Pediatric Nephrology

, 26:1479

Calcium signaling triggered by ouabain protects the embryonic kidney from adverse developmental programming


  • Georgiy R. Khodus
    • Department of women’s and children’s healthKarolinska Institutet
  • Markus Kruusmägi
    • Department of women’s and children’s healthKarolinska Institutet
  • Juan Li
    • Department of women’s and children’s healthKarolinska Institutet
  • Xiao-Li Liu
    • Department of women’s and children’s healthKarolinska Institutet
    • Department of women’s and children’s healthKarolinska Institutet

DOI: 10.1007/s00467-011-1816-y

Cite this article as:
Khodus, G.R., Kruusmägi, M., Li, J. et al. Pediatr Nephrol (2011) 26: 1479. doi:10.1007/s00467-011-1816-y


The kidney is extraordinarily sensitive to adverse fetal programming. Malnutrition, the most common form of developmental challenge, retards formation of the kidney’s functional units, the nephrons. The resulting low nephron endowment increases susceptibility to renal injury and disease. Using explanted rat embryonic kidneys, we found that the sodium-potassium-adenosine triphosphatase (Na, K-ATPase) ligand ouabain triggers, via the Na, K-ATPase/ inositol 1,4,5-trisphosphate receptor signalosome, a calcium-nuclear factor-kappa B (NF-κB) signal that protects kidney development from adverse effects of malnutrition. Serum deprivation resulted in severe retardation of nephron formation and robust increase in apoptotic rate, but in ouabain-exposed kidneys, no adverse effects of serum deprivation were observed. Depletion of intracellular calcium stores and inhibition of NF-κB activity abolished the rescuing effect of ouabain. Proof of principle that ouabain rescues development of embryonic kidneys exposed to malnutrition was obtained from studies on pregnant rats given low-protein diets and treated with ouabain or vehicle throughout pregnancy.


Calcium signalingOuabainMalnutritionDevelopmental programing


Developmental programming of the kidney

The kidney is extraordinarily sensitive to the effects of adverse environmental events during certain critical windows of development. Fetal malnutrition, the most common form of developmental challenge, results in reduced nephron formation [14]. As nephron formation is completed toward the end of the gestational period in humans and within the first postnatal week in rodents, fetal malnutrition will result in a permanent reduction of nephron number. Experimental studies performed primarily on rodents have indicated that loss of nephrons caused by maternal low caloric intake or by placental insufficiency encompasses increased risk for renal disease [57]. Epidemiological and morphological studies of nephron number in autopsy material from individuals of different ages and ethnic backgrounds suggest that this is also the case in humans [8, 9]. Yet, despite overwhelming evidence that fetal malnutrition endangers kidney development and results in irreversible nephron loss and increased risk for renal disease, there is as yet no drug available that would alleviate the effects of fetal malnutrition on nephron formation. In this review article, we present evidence from some of our recent studies that, in the future, ouabain, a cardiotonic steroid, may be used to rescue the development of a fetal kidney exposed to malnutrition.

Tissue-protective effect of calcium signals triggered by ouabain activation of the Na, K-ATPase–IP3R signalosome

Several years ago, our group made the observation that the salt pump sodium, potassium-adenosine triphosphatase (Na, K-ATPase) (NKA) can activate the inositol 1,4,5-trisphosphate receptor (IP3R) via protein–protein interaction [10, 11]. This interaction is triggered by ouabain, a highly specific NKA ligand. Ouabain belongs to the family of cardiotonic steroids, which bind to the catalytic subunit of NKA and can dose-dependently inhibit the function of the salt pump. When ouabain binds to NKA in concentrations that spare pump function, the IP3R is activated, and this results in a series of transient increases in intracellular calcium [12, 13]. The oscillatory nature of the signal triggered by NKA/IP3R interaction is of great significance. Calcium oscillations have emerged as the most versatile of all cell signals, as the cell can decode the frequency of the oscillations [14, 15]. Calcium oscillations generated by the NKA/IP3R complex have a low frequency and will activate the pleiotropic transcriptional factor nuclear factor kappa B (NF-κB). This calcium-dependent NF-κB activation results in protection from apoptosis [16]. In a study performed on primary cultures of proximal tubule cells, we have shown that downstream effects of this calcium response include NF-κB activation , protection from apoptosis, and enhanced cell proliferation. The cells had been challenged with serum deprivation to mimic starvation. We concluded from this study that signaling via the NKA/IP3R complex may have a tissue-protective effect in the kidney as well as in other organs. Figure 1 shows depicts the ouabain/NKA/IP3R signaling pathway.
Fig. 1

