in Refs. 52–54. Li et al. (48) developed and analyzed the first sex-specific computational epithelial transport model for the proximal convoluted tubule (PCT) of the rat. Specifically, model simulations in their study predicted that the lower fractional volume reabsorption in the female proximal tubule may be attributed to lower aquaporin (AQP) expression levels and the smaller transport area due to the significantly smaller proximal tubule size in females compared with males. Later, Hu et al. (49) extended the model to a full superficial nephron in the rat kidney for both males and females. Their simulations were used to analyze the functional implications of sex differences in hemodynamics, size, and renal transporters in male and female rats from experimental results described in Refs. 52–54. The pregnancy-specific superficial nephron models presented here are based on Hu et al. by incorporating pregnancy adaptations reported in the literature to represent superficial nephron function in MP and LP.
The main goal of this study was to assess the extent to which individual pregnancy adaptations, in morphology or transporter activities, contribute to the observed differences in electrolyte and volume reabsorption during pregnancy. Specifically, we identified which renal adaptations may have the largest impact on Na⁺, K⁺, and volume reabsorption using model simulations and sensitivity analysis. Furthermore, transporter activities in pregnant rats have not been fully characterized. We sought to predict additional renal adaptations that may occur during pregnancy. Separate analyses are conducted for MP and LP due to different renal adaptations in these stages that support evolving fetal and placental demands.

MATERIALS AND METHODS

Hu et al. developed an epithelial cell-based model of the superficial nephron of a female rat kidney. This model was used as a control in this study and referred to as the NP model. In this study, we extended the NP model to simulate solute and volume transport along a superficial nephron of 1) a MP kidney and 2) a LP rat kidney. Note that rat gestation is ∼21–22 days (1, 23). The MP rat model represents nephron transport at ∼12–15 days of gestation (i.e., about halfway through gestation). The LP rat model represents nephron transport at ∼19–20 days of gestation (i.e., near the end of rat gestation). As previously noted, we built separate models for MP and LP because there are distinct adaptations during the different stages of pregnancy due to the growing demands of the developing fetus and placenta. Because the original model equations in the study by Hu et al. were based on mass conservation that is similarly valid in pregnancy, those same equations were used here, but appropriate parameter values were changed to account for renal adaptations during MP and LP.
Pregnancy-Specific Models
Using the NP (female) cell-based superficial nephron epithelial transport model developed in the study by Hu et al. we created pregnancy-specific models (i.e., MP and LP) by increasing or decreasing relevant NP model parameter values based on experimental findings in the literature.
The first major change in the MP and LP models is the increase in single-nephron GFR (SNGFR). MP and LP SNGFR were increased by 30% and 20%, respectively, from the NP SNGFR value. These increases were based on findings in the study by Baylis where euvolemic pregnant rats were studied. We note that a previous study has shown that LP rat SNGFR is significantly higher than MP rat SNGFR (13); however, an important note is that this study was done in hypervolemic rats, thus likely giving a differing result (9, 25). As such, we chose to model the euvolemic rat based on the Baylis result. Specifically, MP and LP SNGFR values were 31 and 29 nL/min, respectively, versus 24 nL/min in NP. The elevated SNGFR significantly increases the filtered load at the beginning of the nephron in both MP and LP models. In addition, during pregnancy, plasma osmolality is decreased, where plasma Na⁺ concentration and Cl⁻ concentration slightly decreased, but with slightly elevated plasma K⁺ concentration in LP (10, 11, 13, 17, 55). These changes are small compared with the SNGFR increases, still resulting in substantially increased filtered loads.
During pregnancy, kidney volume increases (20, 23). In particular, it has been shown that the proximal tubule lengthens (19, 23). Accordingly, we increased the proximal tubule length by 14% and 17% in MP and LP models, respectively.
Based on the increased kidney volume and the observed dilation in the collecting system during pregnancy (56), we assumed that the diameter increased by 7% in the proximal tubule and 5% in the distal tubule in both pregnant rat models. Although tubular diameter has not been measured experimentally, without assuming this small tubular dilation, the much-elevated volume flow in pregnancy would cause an excessive drop in tubular fluid pressure.
* Na⁺ Transporters
There have been a multitude of changes found in the activity and expression of Na⁺ transporters, as recently reviewed by de Souza and West. Here, we explain how we implemented experimental results to determine MP and LP model parameter values for Na⁺ transporters.
