S6 kinase 1 knockout inhibits uninephrectomy- or diabetes-induced renal hypertrophy

Abstract

Removal of one kidney stimulates synthesis of RNA and protein, with minimal DNA replication, in all nephron segments of the remaining kidney, resulting in cell growth (increase in cell size) with minimal cell proliferation (increase in cell number). In addition to the compensatory renal hypertrophy caused by nephron loss, pathophysiological renal hypertrophy can occur as a consequence of early uncontrolled diabetes. However, the molecular mechanism underlying renal hypertrophy in these conditions remains unclear. In the present study, we report that deletion of S6 kinase 1 (S6K1) inhibited renal hypertrophy seen following either contralateral nephrectomy or induction of diabetes. In wild-type mice, hypertrophic stimuli increased phosphorylation of 40S ribosomal protein S6 (rpS6), a known target of S6K1. Immunoblotting analysis revealed that S6K1(-/-) mice exhibited moderately elevated basal levels of rpS6, which did not increase further in response to the hypertrophic stimuli. Northern blotting indicated a moderate upregulation of S6K2 expression in the kidneys of S6K1(-/-) mice. Phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1, another downstream target of the mammalian target of rapamycin (mTOR), was stimulated to equivalent levels in S6K1(-/-) and S6K1(+/+) littermates during renal hypertrophy, indicating that mTOR was still activated in the S6K1(-/-) mice. The highly selective mTOR inhibitor, rapamycin, inhibited increased phosphorylation of rpS6 and blocked 60-70% of the hypertrophy seen in wild-type mice but failed to prevent the approximately 10% hypertrophy seen in S6K1(-/-) mice in response to uninephrectomy (UNX) although it did inhibit the basal rpS6 phosphorylation. Thus the present study provides the first genetic evidence that S6K1 plays a major role in the development of compensatory renal hypertrophy as well as diabetic renal hypertrophy and indicates that UNX- and diabetes-mediated mTOR activation can selectively activate S6K1 without activating S6K2.

Fig. 1.

Determination of the genotypes of the offspring derived from S6 kinase 1 (S6K1) heterozygous mice. S6K1^+/− mice were intercrossed to generate S6K1 knockout mice as previously described (52). Genotypes of the offspring were determined by PCR as detailed in materials and methods. A: representative data of the genotyping by PCR using the primer set S6K1-pr. The primer sequences of S6K1-pr were indicated in materials and methods; this primer set was expected to produce a 703-bp single band only from wild S6K1 allele. MK, markers of DNA size in base pairs. B: representative data of the genotyping confirmation by PCR using an additional set of PCR primers Neo-pr (see materials and methods for the sequences of these primers). The Neo-pr primer set was designed to produce a 280-bp single band only from the mutant S6K1 allele, which contains the Neo-cassette (52). C: representative immunoblotting data confirming the genotypes determined by PCR using S6K1-pr and Neo-pr. Specifically, whole kidney homogenates from the same animals shown in A and B were immunoblotted with an antibody that recognizes p70S6K1. +/+, +/−, and −/− indicate the genotypes of S6K1 wild-type, heterozygous, and homozygous knockout mice, respectively.

Fig. 2.

S6K1 knockout blunted compensatory renal hypertrophy induced by unilateral nephrectomy (UNX). S6K1^−/− mice and their wild-type littermates (S6K1^+/+ mice) at 10 wk of age were subjected to right UNX or Sham surgery. UNX-induced compensatory renal hypertrophy 7 days after surgery was assessed by increases in left kidney/body weight ratio (A) and increases in protein/DNA ratio of the kidney (B), compared with their Sham mice, respectively. Values are means ± SE (n = 6 for each group of UNX or Sham; ***P < 0.0001 vs. their corresponding Sham control groups).

Fig. 3.

UNX-induced phosphorylation of the 40S ribosomal protein S6 (rpS6) in the remaining kidney of S6K1^+/+ mice but not in their S6K1^−/− littermates. S6K1^−/− mice and their wild-type littermates (S6K1^+/+ mice) at 10 wk of age were subjected to right UNX or Sham surgery. One week after surgery, the animals were euthanized, and kidney lysates were subjected to immunoblotting analysis with antibodies specifically recognizing Ser235/236-phosphorylated rpS6 (P-rpS6) (A), total rpS6 (B), all of the total 4E-binding protein 1 (4E-BP1) species, α, β, γ, and δ (C), or Ser65-phosphorylated 4E-BP1 (P-4E-BP1) alone (D), respectively. Note the band shift of the multispecies protein, total 4E-BP1, in response to UNX that indicates activation of mammalian target of rapamycin (C). Equal loading was confirmed by immunoblotting with a β-actin antibody (E). All of the blots shown are representative blots of 3 separate experiments with similar results.