Sodium, potassium-adenosine triphosphatase inositol 1,4,5-trisphosphate receptor (NKA/IP3R) signalosome responsible for activating calcium oscillations and the subsequent downstream effects

Ouabain rescues development of the malnourished rat kidney

Although it is well recognized that calcium oscillation is a key regulator of early development [15], little is known about the role of calcium for pattern formation and cell differentiation during later stages of embryonic development. Pattern formation is an essential feature in kidney and brain development, and selective apoptosis is important for the proper sculpting of kidney and brain [17, 18]. An imbalance between pro- and antiapoptotic factors may be the cause of adverse fetal programming [7, 19]. In a recently published study [20], explanted E14 embryonic rat kidneys were exposed to very low concentration of serum on the third day of culture to mimic malnutrition. This caused profound apoptosis and a large reduction in nephron formation. If, however, the kidneys had been exposed to ouabain during the period of serum deprivation, they were protected from apoptosis, and there was no reduction in nephron formation. We excluded the possibility that ouabain acted by increasing intracellular sodium concentration. Could this have been a calcium-dependent effect due to NKA/IP3R signalosome activation? We screened the literature for information about calcium activity in the developing kidney, but surprisingly, we found very little information.

We developed a method to record calcium activity in the mesenchymal cells bordering the ureter buds (Fig. 2). Spontaneous calcium activity can be observed in a majority of these cells. In general, calcium activity is observed as repeated transient increases in intracellular calcium: calcium waves (Fig. 3). Approximately 8% of cells show more than four calcium waves during an observation period of 26 min. Ouabain significantly increases the number of cells exhibiting more than four calcium waves. The transient increases of intracellular calcium can be attributed to release of calcium from the intracellular stores, compatible with the notion that they are triggered by the interaction between NKA and IP3R. Acute removal of extracellular calcium has no appreciable effect on intracellular calcium waves. Inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump will deplete the intracellular stores of calcium. This procedure completely blocked the rescuing effect of ouabain.
Fig. 2

Whole-mount immunostaining of an embryonic kidney showing ureteric bud cells (E-cadherin, green) and mesenchymal cells (Wt1, red)
Fig. 3

Representative calcium recording from the mesenchymal cells of an embryonic kidney

In previous studies, we consistently found that one of the downstream effects of ouabain signaling is NF-κB activation. We showed that this was also the case in the embryonic kidney and that NF-κB inhibition abolished the rescuing effect of ouabain. As our observations had potential clinical implications; i.e., that ouabain or its analogues might be used to prevent adverse developmental programming of the malnourished kidney, we decided to go one step further and try to obtain proof of principle in a live animal study. Pregnant rats were give normal or low-protein diet throughout the gestational period. The dams on a low-protein diet were also given ouabain or vehicle throughout the gestation period. The delivery rate of ouabain was such that it should yield a blood concentration of 1 nM ouabain [21]. The offspring were sacrificed at the age of 4 weeks. The offspring of the low-protein dams had a large reduction in the number of glomeruli compared with controls, but the offspring of the low-protein dams that had been treated with ouabain had a similar number of glomeruli as controls.

Clinical implications

The kidney is extraordinarily sensitive to the effects of an adverse environment during critical windows of development. Our data indicate that ouabain activation of NKA/IP3R signaling completely rescues the development of the malnourished fetal kidney. This has important implications, as there is strong evidence that in humans, the irreversible loss of nephrons, caused by maternal low-caloric intake or by placental insufficiency, poses a major risk for renal disease and hypertension.

Copyright information

© IPNA 2011