Mahaney et al. showed that pregnancy-induced changes in Na⁺-K⁺-ATPase activity and expression are region specific: in the cortex, Na⁺-K⁺-ATPase activity is decreased in both MP and LP, whereas in the medulla, Na⁺-K⁺-ATPase activity is increased in MP but is unchanged in LP relative to NP controls.
Khraibi et al. investigated Na-P_i cotransporter 2 (NaPi2) in the proximal tubule during pregnancy. They found that protein expression of NaPi2 decreased during MP and LP (29).
The relevant parameter value for NaPi2 was slightly decreased in MP and LP based on this study. Na⁺-K⁺-2Cl⁻ cotransporter 2 (NKCC2) activity was increased significantly in LP and slightly in MP based on West et al..
During pregnancy, aldosterone levels are elevated (1), which regulates the fine tuning of Na⁺ handling in the distal segments (57). West et al. reported: 
   1) epithelial Na⁺ channel (EnaC) activity is increased significantly (about double) during both MP and LP, 
   2) protein abundance of the Cl⁻/bicarbonate exchanger pendrin is increased (30),
   3) Na⁺-Cl⁻ cotransporter (NCC) activity is not changed during MP and slightly decreased during LP relative to NP (34). These findings were implemented in the pregnancy models accordingly.
Na⁺/H⁺ exchanger (NHE) activity has not been well characterized during pregnancy. During pregnancy, cortical mRNA expression of NHE3 was found to be increased during pregnancy (32).
However, NHE3 protein abundance was found to be slightly decreased or unchanged in MP and LP rats compared with NP rats (2, 11, 29). We note that protein abundance is not necessarily a good indicator of NHE3 activity. Specifically, it has been found that in female rats (i.e., NP), there is a higher protein expression of NHE3 but lower NHE3 activity in the proximal tubule compared with male rats. Hence, there is reserve NHE3 in female rats that may be activated during pregnancy (49, 50, 52). We assumed that the NHE3 reserve was activated in pregnancy by increasing NHE3 activity in MP and LP models This assumption allows the model to avoid excess natriuresis and diuresis in pregnancy Water Channels
* Water channel
AQP2 is significantly upregulated during MP and LP (27, 28, 32). It has also been hypothesized that AQP1 in the descending limb is upregulated during LP but largely unchanged during MP (27). Water permeability in the appropriate segments was adjusted to reflect increased AQP1 and AQP2 based on these finding
* K⁺ Transporters
Unlike Na⁺ retention, which starts from early pregnancy, K⁺ retention is not observed until LP (17). Indeed, K⁺-specific renal transporter changes have largely only been studied during LP to date (2, 17). West et al. found that the renal outer medullary K⁺ (ROMK) channel and large-conductance K⁺ (BK) channel are significantly downregulated in the LP rat. This result is represented in the LP model by lowering the K⁺ apical permeability in the appropriate distal segments. In addition, West et al. found that H⁺-K⁺-ATPase type 2 activity in the connecting tubule (CNT) and CD is substantially increased in the LP rat. Salhi et al. reported an increase in H⁺-K⁺-ATPase activity in LP mice as well.
Elevated progesterone levels during pregnancy activate H⁺-K⁺-ATPase (58). Based on these experimental results, we significantly increased H⁺-K⁺-ATPase activity in the CNT and CD of the LP model Similar changes were made in the MP model to avoid excessive kaliuresis  We note that Abreu et al. (32) showed that mRNA expression of ROMK2 is significantly downregulated during MP, but no study has characterized H⁺-K⁺-ATPase activity and BK channels during MP. In addition, to avoid excessive kaliuresis and natriuresis in the MP and LP models, we hypothesized that the K⁺-Cl⁻ cotransporter in the ascending limb and distal convoluted tubule (DCT) is also upregulated during pregnancy
* Epithelial Transport Model
The cell-based epithelial transport model represents functionally distinct segments as follows: the PCT, proximal straight tubule (also known as the S3 segment), short descending limb, thick ascending limb (TAL) divided into the medullary and cortical parts (medullary TAL and cortical TAL), DCT, CNT, and CD divided into the cortical and medullary segments (cortical CD, outer medullary CD, and inner medullary CD). Each nephron segment other than the proximal tubule (which is compliant, as discussed in the following section) is represented as a rigid tubule lined by a layer of epithelial cells, with apical and basolateral transporters that vary according to cell type. Each nephron segment has distinct transporters, permeabilities, and morphological properties. The model accounts for the following 15 solutes: Na⁺, K⁺, Cl⁻, HCO−3, H₂CO₃, CO₂, NH₃, NH+4, HPO2−4, H2⁢PO−4, H⁺, HCO₂⁻, H₂CO₂, urea, and glucose.