Fig. 4.

S6K1 knockout upregulated the expression of S6K2 in the kidney. S6K1^−/− mice and S6K1^+/+ mice at 10 wk of age were euthanized, and total kidney RNA was isolated. The mRNA expression level of S6K2 was determined by Northern blot analysis. To ensure equal RNA loading, the blots were stripped and reprobed with a cDNA probe for GAPDH (A). Shown are representative data from n of 6 for each genotype. Upregulation of S6K2 expression in response to S6K1 knockout was quantitatively confirmed to be statistically significant by densitometric analysis. Densitometric values are expressed as ratios of S6K2/GAPDH densitometry quantification (B). n = 6; **P < 0.005 vs. their wild-type littermates.

Fig. 5.

Rapamycin (Rapa) inhibition of UNX-induced phosphorylation of rpS6 and 4E-BP1. S6K1^−/− mice and their S6K1^+/+ littermates at 10 wk of age were administered either rapamycin (1 mg/kg body wt ip) or vehicle (Veh) alone for 2 h before right UNX or Sham surgery, followed by administration of rapamycin or vehicle once a day. The animals were euthanized 7 days after surgery, and left kidney homogenates were subjected to immunoblotting analysis with an antibody that recognizes only Ser235/236-phosphorylated rpS6 (A) and an antibody specifically recognizing 4E-BP1 that was phosphorylated at serine 65 (B). Equal loading was confirmed by stripping and reprobing of the blot with a β-actin antibody (C). Each lane sample was from pooled kidney lysates of 3 mice in the same group (thus two lanes represent a total n of 6 animals for each group). Shown are representative blots from 1 of 3 separate experiments with similar results.

Fig. 6.

Inhibition of rapamycin on UNX-induced compensatory renal hypertrophy in S6K1^+/+ mice but not in S6K1^−/− littermates. Male, 10-wk-old S6K1^+/+ mice and their S6K1^−/− littermates were pretreated with rapamycin (1 mg/kg body wt ip) or vehicle alone for 2 h before right UNX or Sham surgery, followed by administration of rapamycin or vehicle once a day. Seven days after the surgery, UNX-induced compensatory renal hypertrophy was assessed by increases in kidney/body weight ratios (A) as well as increases in protein/DNA ratios (B), compared with Sham-operated/vehicle-treated mice. Values in both A and B are means ± SE [n = 6 for each group; ***P < 0.001 vs. the increases in kidney/body weight ratios (A) or protein/DNA ratios (B) of vehicle-treated S6K1^+/+ mice]. The increases in kidney/body weight ratios and protein/DNA ratios did not show significant statistical difference among rapamycin-treated S6K1^+/+ mice, vehicle-treated S6K1^−/− mice, and rapamycin-treated S6K1^−/− mice.

Fig. 7.

S6K1 knockout had no significant effect on hyperglycemia but inhibited kidney hypertrophy in streptozotocin (STZ)-induced diabetes. A: S6K1 knockout did not affect the development of hyperglycemia in response to STZ injection. Male, 10-wk-old S6K1^−/− mice and S6K1^+/+ littermates were injected daily with STZ or vehicle (0.1 M citrate buffer, pH 4.5) alone for 5 consecutive days, and fasting blood glucose was measured as described in materials and methods. Ten days after completion of the STZ injections, which was equivalent to 7 days after diabetes, the animals were euthanized for the assessment of renal hypertrophy by increases in kidney/body weight ratios (B) as well as increases in protein/DNA ratios (C), compared with their respective mice injected with vehicle alone. Values are means ± SE. (n of 6 for each group; ***P <0.001 vs. S6K1^+/+ mice).

Fig. 8.

Diabetes stimulated rpS6 phosphorylation in S6K1^+/+ mice but not in S6K1^−/− mice. Samples from the kidneys described in Fig. 7 were subjected to immunoblotting analysis with an antibody specific for Ser235/236-phosphorylated rpS6 (A), followed by stripping and reprobing with antibody against total rpS6 (B). Another aliquot of the samples was immunoblotted with an antibody that recognizes the α, β, γ, and δ species of total 4E-BP1 (C) or an antibody specifically recognizing serine 65-phosphorylated 4E-BP1 (D). Equal loading was confirmed by immunoblotting with a β-actin antibody (E). Shown are representative blots from 1 of 3 separate experiments with similar results.