The model is a large system of coupled ordinary differential and algebraic equations, solved for steady state, and predicts luminal fluid flow, hydrostatic pressure, luminal fluid solute concentrations, cytosolic solute concentrations, membrane potential, and transcellular and paracellular fluxes.
Model Equations is described in more detail in the studies by Layton et al. (41) and Layton and Edwards (59).

RESULTS

Baseline Model Results
Model simulations were conducted for the NP, MP, and LP models to determine how pregnancy-induced changes in renal hemodynamics, morphology, and transporter activity together modify tubular transport in the superficial nephrons of the rat kidney. Delivery of key solutes and volume to each segment is shown in  with transport for each segment and accumulated along the nephron .
In the NP model, 58% of the filtered Na⁺ is reabsorbed along the proximal tubule, primarily via the coordinated transport of apical NHE3 and basolateral Na⁺-K⁺-ATPase. In MP and LP, because of pregnancy-induced hyperfiltration and enhanced tubular transport capacity, net Na⁺ reabsorption along the proximal tubule increases by 27% and 18%, respectively. Interestingly, those net increases in reabsorption correspond to only minor increases in fractional reabsorption, to 60% in both MP and LP models, due to increased filtered Na⁺ load. (recall that SNGFR increases in MP and LP by 30% and 20%, respectively, or to 31 and 29 nL/min vs. 24 nL/min in NP.) The relative stability of the fractional reabsorption is reminiscent of the glomerulotubular balance observed in the proximal tubules.
The marked increase in net Na⁺ proximal tubule transport in pregnancy can be attributed to the increase in the length and diameter and elevated NHE3 activity in the proximal tubule, despite the reduction in Na⁺-K⁺-ATPase activity.
Most of the remaining Na⁺ is reabsorbed downstream along the TAL. Compared with the NP model, Na⁺ transport along the TAL is predicted to be 18% and 8% higher in the MP and LP models, respectively. This enhanced transport is facilitated by increased NKCC2 and NHE activity. Downstream of the TAL (i.e., segments after the macula densa), Na⁺ reabsorption is predicted to be about the same in each of the NP, MP, and LP models. Urine Na⁺ excretion is 32, 34, and 35 pmol/min for the NP, MP, and LP models, respectively. Urinary Na⁺ excretion is the highest in LP, at 10% above the NP model. The predicted natriuresis is consistent with reported ranges (11, 12, 63).
Like Na⁺, 55% of the filtered Cl⁻ is reabsorbed along the proximal tubule in the NP, MP, and LP models, with most of the remaining Cl⁻ reabsorbed along the TAL. Along the TAL, Cl⁻ reabsorption is increased by 19% and 9% in the MP and LP models, respectively. In both pregnant models, increased transport in the TAL is due to the increased NKCC2 activity. In addition, K⁺-Cl⁻ cotransporter activity is increased, resulting in increased Cl⁻ reabsorption. Urinary Cl⁻ excretion for the NP, MP, and LP models is 7.1, 8.0, and 7.4 pmol/min, respectively. We note that Cl⁻ urinary excretion is slightly increased during pregnancy, and the increase in predicted urinary Cl⁻ excretion is within reported ranges (13, 63).
The model predicts that 54% of filtered K⁺ is reabsorbed along the proximal tubule of the NP model. Net K⁺ reabsorption along the proximal tubule increases by 36% in MP and 52% in LP. Together with the increases in filtered K⁺ load, due to increased SNGFR, and in LP increased K⁺ plasma concentration, the net increases in K⁺ reabsorption yield increases in fractional reabsorption, at 57% in MP and 60% in LP, along the proximal tubule. Like Na⁺ and Cl⁻, most of the remaining K⁺ is also reabsorbed along the TAL. Downstream of the loop of Henle, the DCT and CNT vigorously secrete K⁺ . Consequently, 22% of the filtered load of K⁺, or 21 pmol/min per (superficial) nephron, is excreted in urine in the NP model Interestingly, K⁺ transport along the distal segments differs significantly in pregnancy; H⁺-K⁺-ATPase activity is increased and K⁺ secretion in distal segment K⁺ channels is reduced. As a result, in MP, K⁺ secretion along the DCT and CNT is decreased by 10% despite increased ENaC activity that would, in isolation, increase K⁺ secretion. In addition, reabsorption in the CD is increased by 54% compared with the NP model. Similar changes occur in LP. These changes lead to 29% and 42% more accumulated K⁺ reabsorption along the full nephron during MP and LP, respectively.
Renal NH+4 handling is predicted to be qualitatively similar in NP and pregnancy. In all three cases considered, the proximal tubule is a major site of NH+4 secretion via substitution of H⁺ in the NHE3 transporter, whereas a substantial fraction of NH+4 is reabsorbed along the TAL by substituting for K⁺ in the Na⁺-K⁺-2Cl⁻ cotransporter. The proximal tubule also serves as the major site for HCO−3  reabsorption, with the remainder reabsorbed along the TAL.
The majority (65%) of the filtered volume is reabsorbed along the proximal tubule in the NP model Net water reabsorption along the proximal tubule increases by 34% and 25% in MP and LP, respectively. This yields a similar fractional reabsorption of ∼68% in both MP and LP models along the proximal tubule. Enhanced water reabsorption is primarily due to the increase in proximal tubule length. More water is reabsorbed downstream, albeit at a slower rate. The models, which represent a superficial nephron of the kidney in an antidiuretic state, predict a urine output of 0.25 nL/min/nephron (superficial) in the NP model and 0.30 nL/min/nephron (superficial) in both MP and LP models.
The increase in volume excretion during pregnancy is within reported ranges (11, 13, 63).
Taken together, the baseline results suggest that the adaptations represented in the MP and LP models yield an appropriate response to the marked elevation in filtered loads of solutes and volume during normal pregnancy.
* Na⁺ transporters.
Without increased NHE activity, urinary Na⁺ excretion increased by 21% and 10% in the MP and LP models, respectively. This change had one of the largest impacts on urinary Na⁺ excretion for any of the Na⁺ transporter pregnancy changes for the MP and LP models. In addition, NHE activity had the second largest impact on urinary volume excretion in both MP and LP models, with an increase of 9% and 7%, respectively. These results indicate that upregulation of NHE activity may play a key role in Na ⁺ and fluid retention in pregnancy.
Mahaney et al. (26) observed that changes in Na⁺-K⁺-ATPase activity during pregnancy are region dependent. Specifically, cortical Na⁺-K⁺-ATPase activity is decreased in both MP and LP, whereas medullary Na⁺-K⁺-ATPase activity is increased during MP and unchanged during LP (26). In the LP model, resetting Na⁺-K⁺-ATPase activity to the higher NP value led to a 17% increase in Na⁺ reabsorption along the proximal tubule and ascending limb, resulting in lower Na⁺ delivery to segments after the macula densa so that urinary Na⁺ excretion was decreased by 19%. In the MP model, the opposite changes in cortical and medullary Na⁺-K⁺-ATPase activity resulted in a smaller decrease of 12% in urinary Na⁺ excretion.
Because water transport is largely driven by Na⁺ reabsorption, volume excretion was decreased by ∼7% in both MP and LP models And because distal K⁺ secretion is driven by Na⁺ reabsorption, the reduced luminal Na⁺ flow and Na⁺ transport decreased urinary K⁺ excretion by 6% and 3% in the MP and LP models, respectively NKCC2 activity in the TAL was increased by 15% and 50% in the MP and LP models, respectively, relative to the NP value. However, NKCC2 activity did not seem to have a large effect on the MP and LP models. Without NKCC2 upregulation, net Na⁺ transport along the TAL decreased by about ∼1% in both MP and LP models. This resulted in a change of <1% in urinary Na⁺ excretion in both MP and LP models.
ENaC activity in the distal segment was increased by 85% and 115% in the MP and LPmodels, respectively, based on experimental data from Refs. 11, 31, and 33. Our analysis indicated that increased ENaC activity had the largest impact on predicted urinary Na⁺ excretion in both MP and LP ENaC plays a role in fine tuning Na⁺ transport in the distal segments, so without the increase during pregnancy, our models predicted a ∼5% decrease in reabsorption in the distal segments (downstream of the macula densa) in both MP and LP models. This, in turn, resulted in urinary Na⁺ excretion increasing by about ∼21%, together with an 3% increase in urine output in both MP and LP models.
We also noted that since ENaC is a known potent kaliuretic factor, K⁺ excretion was significantly decreased without its upregulation
Other Na⁺ transporters, including NaPi2, NCC, and NHE1, are all downregulated during pregnancy. An analysis of the impact of these changes on Na⁺, K⁺ and volume excretion revealed a <2% change for each of these transporters in both MP and LP models.
In summary, our analysis showed that increased activities of NHE and ENaC play key roles in the increased Na⁺ reabsorption of the kidneys and subsequent Na⁺ retention observed during pregnancy. These transporters also have a significant impact on volume retention and thus pregnancy-induced plasma volume expansion.
* Water channels.
The CD water channel AQP2 is upregulated during MP and LP (28, 32). Without this adaptation, volume excretion increased by 8% and 5% in MP and LP models, respectively, due to decreased (2% in both MP and LP) CD water reabsorption. It has also been hypothesized that the water channel AQP1 is upregulated in the descending limb during LP (27). We found that this change did not have as significant an impact on urinary excretion as AQP2; downregulation of AQP1 to the NP value increased volume excretion by <1%. Therefore, our analysis suggests that the upregulation of AQP2 may play a key role in volume reabsorption during pregnancy.
* K⁺ transporters.
Apical K⁺ permeability in the distal segments was significantly lowered to account for downregulated major K⁺ secretory channels (ROMK and BK channels) in the distal tubule during pregnancy. In the LP model, without this downregulation, K⁺ secretion along the DCT and CNT was increased by 21%, resulting in a 10% increase in urinary K⁺ excretion. In the MP model, without downregulated secretion in the distal segments, K⁺ secretion along the DCT and CNT increased by 25%, resulting in K⁺ excretion predicted to increase by 9%. We also noted that when the apical K⁺ permeability was increased to NP levels, Na⁺ excretion greatly decreased due to the natriuretic effect of lower K⁺ secretion.
H⁺-K⁺-ATPase type 2 activity in the CNT and CD is substantially increased during pregnancy (17, 58). Without the increase in H⁺-K⁺-ATPase activity, urinary K⁺ excretion increased by ∼18% in both MP and LP models due to a 40% and 26% decrease in K⁺ reabsorption along the CD in the MP and LP models, respectively. This result shows that increased H⁺-K⁺-ATPase activity during pregnancy is essential to prevent excess kaliuresis in both MP and LP.
Activity of the K⁺-Cl⁻ cotransporter (KCC) has yet to be characterized experimentally in the pregnant rat. We estimated that KCC activity should be increased by ∼40% in MP and 35% in LP to avoid excessive water and electrolyte loss. In the absence of KCC upregulation, the predicted K⁺ excretion increased by 17% and 8%, Na⁺ excretion increased by 21.5% and 10%, and volume excretion increased by 13% and 7% in both MP and LP models, respectively. This result suggests that KCC may play a role in electrolyte and water retention during pregnancy.
In summary, model analysis supports the hypothesis by West et al. (17) that K⁺ retention in LP is driven by decreased secretion due to decreased ROMK and BK channel expression in conjunction with significantly increased H⁺-K⁺-ATPase activity.
The model analysis also suggests that similar adaptations are made during MP to prevent excess kaliuresis. In addition, the simulation results suggest that upregulation of KCC may be necessary to prevent excessive diuresis, natriuresis, and kaliuresis in pregnancy.

DISCUSSION

Pregnancy induces major changes in the structure and function of the kidney, resulting in kidney growth as well as elevated blood flow, in a process that changes continually throughout pregnancy (1, 2, 13, 23). A particular drastic change is the ∼30% increase in GFR in pregnant rats (9, 21). Although osmolality of the plasma is slightly decreased, this increase in GFR results in an increased filtered load to the nephrons during pregnancy. How do the nephrons of a pregnant rat handle the increased filtered load? Pregnancy-induced renal adaptations, as recently reviewed by de Souza and West (2), are complex and extensive. How might those coordinated changes not only meet that increased demand but retain electrolytes? These are the questions that the present study sought to answer.
The increase in plasma volume required to supply the developing fetus and placenta is largely driven by Na⁺ retention (1, 2, 11). During pregnancy, aldosterone, angiotensin II, estrogen, and insulin are elevated; all these hormones are known to stimulate renal Na⁺ retention (2, 64, 65). However, progesterone, nitric oxide, and GFR are also elevated; these are factors known to promote Na⁺ excretion (2, 19, 26, 64–66). Thus, Na⁺ retention in pregnancy is a result of several competing factors.
Located on the apical side of proximal tubular cells, NHE3 drives most renal Na⁺ transport in the proximal tubule. The protein abundance of NHE3 has been reported to decrease in MP and LP rats (29) or remain unchanged in whole kidney homogenates (11). That said, the difference between transporter protein abundance and activity must be appreciated, and it is the latter, i.e., activity, that ultimately determines the transport capacity of the cell. For example, proximal tubules in female rats exhibit a higher protein expression of NHE3 compared with male rats but lower NHE3 activity due to a higher level of phosphorylation (49, 52). These two competing effects result in lower Na⁺ transport along female rat proximal tubules (49, 52). The mystery of the “untapped” NHE3 in the female rat kidney has led to the hypothesis that it gives females the reserved capacity to handle the increased transport demand in pregnancy and lactation (49, 50). No known study to date has determined NHE3 activity during pregnancy. We have postulated that NHE3 is elevated in MP and LP, making it possible for pregnant females to reabsorb the enhanced filtered Na⁺ load (50). In the absence of this adaptation, model analysis suggests that the water and electrolyte retention required in pregnancy would not have been possible.
Although the proximal tubules reabsorb most of the filtered Na⁺ and fluid, the distal tubular segments are responsible for fine tuning the remaining filtrate so that urinary excretion approximates equal intake.
West et al. (11) reported an increase in renal α-ENaC activity during MP and LP. In addition, Fu et al. (35) found a significant increase in ENaC protein expression during pregnancy and hypothesized that this was driven by activation of the intrarenal renin-angiotensin-aldosterone system via the prorenin receptor. West et al. (31) found that chronic ENaC blockade in pregnant rats resulted in significantly reduced Na⁺ retention and plasma volume expansion and pups with a lower birth weight. Our sensitivity analysis indicated that without the ENaC activity increase, excess natriuresis occurs. Together, model analysis and previous experimental reports have demonstrated the importance of massively increased ENaC activity during pregnancy.
Despite Na⁺ retention occurring in pregnancy, some renal Na⁺ transporters are down regulated. Mahaney et al. showed that Na⁺-K⁺-ATPase activity is significantly decreased in the renal cortex during both MP and LP. In the medulla, Na⁺-K⁺-ATPase activity is slightly increased during MP and then largely remains unchanged during LP (26). It is likely that the increased progesterone and nitric oxide during pregnancy decrease the activity of cortical Na⁺-K⁺-ATPase (26, 67). These findings are initially surprising as an increase in Na⁺-K⁺-ATPase activity would likely be expected to facilitate Na⁺ retention. Also notable is the finding that renal NCC is essentially unchanged during MP and decreased during LP (34). That observation appears to be counterintuitive to the upregulation of aldosterone during pregnancy, which normally activates NCC (2, 34). Nonetheless, model simulations indicated that, with the other renal adaptations, the kidney can meet the Na⁺ transport demand in pregnancy via Na⁺ reabsorption through other transporters and increased nephron size.
The kidney is larger in both MP and LP rats (20, 23). In particular, the proximal tubule significantly increases in length (13, 19, 23); its diameter likely increases as well. Together, these imply a significantly larger transport area. For example, in the LP model, the 17% and 7% increases in proximal tubule length and diameter, respectively, imply a 25% increase in transport area, which has a major impact on electrolyte and water transport. The effect of larger transport area has been previously assessed in a modeling study that focused on sex difference (not pregnancy). Li et al. (48) suggested that the larger transport area of the proximal tubule of the male rat compared with the female rat (∼50%) led to a corresponding ∼50% higher Na⁺ reabsorption in the proximal tubule. In the case of the pregnant rat, its larger proximal tubule may be what drives overall increased Na⁺ retention despite down regulated key Na⁺ transporters. It is also noteworthy that to allow for the significant pregnancy-induced increase in proximal tubule length and likely diameter size, the cortex of a virgin female kidney may have developed to be smaller, which may explain the observation that much of the difference in size between female and male rat kidneys can be attributed to the cortex (68).
Pregnancy is marked by an increased water reabsorption and water retention. Joyner et al. (27) found that AQP1, expressed along the descending thin limb, is unchanged during MP but significantly upregulated during LP. Ohara et al. (28) found that in the CD, AQP2 was significantly upregulated during both MP and LP. As noted previously, the distal tubular segments are responsible for fine tuning electrolyte and water transport. Our sensitivity analysis indicated that the marked increase in water transport along the proximal tubules alone was not enough to achieve the water retention required in pregnancy; additional upregulation of AQP2 is necessary to avoid excessive water loss.
Unlike Na⁺ retention, which begins at early gestation, K⁺ retention is only observed in LP (2, 17, 63). K⁺ is retained despite known kaliuretic factors, including elevated levels of circulating aldosterone (64), increased plasma K⁺ concentration (17, 63, 64), decreased NCC activity (34), and increased ENaC activity (11) that, when taken in isolation, tend to increase K⁺ secretion. In contrast, K⁺ secretion along much of the distal nephron segments is mediated by apical ROMK channels, which are significantly downregulated in LP, promoting K⁺ retention (17). In addition, West et al. (17) found that H⁺-K⁺-ATPase expression is highly upregulated during LP. Salhi et al. (58) showed that in LP mice, elevated progesterone levels activate H⁺-K⁺-ATPase and attenuate CD K⁺ secretion. Taken together, these competing changes reduce K⁺ excretion leading to K⁺ retention in LP. Our sensitivity analysis indicated that upregulation of H⁺-K⁺-ATPase activity and significantly decreased K⁺ secretion along the distal nephron are needed for K⁺ retention, consistent with the hypothesis by West et al. (17).
Abreu et al.showed major decreased ROMK2 mRNA expression in MP rats, but no other study to date has studied K⁺ transporter activity during MP specifically. However, kaliuretic factors have been found during MP, including increased aldosterone and ENaC activity (1, 11). In addition, there are high progesterone levels, which are known to increase H⁺-K⁺-ATPase activity (58, 64). Our model analysis found that without decreased K⁺ secretion along the distal segments and increased H⁺-K⁺-ATPase activity, excessive kaliuresis is predicted in MP Therefore, we hypothesize that the K⁺ transport adaptations that occur during LP also occur in MP (likely to a lesser extent) despite K⁺ retention occurring only in LP.
This study sought to answer this question: What is the physiological implication of the differences in renal transporter distribution between female and rat male nephrons: lower Na⁺ and water transporters and lower fractional reabsorption in the proximal nephron of female versus male rats, coupled with more abundant transporters in renal tubule segments downstream of the macula densa (52)? Our findings suggest this answer: to better prepare females for the electrolyte and fluid retention adaptations required in pregnancy. More specifically, a key finding is that the relatively large fraction of inactivated NHE3 in the proximal tubule of the female rat (52) makes it possible for increased Na⁺ transport capacity to meet the higher demands in pregnancy, by activating reserved NHE3 without increasing the overall abundance of NHE3 proteins. Moreover, compared with males, females exhibit more abundant transporters along the distal nephron segments, which are responsible for the fine control of electrolyte excretion (69). Indeed, simulation results indicated that Na⁺ and K⁺ retention in pregnancy is accomplished, in large part, by the changes in transporters along the distal nephron. Taken together, the model simulations indicated that the pregnancy-induced morphological and molecular changes along the nephron allow the kidneys of a pregnant rat to meet the marked increase in filtered load and to retain Na⁺ and K⁺ necessary for supporting rapid fetal growth.

PERSPECTIVES AND SIGNIFICANCE

A healthy pregnancy requires a myriad of physiological adaptations. Due to an essential increase in plasma volume and electrolyte retention, major adaptations in the kidney are required. Experimental studies have shown several major adaptations specifically in kidney morphology and renal transporter activities. To investigate the functional implications of these changes, we developed the first computational models of nephron function during MP and LP in the rat. Model simulations quantify which adaptations play major roles in electrolyte and volume homeostasis during pregnancy, analyze the synergy of these changes, and predict additional adaptations necessary for homeostasis. The insights provided by this study into renal physiology during normal pregnancy are a step toward understanding pregnancy disorders that may alter renal transport.

NOTE: This is an extensive summary. The full text, figures, tables, and complete references can be found in the publication mentioned at
           the beginning.

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